DNA encoding a cell membrane glycoprotein of a tick gut

The invention relates to antigens derived from ticks and to their purification. It also relates to genes encoding such antigens and to their cloning and expression from recombinant DNA molecules. Further, the invention describes the use of purified antigens and recombinant expression products having similar biological activity to those purified antigens to provide vaccines to protect cattle against tick infestation.

TECHNICAL FIELD 
This invention relates to an antigen isolated from the cattle tick 
Boophilus microplus and to the gene coding for that antigen and to the 
protein product of that gene. The antigen when used in part or in entirety 
as an immunogen administered to cattle as a vaccine results in the 
production by the cattle of an immune response which is capable of 
damaging ticks feeding on vaccinated cattle to such an extent that the 
survival of such ticks is decreased and/or the reproductive capacity of 
the ticks is decreased to such an extent that the antigen coded for by the 
gene can be used as an effective vaccine against said ticks. 
BACKGROUND ART 
On first infestation with ticks such as the cattle tick, Boophilus 
microplus, animals such as cattle are very susceptible to the parasite. 
Typically about 50% of the tick larvae which attach, complete the full 
life cycle to eventually drop off as engorged adults. On prolonged 
exposure to the parasite, cattle acquire some degree of immunological 
resistance to it, but this resistance reaches a relatively stable level at 
which economically important losses to cattle production still occur. The 
losses to production are largely due to losses of blood and tissue fluid 
taken up by the parasite during feeding. Additional losses are due to the 
hypersensitive or allergic response which animals develop to tick salivary 
and cement antigens in conjunction with natural immunity, a condition 
known as tick worry. 
A large number of approaches are used to control ticks. The most widely 
used is treatment of cattle with acaracides -chemicals which kill ticks. 
This approach has several short comings. For example resistance to the 
chemicals arises in the tick population and new classes of chemicals must 
be introduced frequently. The chemicals have little residual effect so 
cattle must be treated frequently in order to control the ticks 
effectively. The chemicals may have detrimental effects on the cattle, 
personnel and the environment. A second method for control of ticks is to 
breed for host resistance. Zebu breeds and Zebu cross breeds are more 
resistant to ticks than the highly susceptible British breeds. However 
Zebu crosses have behavioural problems, are less productive than pure 
British breeds and, even with the use of chemicals, the degree of 
resistance to ticks is far from ideal. Other methods of tick management 
such as pasture spelling and tick eradication present practical problems 
in most cattle producing areas throughout the world. An effective vaccine 
against ticks would provide a highly attractive alternative to the 
currently available methods of tick control. 
Intermittent attempts have been made in the past to immunise animals 
against ticks. (1-5, see 13 for review) The majority of these studies have 
used tick-host systems in which strong immunity seems to develop 
naturally, and have usually used laboratory animals as hosts. Usually the 
effects observed have been some reduction in engorgement weights and egg 
masses of adult ticks and some decrease in the viability of those eggs 
(1-5) although in two reports some decrease in the viability of engorging 
adults has been reported (3,4). Many of these studies have used antigens 
derived from salivary glands in order to attempt to mimic natural 
immunity. However, it is unlikely that a vaccine which mimics natural 
immunity would be of great commercial benefit due to the economic losses 
which still occur once natural immunity has been expressed and the 
deleterious effect of hypersensitivity responses to ticks. 
The alternative approach is to vaccinate animals with "concealed" or 
"novel" antigens, "Concealed" or "novel" antigens are, in this context, 
components of the parasite which can be used to raise a protective immune 
response in animals when used (in partially or fully purified form) to 
vaccinate those animals, but are antigens which are not involved in 
naturally acquired immunity. 
The successful vaccination against ticks using concealed or novel antigens 
has been reported (2,5). Animals were immunised with extracts of whole 
ticks or tick midgut. Immunization led to reductions in tick engorgement 
weights, feeding period, egg masses and egg viability but no significant 
increase in tick mortality was observed. However, the antigen fractions 
used in these experiments were so complex that it was not possible to 
identify the individual tick antigens which were responsible for the 
effects noted and the reasons for the effects were not investigated in 
detail. 
In a recent patent application (Australian Patent Application No. 
59707/86), claims are made that antigens derived from the synganglia of 
ticks can act as effective vaccines against tick infestation. However, 
there is no evidence presented in that patent that synganglia antigens can 
be effective alone. In this work dissected guts and synganglia were 
isolated, the gut cells were lysed, centrifuged and both the supernatant 
and pellet were used to vaccinate the same animals together, in some 
cases, with a cell suspension of synganglia. All cattle in the experiments 
reported were vaccinated with tick gut components and some received 
synganglia in addition. Therefore, it is clearly implicit in the 
experimental design that gut damage as a result of an immune response 
against gut components of ticks such as the gut cell antigens described 
herein and in the CSIRO patent application (45936/85), is an essential 
prerequisite for any secondary protective effects which may possibly 
result from an immune response against synganglia-specific antigens. 
In all of the examples cited above, the tick extracts which were used to 
vaccinate the animals were extremely complex. In the majority of the 
reports the fractions used were homogenates of tick organs and in some 
cases, the pellets derived therefrom by centrifugation. In this and the 
other studies, no data on the complexity of the fractions is presented but 
it is certain that they must contain many hundreds and probably thousands 
of components. In the one study where any purification and 
characterization of the protective fraction was carried out (Australian 
patent application No. 45936/85) the most highly purified fraction, GF 5/6 
was still very complex as will be shown below and it was not possible from 
this work to identify the individual component(s) of this fraction which 
were responsible for the protective immune response. In the present 
invention one such antigen is purified and characterized. 
Boophilus microplus presents a particularly challenging problem. Since the 
naturally-acquired immunity is only partially effective, duplication of 
natural immunity by artificial immunization would be of comparatively 
little commercial value. Boophilus microplus is a parasite of cattle and 
does not feed readily on laboratory animals. The possibility of inducing 
"unnatural immunity" to Boophilus microplus has been examined and shown to 
be possible (6, 7, 8, Australian Patent Application No. 45936/85). The 
practical exploitation of this, however, would require as a first step the 
isolation of the antigen or antigens responsible, and as a second step, 
the development of means by which the effective antigens could be produced 
in quantities which would be sufficient for commercial uses. 
The initial steps in the purification of the antigens in question and the 
demonstration of the efficacy of these antigens has been described 
previously (Australian Patent Application No. 45936/85). Briefly, ticks 
removed from cattle were disrupted, and sonicated, the cuticles and debris 
removed by low speed centrifugation, the supernatant was subjected to high 
speed centrifugation at 100,000.times. g for 1 hour, the membrane enriched 
pellet was extracted with a non-ionic detergent, the extract was subjected 
sequentially to chromatography on Sephacryl S-300 columns, broad range 
isoelectric focussing, narrow range isoelectric focussing and gel 
filtration chromatography on HPLC. At each step, fractions obtained were 
tested for efficacy as immunogens and the most highly protective fractions 
subjected to the next purification step. The most highly protective 
antigens were thus identified as being membranous, possessing an 
isoelectric point (pI) of between 5.05 and 5.65 and molecular weights in 
the range 205 to 79 kilodaltons. Other less highly protective fractions 
were also described and are of interest in both this and the preceding 
Australian Patent Application 45936/85. 
Further development of the purification procedure as described herein has 
enabled the most highly protective antigens to be more clearly defined and 
characterised more precisely and has enabled animals to be vaccinated with 
more highly purified immunogen preparations. One such antigen has been 
purified to near homogeneity and it has been shown that when cattle are 
vaccinated with this tick component an immune response is generated in 
those cattle which results in the death of the majority of ticks used to 
challenge those vaccinated animals. The antigen isolated from ticks has 
been shown to be a glycoprotein with a molecular weight of approximately 
89 kilodaltons and an isoelectric point in the range of 5.30 to 5.67. The 
method for the purification of this glycoprotein (referred to hereafter as 
the WGL.sup.+ antigen or WGL.sup.+) has been improved and a method is 
disclosed herein which results in a much larger yield of the antigen than 
could be obtained by the method previously described (Aust. Patent 
Application No. 45936/85). During this and previous work, other fractions 
which give protection have been identified. 
Having devised means by which the WGL.sup.+ antigen can be obtained in 
larger amounts (not sufficient for commercial uses), experiments have been 
performed to analyse the structure of parts of the protein portion of the 
antigen. The purified preparation was reduced and carboxy-methylated and 
digested with endoproteinase lys-C. The peptide fragments so produced were 
purified and the partial amino acid sequence determined for some peptides. 
This amino acid sequence data has enabled the design of oligonucleotides 
which have been used to isolate bacterial cells containing cDNA coding for 
the WGL.sup.+ antigen. 
Analysis of the DNA from these bacterial cells leads to the unambiguous 
identification of the gene coding for one protective antigen and the 
production of recombinant proteins which can be used as effective vaccines 
against ticks. These developments are the subject of the present 
invention. 
DEFINITIONS 
Whilst the invention provides products and processes suitable for the 
protection of cattle against tick infestation, it is to be understood that 
the principles of the invention can be equally applied to the protection 
of other animals such as horses, deer, goats, sheep, dogs, cats and pigs 
against tick infestation. 
It is recognised that the tick population worldwide is genetically diverse 
as is the case for all organisms which reproduce sexually. Each individual 
of a population differs subtly from the others in the population and these 
differences are a consequence of differences in the sequence of the DNA 
which each individual inherits from its parents. 
Further, random mutational events which can occur in either sexually or 
asexually reproducing organisms are a further source of genetic variation. 
Thus for each gene encoding a particular protein, there are likely to be 
differences in the sequence among the population of individuals. 
Such related molecules are referred to herein as homologues of antigens 
according to the invention and to the extent that they fulfill the 
functions of immunogens as defined herein they are included within the 
scope of the invention. 
Homologous antigens may be defined as antigens related by evolution but not 
necessarily by function. Similar but not necessarily identical DNA or 
protein sequences may be provided. It should be noted however that 
function in this sense relates to the natural in vivo function of the 
protein. 
Illustration of this point is provided by considering: 
1. WGL.sup.+ form Boophilus microplus and other tick species 
2. WGL.sup.+ from variants or different individuals of the Boophilus 
microplus population 
3. WGL.sup.+ and related gut cell plasma membrane glycoproteins from ticks 
which are homologues of the WGL.sup.+ antigen defined herein. 
It is stressed that for the purposes of this invention, homologues include 
only those WGL.sup.+ related plasma membrane glycoproteins which function 
as immunogens as defined herein. 
Such homologous WGL.sup.+ related plasma membrane glycoproteins may exist 
in the tick population worldwide and will be capable, when incorporated 
into a vaccine, of eliciting in animals vaccinated with those antigens an 
immune response which is capable of killing ticks, by damaging tick gut 
cells and which additionally results in a reduction in tick engorgement 
weights or otherwise damaging the surviving ticks in such a way that for 
example egg production by those ticks is decreased to such an extent that 
the vaccine can be used commercially agains infestation by tick species 
such as Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, 
Ambylomma spp, Dermacentor spp, Ixodes spp and Hyalomma spp, and 
especially from B. annulatus, B. decoloratus, Otobius megnini, 
Rhiphicephalus appendiculatus, Dermacentor andersoni, D. variabilis, 
Haemaphysalis longicornis, Ambylomma variegatum and Ixodes holocyclus. 
Further, it should be recognised that it is possible to generate chemicals 
which are not related to the WGL.sup.+ antigen by evolution or 
necessarily by structure but which may serve as immunogens to generate an 
immune response against protective epitopes on the WGL.sup.+ antigen and 
thereby act as effective vaccines. These molecules are referred to herein 
as analogues and to the extent that they fulfill the functions of 
immunogens as defined herein, they are included within the scope of the 
invention. Such analogues include chemically synthesized oligopeptide 
molecules with sequences corresponding to portions of the amino acid 
backbone of the WGL.sup.+ molecule, oligopeptides which when used as 
immunogens elicit an immune response which recognises native WGL.sup.+ 
antigen in ticks, carbohydrate structures from whatever source which when 
used as antigens elicit an immune response which recognises the WGL.sup.+ 
antigen in ticks, and anti-idiotype antibodies raised against the variable 
region of antibodies which recognise the epitope(s) of the WGL.sup.+ 
antigen. 
DISCLOSURE OF THE INVENTION 
In a first embodiment the invention provides an immunogen comprising an 
antigen derived from a tick species or tick cell line which antigen is 
capable of inducing immunity to tick infestation of a mammalian host to 
which said immunogen has been administered characterised in that said 
immunity results in the mammalian host producing an immune response which 
is capable of damaging the plasma membrane of the gut cells of ticks 
feeding on said host to such an extent that the majority of said ticks 
fail to survive to adult stage or surviving ticks become red in colour and 
the reproductive capacity of said surviving ticks is substantially 
decreased wherein said immunogen includes immunogens displaying similar 
immunological activity to said antigen including parts, analogues, 
homologues, derivatives and combinations thereof of said antigen. 
Preferably the antigen is derived from Boophilus microplus. 
In a preferred embodiment the immunity induced is immunity to infestation 
by a Boophilus species. 
More preferably the immunity induced is immunity to B. microplus 
infestation. 
However, immunity may also be induced to other species of ticks, including 
Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma spp, 
Dermacentor spp, Ixodes spp and Hyalomma spp and especially to other 
species of Boophilus such as B. annulatus or B. decoloratus. 
Of the other species of ticks against which immunity can be induced 
preferred species include Otobius megnini, Rhiphicephalus appendiculatus, 
Dermacentor andersoni, D. variabilis, Haemaphysalis longicornis, Ambylomma 
variegatum and Ixodes holocyclus. 
By immunization with related antigens isolated from other species of ticks 
including Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus 
spp, Ambylomma spp, Dermacentor spp, Ixodes spp and Hyalomma spp., 
immunity to infestation by other ticks may also be induced. Preferred 
species from which the related antigens are isolated include B. annulatus, 
B. decoloratus, Otobius megnini, Rhiphicephalus appendiculatus, 
Dermacentor andersoni, D. variabilis, Haemaphysalis longicornis, Ambylomma 
variegatum and Ixodes holocyclus. By protecting against infestation with 
ticks, the antigen may also provide protection against diseases caused by 
agents such as Babesia bovis, Babesia bigemina, Anaplasma marginale, 
Cowdria ruminantium, Theileria parva parva, T. parva lawrencii, T. 
annulata and T. hirci. 
In a second embodiment the invention provides a polynucleotide sequence 
comprising a first polynucleotide sequence which acts as a coding sequence 
for amino acid sequences of an immunogen according to the invention, a 
polynucleotide sequence which hybridises to said first sequence or a 
polynucleotide sequence related to said first sequence or hybridising 
sequence by mutation including single or multiple base substitutions, 
deletions, insertions and inversions. 
Preferably the polynucleotide sequence is a DNA sequence. 
In a further preferred form of the invention the DNA sequence is a cDNA 
sequence. 
The DNA sequence coding for part or all of the protective antigen isolated 
from Boophilus microplus can be used in DNA hybridization experiments to 
identify related DNA sequence from other species of ticks. These latter 
DNA sequences can be constructed by genetic engineering techniques to 
obtain the expression by bacterial or eukaryote cells such as yeast, 
plant, insect, tick or mammalian cell lines of all or parts of the antigen 
from other species of ticks and provide an effective vaccine against those 
tick species which are responsible for morbidity or economic losses to man 
or morbidity and productivity losses to animals. 
The invention also provides recombinant DNA molecule which comprises at 
least one DNA sequence according to the invention and vector DNA. 
In a preferred form the vector DNA comprises plasmid, phage or viral DNA. 
Preferred vectors include lambda gt11, pUR290, pUR291, pUR282, pUK270, 
pUC8, pUC9, baculovirus, pZipNeo, an SV40 based vector, lambda gt10, an 
EMBL vector, pBR327, pBR329, and pBR329 containing a par locus. 
The invention further provides a transformant cell line, said transformant 
carrying at least one recombinant DNA molecule according to the invention. 
In a further embodiment the invention provides a vaccine comprising at 
least one immunogen according to the invention together with a 
pharmaceutically acceptable carrier, adjuvant, immunopotentiator or 
diluent. 
In accordance with the present invention an antigen derived from a tick 
species which antigen is capable of inducing a highly significant degree 
of immunity to tick challenge when used to vaccinate cattle has been 
purified and characterised. Further, bacterial cells which contain DNA 
sequences derived from a tick species have been produced and those 
bacterial cells which contain DNA sequences encoding portions of the tick 
protective antigen have been identified. The DNA sequence of the tick gene 
encoding that antigen has been determined, the resulting DNA sequence has 
been used to identify further bacterial cells containing related genes 
from other species of ticks. Expression of the antigen or portions of the 
antigen by bacteria or other microorganisms or by eukaryotic cells such as 
yeast, insect, tick, plant and mammalian cells grown in vitro provides a 
large amount of the antigen effective as an immunogen for the protection 
of cattle and other domestic animals against infestation by Boophilis 
microplus and other tick species. 
The invention also includes within its scope the epitope or the epitopes of 
immunogens of the invention which are responsible for the protective 
immune response. These epitopes may be created artificially by the 
synthetic production of oligopeptides which contain sequences of portions 
of the protective antigen which can be predicted from the results of 
immunochemical tests on fragments of the protective antigen produced in 
bacteria or generated as a result of chemical or enzymatic cleavage of the 
native or recombinant peptides and includes relevant epitopes from those 
protective antigens, oligopeptides, idiotypes and anti-idiotypes which 
resemble or recognise those epitopes which may have protective effects 
when used to actively or passively immunise animals. 
In a further embodiment the invention provides methods for the purification 
of immunogens according to the invention and particularly protective 
antigens derived from ticks. 
The invention provides a process for the preparation of an immunogen 
according to the invention which process comprises a chromatographic step 
performed on wheat germ lectin or on a lectin having the same or similar 
terminal sugar specificity as wheat germ lectin. 
Preferably the invention provides a process for the preparation of an 
immunogen according to the invention said process comprising extracting 
membrane enriched fractions obtained from homogenised ticks with detergent 
and subjecting the solubilised material to wheat germ lectin sepharose 
chromatography and elution with N-acetylglucosamine or chromatography 
using a lectin having the same or similar terminal sugar specificity to 
wheat germ lectin. 
Preferably said detergent is selected from NP40, an NP40 derivative, 
Zwittergent 3-14 or SDS. 
The process may further comprise Concanavalin-A sepharose chromatography 
and elution with methyl-.alpha.-D-mannopyranoside, a preparative 
isoelectrofocussing step or size exclusion chromatography. 
In a preferred form said methods include preparation of an homogenate of 
ticks, centrifugation to produce membrane enriched fractions, treatment of 
those membranes with detergents such as Zwittergent 3-14, chromatography 
of the detergent soluble material on lectin affinity columns such as wheat 
germ lectin-Sepharose 6B columns, separation of the lectin binding 
antigens by isoelectric focusing in buffers containing detergent such as 
Zwittergent 3-14, chromatography of these antigens by size exclusion HPLC 
on columns such as Bio-Sil TSK 4,000 and PP 300 SW columns in series in 
buffers containing detergents and analysis of various fractions produced 
by SDS-polyacrylamide gel electrophoresis. 
The invention also provides an immunogen produced by a process according to 
the invention. Included within the scope of an immunogen produced by a 
process according to the invention are those immunogens produced as a 
result of purification schemes performed on native materials and 
recombinant or synthetic immunogens produced as a result of recombinant 
DNA or chemical synthetic methods respectively. 
In a further embodiment, the invention provides examples of methods for the 
treatment of the purified antigens with proteolytic enzymes such as endo 
lys-C, the purification of oligopeptide fragments produced as a result of 
proteinase digestion by HPLC chromatography on columns such as Aquapore 
RP-300 C-8 or Aquapore RP-318 columns and determination of the amino acid 
sequence of some of the oligopeptides so produced and purified. 
The invention further provides the peptide sequence information for such 
peptide fragments including: 
______________________________________ 
FRAGMENT NUMBER 
______________________________________ 
F1 (SEQ ID NO: 1) 
(K) D P D P G K 
F2 (SEQ ID NO: 2) 
(K) W Y E D (G) V L E A I (X) T S I G K 
F3 (SEQ ID NO: 3) 
(K) (X) Q A C E (H) P I G E (W) C M M Y P K 
F4 (SEQ ID NO: 4) 
##STR1## 
F5 (SEQ ID NO: 5) 
##STR2## 
F6 (SEQ ID NO: 6) 
##STR3## 
F7 (SEQ ID NO: 7) 
##STR4## 
F8 (SEQ ID NO: 8) 
##STR5## 
F9 (SEQ ID NO: 10) 
##STR6## 
F10 (SEQ ID NOS: 11 and 12) 
##STR7## 
F11 (SEQ ID NO: 13) 
(K) W Y E D R V L E A I R T S I G K 
F12 (SEQ ID NO: 14) 
(K) E S S I C X D F G N E F C R N A E C E V V P 
F13 (SEQ ID NO: 15) 
(K) T R E C S Y G R C V E S N P S K 
F14 (SEQ ID NO: 16) 
(K) A Y E C T C P R A F T V A E D G I S/H C K 
F15 (SEQ ID NOS: 17-19) 
##STR8## 
F16 (SEQ ID NO: 20) 
##STR9## 
F17 (SEQ ID NO: 21) 
(K) -- Q A C E H P I 
______________________________________ 
NOTE: 
Amino acids which were ascribed with low confidence are bracketed. 
X indicates no amino acid could be ascribed to this position; 
[ ] denotes mixed sequences. 
In a preferred embodiment of the invention, these peptide sequences are: 
__________________________________________________________________________ 
F1 (SEQ ID NO: 1) 
K D P D G K 
F2, F11 (SEQ ID NO: 13) 
K W Y E D R V L E A I R T S I G K 
F3, F17 (SEQ ID NO: 22) 
K L Q A C E H P I G E W C M M Y P K 
F4 (SEQ ID NO: 23) 
K E A G F V C K 
F5 (SEQ ID NO: 24) 
K G P D G O C I N A C K 
F6 (SEQ ID NO: 25) 
K A G V S C N E N E Q S E C A D K 
F8 (SEQ ID NO: 26) 
K D Q E A A Y K 
F9, F10 (SEQ ID NOS: 27-29) 
K C P R D N M Y F N A A E K 
K A N C Q C P P D T K P G E I G C I E 
K A N C Q C P P D T R P G E I G C I E 
F12 (SEQ ID NO: 30) 
A E S S I C S D F G N E F C R N A E C E V V P G 
F13 (SEQ ID NO: 15) 
K T R E C S Y G R C V E S N P S K 
F14 (SEQ ID NOS: 31 and 32) 
K A Y E C T C P S G S T V A E D G I T C K 
K A Y E C T C P R A F T V A E D G I T C K 
F15 (SEQ ID NO: 33) 
K N L L Q R D S R C C Q 
F16 (SEQ ID NO: 34) 
K G T V L C E C P 
__________________________________________________________________________ 
The invention also provides examples of methods which can be used to design 
from the amino acid sequence data oligonucleotide sequences which are 
suitable for use as hybridization probes to identify nucleic acids 
sequences (DNA or RNA) coding for the polypeptide containing those amino 
acid sequences, methods for the construction of bacterial cells containing 
complementary DNA and genomic DNA fragments from ticks, the use of the 
oligonucleotides to identify bacterial cells containing complementary and 
genomic DNA fragments coding for that antigen, the DNA sequence of one 
such cDNA fragment, methods by which recombinant DNA technology can be 
used to produce bacterial or eukaryote cells which synthesize the protein 
or parts of that protein and methods for culturing those cells and for 
purification of the tick antigen or parts thereof to be incorporated into 
effective vaccines against ticks. 
In a preferred model, the mechanism of action of the vaccine is one in 
which an immune response is generated in vaccinated animals which results 
in ticks feeding on those animals ingesting components of the host immune 
system such as antibodies which interact with the surface of tick gut 
cells and either alone, or together with other factors in the host blood 
such as components of complement result in damage occuring such as lysis 
of the tick gut cells which in turn results in the ticks becoming unable 
to effectively digest blood, the tick gut becoming permeable to host blood 
components, to such an extent that host blood components such as albumin, 
haemoglobin, immunoglobulin and blood cells can be identified in the 
haemoloymph of the ticks and the ticks appear red in colour. This gut 
damage in turn results in the death of the majority of the ticks feeding 
on vaccinated animals before they reach engorgement stage and those few 
which may survive are so badly damaged that their engorgement weight is 
decreased and/or reproductive capacity is impaired (6,7,8). 
The invention also relates to antibodies generated against epitopes on the 
antigens according to the invention (so called idiotype antibodies) and to 
antibodies generated against the variable region of those first 
antibodies, (so called anti-idiotype antibodies) which mimic the 
protective epitopes on the antigen and may be used as effective vaccines 
in either passive protection of animals (idiotypes) or active immunization 
of animals (anti-idiotypes) and thereby result in effective protection.

BEST MODE OF CARRYING OUR THE INVENTION 
The invention is further described in the following examples which are 
illustrative of the invention but in no way limiting on its scope. 
______________________________________ 
SOURCE OF REAGENTS 
______________________________________ 
Sephacryl Pharmacia 
Sepharose 6 MB Pharmacia 
Zwittergent 3-14 Calbiochem 
Sephadex Pharmacia 
Brij 35 Sigma 
Bio-gel Bio Rad 
Cyanogen Bromide Sigma or Ajax 
Sarkosyl Sigma 
Endoproteinase lys-c 
Boehringer 
Triflouroacetic acid 
Pierce 
HFBA Pierce 
Acetonitrile Mallinckrodt 
Columns for HPLC Waters, BioRad, Beckman 
Poly U Sepharose Collaborative research 
Oligo dT cellulose Collaborative research 
dATP, dCTP, dGTP and dTTP 
Boehringer 
.sup.32 P-labelled deoxynucleic acid 
Amersham 
triphosphates 
Spermidine Calbiochem 
PEI cellulose Merck 
Concanavalin A-Sepharose 
Pharmacia 
CNBr-Sepharose Pharmacia 
Other chemicals used were of 
reagent grade. 
______________________________________ 
ABBREVIATIONS 
______________________________________ 
HPLC High performance liquid chromatography 
SDS Sodium dodecylsulfate 
EDTA Ethylenediaminetetraacetic acid 
WGL Wheat germ lectin 
WGL 1 Wheat germ lectin bound antigen pool 1 
WGL 2 Wheat germ lectin bound antigen pool 2 
WGL.sup.+ Wheat germ lectin bound antigen 
WGL.sup.- Wheat germ lectin unbound 
IEF Iso electric focussing 
LL Lentil lectin 
LL.sup.+ Lentil lectin bound antigen 
LL.sup.- Lentil lectin unbound 
HEPES N-2-Hydroxyethylpiperazine-N.sup.1 -2-ethane- 
sulfonic acid 
Endo lys C Endoproteinase lys C 
DTT dithiothreitol 
pI isoelectric point 
HFBA heptafluorobutytic acid 
BSA bovine serum albumin 
MMLV murine maloney leukemia virus 
dNTP deoxy nucleotide triphosphate 
dATP deoxy adenosine triphosphate 
dCTP deoxy cytidine triphosphate 
dGTP deoxy guanidine triphosphate 
dTTP deoxy thymidine triphosphate 
d(GCT)TP a mixture of dGTP, dCTP, and dTTP 
NAD nicatinamide adenine dinucleotide 
ATP adenosine triphosphate 
PEI polyethyleneimine 
BRL Bethesda Research Laboratories 
IBI International Biotechnologies Inc. 
A260, A280 Absorbance at 260 or 280 nm 
cDNA Complementary DNA 
ds double stranded 
g gram 
g.sub.av average gravity units 
m,.mu.,n,p (prefixes) 
milli, micro, nano, pico 
M Molar 
l liter 
U Units of activity (restriction enzymes) 
bp base pairs 
Kb Kilobase pairs (thousand base pairs) 
TLC Thin layer chromatograph 
ELISA enzyme linked immunoabsorbent assay 
______________________________________ 
BUFFERS 
______________________________________ 
10 .times. 1st strand 
0.5M Tris pH 7.5 
0.75M KCl 
0.03M MgCl.sub.2 
5 .times. 2nd strand (RNase H) 
0.2M Tris pH 7.5 
0.05M MgCl.sub.2 
0.1M (NH.sub.4).sub.2 SO.sub.4 
1M KCl 
1.5 mm B-NAD 
10 .times. Methylase Buffer 
0.5M Tris pH 7.5 
0.01M EDTA 
10 .times. TA Buffer 
0.33M Tris-Acetate pH 7.9 
0.66M K-Acetate 
0.1M Mg-Acetate 
5 .times. Kinase Buffer 
0.05M Tris pH 7.5 
0.05M Mg Cl.sub.2 
0.05M DTT 
0.5 mM Spermidine 
10 .times. Ligation Buffer 
0.3M Tris pH 8 
17 mM EDTA 
70 mM MgCl.sub.2 
10 mM ATP 
0.1M DTT 
20 ug/ml BSA 
1 mM Spermidine 
10 .times. High Salt Buffer 
1M NaCl 
0.5M Tris pH 7.5 
0.1M MgCl.sub.2 
10 mM DTT 
10 .times. S1 Buffer 
0.3M NaAcetate pH 4.4 
2.5M NaCl 
10 mM ZnCl.sub.2 
TE 10 mM Tris pH 7.5 
1 mM EDTA 
PEI cellulose buffer 
0.75M KH.sub.2 PO.sub.4 pH 3.5 
Buffer A 0.05M Tris 
0.03M acetic acid 
0.1M NaCl 
______________________________________ 
TEAB buffer 
TEAB is a solution of triethylamine equilibrated to pH 7 by bubbling 
CO.sub.2 through the solution. It is prepared as a 1M stock solution which 
is stored at 4.degree. C. The pH is checked before use, the solution being 
re-equilibrated with a CO.sub.2 pellet if required. 
EXAMPLE 1 
(a) Demonstration that at least some of the protective antigens are 
glycoproteins. 
Since the majority of plasma membrane proteins are glycoproteins, initial 
attempts at further characterization of the protective antigen(s) focussed 
on lectin affinity. 
It was found that wheat germ lectin and Concanavalin A bound to several 
components of tick antigen preparation B4/B5. Thus it appeared that a 
number of antigens in the tick preparation bear terminal 
N-acetylglucosamine residues and it is recognised that wheat germ lectin 
could be replaced in the purification scheme by other lectins with the 
same or similar terminal sugar specificity. 
Approximately 2.1 mg of antigen B4/B5 purified by the narrow range 
isoelectric focussing procedure (described in Australian Patent 
Application No. 45936/85) was applied to a column of WGL-Sepharose 6 MB 
(14) in 0.05M Tris chloride buffer, 1% Zwittergent 3-14, pH 8 and washed 
with the same buffer. Bound glycoproteins were then eluted with 100 mg/ml 
N-acetylglucosamine in the same buffer. Bound and unbound material was 
used to immunize sheep (Two vaccinations in Freunds incomplete adjuvant 
using five sheep per group). Induced immunity was estimated by applying 
freshly moulted adult ticks to the sheep and measuring the success of 
engorgement by the proportion of female ticks which finally engorged, 
relative to the number attached to the sheep skin three days after initial 
application of the young adults (Table 1). 
TABLE 1 
______________________________________ 
Immunization of Sheep with Glycoprotein Preparations 
Percentage of Ticks 
Group Engorging 
______________________________________ 
Controls 100, 100, 100, 100, 100 
Material not binding to WGL 
100, 6, 93, 100, 100 
Material binding to WGL 
0, 93, 0, 83, 28 
______________________________________ 
It is clear that some animals in each vaccinated group were highly 
protected from tick challenge. Serum was obtained from each sheep in this 
experiment after vaccination but before tick challenge and the antibody 
titres of each serum sample against the antigens used in the vaccine were 
measured by radioimmunoassays. The animals in each group which showed tick 
damage had high antibody titres against the antigen preparation injected 
whereas those which had low titres allowed large numbers of ticks to 
engorge without any visible signs of damage (data not shown). It appears 
that protective antigens were present in both fractions used in this 
experiment but failure to observe tick damage with some animals was due to 
the failure of those animals to respond vigorously to vaccination for 
reasons which are currently unclear. 
(b) In a subsequent experiment with sheep, fraction GF5 and 6, the more 
highly purified gel filtration fractions (Australian Patent Application 
No. 45936/85) were chromatographed on a WGL-sepharose affinity column and 
the specifically bound and the unbound material was used to vaccinate 
sheep in a similar way to that described above. Again, for some animals in 
each group, the immune response generated by vaccination with either 
fraction was capable of producing damage to ticks feeding on those animals 
as demonstrated by the lower numbers of viable ticks recovered from the 
sheep (% surviving), the percentage of those ticks where were red in 
colour (% damage) and the lower weight of those ticks which survived or 
engorged (Table 2). 
TABLE 2 
__________________________________________________________________________ 
Number of Ticks 
Surviving/Number 
% Surviving Mean 
Group Animal No. 
Applied Ticks % Damage 
Weight 
__________________________________________________________________________ 
Controls 
181 36/40 90% 0 254 
182 45/50 90% 4 224 
183 32/40 80% 3 214 
WGL 121 27/40 67.5% 19 182 
unbound 
122 27/40 67.5% 41 179 
123 30/40 75% 3 223 
WGL bound 
124 27/40 67.5% 52 156 
125 7/40 17.5% 100 11 
180 9/40 22.5% 100 11 
__________________________________________________________________________ 
In particular the material which was specifically bound to the affinity 
column is to be characterized herein but the protective antigens in the 
unbound fraction are also clearly capable of giving protection. 
(c) An experiment similar to that described above was performed in which 
cattle were vaccinated with material which had specifically bound and 
material which failed to bind to a WGL-Sepharose column (Table 3). Again, 
the immune response generated by both fractions following vaccination gave 
indications of damage to ticks feeding on vaccinated cattle. The material 
which failed to bind to the WGL-Sepharose column was particularly 
effective in this experiment. 
TABLE 3 
______________________________________ 
Animal 
Group No. Tick No. % Damage 
Weight (mg) 
______________________________________ 
Controls 943 239 2 226 
944 190 7 216 
957 282 1 245 
WGL bound 945 214 22 202 
960 125 64 183 
962 188 5 218 
WGL unbound 
938 19 84 148 
950 10 97 164 
959 25 92 140 
______________________________________ 
NOTE: Tick no. in this and subsequent experiment refers to the average 
number of engorged female ticks dropping from each animal per day. Three 
weeks after vaccination, cattle are challenged with approximately 1000 
larvae per day for a period of at least 16 days. When the ticks mature an 
engorged female ticks are observed, the engorged female ticks are 
collected each day and counted for a period of at least 16 days. This 
number is averaged over that period and presented in the Tick no. column. 
On each day during this period, the number of ticks which are visibly 
damaged are scored (red ticks) and that proportion listed in the % damage 
column. The average weight of the engorged females is also determined. 
(d) Concurrently with this experiment, the material which had specificall 
bound to the WGLsepharose column was fractionated on SDS polyacrylamide 
gels. Silver stains of these gels showed two major staining components 
which were excised (fractions S2 and S4) and these, as well as the 
intermediate portions of the gels (fractions S1, S3, S5 and S6) were used 
to vaccinate cattle. The most highly protective fraction was S2 (Table 4) 
which corresponds to one of the bands observed in stained gels which has 
an apparent molecular weight of approximately 80-90 kilodaltons in this 
gel system compared with Pharmacia and BRL molecular weight markers. 
In this experiment, the number of ticks surviving on the cattle vaccinated 
with S2 was reduced compared with the other groups (Tick No column--the 
average number of engorged adult female ticks dropping from each animal 
per day over the 21 day period studied). In addition, the majority of the 
surviving ticks were red or appeared to be otherwise abnormal when 
examined visually (% damage) and the weight of those surviving ticks in 
the S2 group was reduced compared to the ticks from the animals in the 
other groups (Table 4). 
TABLE 4 
______________________________________ 
Group Animal No. Tick No. % Damage Weight 
______________________________________ 
S1 947 196 10 215 
951 194 1 230 
963 243 30 198 
S2 941 115 66 147 
942 86 87 150 
953 173 32 192 
S3 961 166 3 212 
967 240 4 243 
968 193 4 233 
S4 939 163 1 229 
940 155 4 229 
952 149 9 258 
S5 937 276 3 248 
955 232 2 225 
956 160 5 221 
S6 946 269 12 222 
954 157 19 297 
958 281 1 245 
______________________________________ 
(e) In vitro experiments were conducted in which a range of lectins were 
tested to determine which were capable of reacting with the material not 
retained on the WGL-sepharose column. Lentil lectin was found to be 
reactive and therefore the material not bound to the WGL-Sepharose was 
fractionated on a lentil lectin column (15). Cattle were vaccinated with 
these fractions, and the immune response generated against the material 
not bound to WGL-sepharose but bound to the LL-sepharose was found to 
result in some small indication of damage to ticks feeding on vaccinated 
cattle (Table 5). SDS gel analysis of this fraction shows a band which has 
a molecular weight which is in the same range as the S2 antigen identified 
in the previous experiment. 
TABLE 5 
______________________________________ 
Group Animal No. Tick No. % Damage 
Weight 
______________________________________ 
Controls 990 195 0.7 252 
980 220 0.7 243 
979 248 0.6 252 
WGL unbound 
1006 183 6.2 196 
LL unbound 
1002 188 0.3 233 
988 185 30.8 197 
WGL unbound 
1001 270 3.9 244 
LL bound 996 267 1.1 252 
994 249 16.1 206 
______________________________________ 
Both fractions used in this experiment were capable of generating an immune 
response which was capable of giving some indication of protection in this 
experiment. 
The lentil lectin chromatography step produced a far greater yield of 
material having a similar molecular weight to the S2 antigen than was 
produced by the wheat germ lectin chromatography step. 
This similarity in molecular weight and difference in lectin affinity 
suggested that the molecules may have been related by a common peptide 
backbone but differed in glycosylation. 
This was later disproved (Example 2g). 
Due to the presumed similarity to S2 and greater abundance of the LL bound 
material it was proposed that this material be used as a starting material 
for further purification. 
However, subsequent poor vaccination results with this material in the 
light of good vaccination results with WGL bound material (Example 3) and 
demonstrated differences in amino acid composition have led to further 
purification schemes and cloning schemes being developed for the S2 or 
WGL.sup.+ material. 
EXAMPLE 2 
Knowing from the above results the iso-electric point, molecular weight and 
lectin binding characteristics of the major protective antigen (referred 
to above as S2), a number of experiments were performed in order to 
improve the efficiency of the isolation procedure. The following method 
has been devised which yields at least 10 times more of the S2 antigen 
(later referred to as the wheat germ lectin bound antigen, WGL.sup.+ 
antigen or WGL.sup.+) and lentil lectin bound antigen (later referred to 
as the LL.sup.+ antigen or LL.sup.+) than the methods described in 
Australian Patent Application No. 45936/85. 
The procedure is outlined in the flow charts (FIGS. 1, 2, 3, and 4). 
Improvement of the Procedures for Isolation of the Major Protective Antigen 
(a) Isolation and Extraction of Tick Membrane and Particulate Material 
1290 grams of semi-engorged adult female Boophilus microplus were picked 
from cattle on the day prior to the completion of engorgement. They were 
homogenised in 0.05M Tris, 0.025M acetic acid, 0.1M sodium chloride, 1 mM 
EDTA, the homogenate strained through fine gauze and the retained 
material, which was mostly cuticle fragments, was rinsed with buffer. A 
total of 3 ml of buffer per gram of ticks was used in the extraction. The 
suspension of tick material was then mixed with 350 mg 
phenylmethanesulfonyl fluoride per liter and centrifuged at 600.times. 
g.sub.av for 15 min. The supernatant was then centrifuged at 20,000.times. 
g.sub.av for 30 min and the supernatant from that, centrifuged for 
100,000.times. g.sub.av for 1 h. Precipitates were collected from each of 
these centrifugation steps and frozen at -20.degree. C. until used. 
The 600.times. g, 20,000.times. g and 100,000.times. g precipitates were 
thawed, suspended in buffer A (0.05M Tris, 0.03M acetic acid) and the 
protein concentration was measured. The suspension was diluted in buffer A 
containing Brij 35 to final protein and detergent concentrations of 5 and 
10 mg/ml respectively. The tick material was extracted at 37.degree. C. 
for 1 h then centrifuged at 3,300.times. g.sub.av for 30 min at 20.degree. 
C. The precipitate was resuspended in buffer A and the protein 
concentration re-assayed. Extraction was repeated at the protein and 
detergent concentrations used before, substituting Zwittergent 3-14 for 
Brij 35, while the extraction time was lengthened to 90 min. The 
suspension was centrifuged as before and the supernatant was retained. 
(b) Lectin Affinity Chromatography and Isoelectric Focussing (FIG. 1) 
The supernatant from the Zwittergent 3-14 extraction, (3255 ml), was 
stirred with 90 ml of WGL Sepharose for 16 h at 20.degree. C., filtered 
and the WGL-Sepharose conjugate was poured into an 18.times.2.5 cm column, 
washed with buffer A containing 1% Zwittergent 3-14 then eluted in buffer 
A containing 1% Zwittergent 3-14 and 100 mg/ml N-acetylglucosamine. 
Fractions were pooled on the basis of the A280 absorption of specifically 
eluted material to give wheat germ lectin bound pool 1 (WGL1). 
The adsorption of the detergent supernatant with WGL-Sepharose and 
subsequent elution of bound material was repeated as described above to 
give WGL2. The two eluates were then pooled (WGL pool). 
The WGL pool was dialysed against 2.times.2.5 liters of water, then against 
0.05M Tris-chloride buffer pH 7.5 containing 0.1M ammonium thiocyanate. 
Concanavalin A-Sepharose (Pharmacia) was poured as a 2.5.times.11 cm 
column and washed in buffer containing 0.05M Tris, 1% Zwittergent 3-14, 
0.1 mM calcium chloride, 0.1 mM manganese chloride, 0.1M ammonium 
thiocyanate, adjusted to pH 7.5 with hydrochloric acid. The WGL pool was 
loaded on this column, washed and the specifically bound material was 
eluted in the same buffer to which had been added 50 mg/ml 
methyl-.alpha.-D-mannopyranoside. Fractions were pooled, dialysed against 
water then subjected to preparative isoelectricfocussing. 
Isoelectricfocussing was carried out in a flat bed of IEF Sephadex 
containing 1% (w/v) Zwittergent 3-14 and Pharmalyte 4-6.5 diluted 1 to 15 
(v/v) for 10,000 Vhr. Individual fractions were analysed by SDS gel 
electrophoresis. The required protein appeared to be present in fractions 
with pI's of 5.3 to 5.7 though, for the sake of better purification, only 
those fractions with pI's of 5.4 to 5.6 were pooled to give "WGL post IEF 
pool". 
The Zwittergent 3-14 soluble material left after the second extraction with 
WGL-Sepharose was mixed with 70 ml of LL-Sepharose and stirred for 24 h at 
20.degree. C., the suspension was filtered and the collected Sepharose 
conjugate was poured as a 2.5.times.14 cm column. This was then washed 
with Tris-acetate, 1% Zwittergent 3-14 buffer and eluted in the same 
buffer containing 50 mg/ml methyl-.alpha.-D-manno-pyranoside. Fractions 
were pooled on the basis of their A280 and dialysed against water. Further 
fractionation was carried out by preparative isoelectric focussing using 
the conditions already described for material which bound to WGL. 
Fractions were analysed by SDS polyacrylamide gel electrophoresis. The 
protein being isolated focussed over a pI range of 4.8 to 5.2 though the 
fractions which were pooled for further purification covered the range of 
4.8 to 5.0. 
The LL unbound material from the first affinity chromatography was 
readsorbed to LL-Sepharose and the material specifically eluted with 
methyl-.alpha.-D-mannopyranoside was separated by IEF. Material with the 
same pI range of 4.8 to 5.0 was pooled, then the products of the two 
experiments mixed to give "LL post IEF pool". 
The method for the isolation of "WGL post IEF pool" and "LL post IEF pool" 
is shown schematically in FIG. 1. 
(c) Hydrophobic Chromatography of LL Post IEF Pool (FIG. 2) 
A 1.6.times.6.5 cm column of LL-Sepharose was equilibrated in 0.1M 
Tris-acetate buffer, 1% Zwittergent 3-14 pH 8.0. The "LL post IEF pool" 
was adjusted to pH 7.1 and applied to this column which was subsequently 
washed with buffer, then with 0.1M Tris-acetate buffer, 0.1% Brij pH 7.5. 
Bound material was then eluted with 0.1M Tris-acetate-Brij buffer 
containing 50 mg/ml methyl-.alpha.-D-mannopyranoside. 
Eluted material was dialysed against 0.1M Tris-acetate-Brij buffer then 
ammonium sulfate was added to a final concentration of 0.5M. The sample 
was applied to a 7.5.times.75 mm TSK phenyl-5-PW column which had been 
equilibrated in 0.1M Tris-acetate, 0.5M ammonium sulfate, 0.1% Brij, pH 
7.5 and, after washing, the column was resolved with a linear gradient 
from this starting buffer to a buffer containing 0.1M Tris-acetate, 0.1% 
Brij pH 7.5. Fractions were analysed by SDS gel electrophoresis and those 
containing the required protein pooled to give "LL.sup.+ antigen" or 
LL.sup.+. 
This procedure is shown schematically in FIG. 2. 
(d) Size Exclusion Chromatography of WGL Post IEF Pool (FIG. 3) 
The pH of the "WGL post IEF pool" was increased to 7.3 and the material 
then loaded on a column of WGL-Sepharose equilibrated in 0.05M 
Tris-chloride, 0.2% Zwittergent 3-14 pH 7.5. The column was washed with 
0.05M Tris-chloride, 0.1% SDS, then bound material eluted in 
Tris-chloride-SDS buffer containing 100 mg/ml N-acetylglucosamine. 
Fractions were analysed by SDS electrophoresis and those containing the 
required protein pooled, dialysed against 0.05M Tris-chloride buffer pH 
7.5 and concentrated on a Savant Speedvac. 
Size exclusion chromatography was carried out using a Waters HPLC system 
and, in sequence, an Si200 Polyol guard column (Serva, Heidelberg), a 
7.5.times.30 cm Bio-Sil TSK 4000 and a 7.5 mm.times.30 cm PP 300 SW 
(Waters). Chromatography was carried out in a buffer containing 0.05M 
HEPES, 0.1M sodium thiocyanate, 0.1% SDS, the pH adjusted to 7.0 with 
sodium hydroxide, at a flow rate of 1 ml/min and a column temperature of 
37.degree. C. In this system, bovine serum albumin had an elution time of 
13.8 min and ribonuclease A of 17.7 min. Fractions were analysed by SDS 
gel electrophoresis. The material of interest was found to elute from the 
HPLC column at between 14.0 and 15.0 min and these fractions were pooled. 
The product of this step still contained some impurity of lower molecular 
weight. It was therefore loaded on a 0.6.times.10 cm column of 
WGL-Sepharose in 0.05M Tris-chloride, 0.1% SDS pH 7.5, washed in this 
buffer, then the bound material was eluted in the same buffer containing 
firstly 20 mg/ml then 100 mg/ml N-acetylglucosamine. Fractions were 
analysed by SDS gel electrophoresis and pooled on a basis of the amount 
and purity of the desired protein in each. They were concentrated and 
re-chromatographed on HPLC size exclusion chromatography as described 
above. The final pool of fractions containing the desired antigen 
("WGL.sup.+ antigen") was made after analysis by SDS gel electrophoresis 
as described above. 
This procedure is shown schematically in FIG. 3. 
(e) Protein Determination 
Four methods of protein determination were used during antigen isolation, 
the methods being chosen on a basis of sensitivity required and the nature 
of expected interfering substances. These methods, and the abbreviations 
used for them in FIGS. 1, 2 and 3 were: 
1. Biuret method; abbreviated (B) 
2. Spectrophotometric method, from A280 and A260 measurements; abbreviated 
(S). 
3. Fluorescence method, from the integrated fluorescence of high molecular 
weight material after derivatization with o-phthalaldehyde; abbreviated 
(F). 
4. Absorbance method, based on the integrated A280 from HPLC 
chromatographic runs, assuming that a 1 mg/ml solution of the protein in a 
1 cm light path had an absorbance at 280 nm of 1; abbreviated (A). 
(f) Comments on the Isolation Procedure 
The major residual problem with the procedure described above is that in 
some preparations of the WGL.sup.+ antigen, a contaminant of lower 
molecular weight was observed as judged by SDS polyacrylamide gel 
electrophoresis. This contaminant could be partially, though not entirely, 
removed by repeating the affinity chromatography on WGL-Sepharose in SDS 
buffer and elution at two concentrations of N-acetylglucosamine. 
The amounts of this impurity are variable from preparation to preparation. 
In a subsequent antigen isolation it was present in minor amounts and good 
antigen purity was obtained after pooling fractions with pI's in the range 
5.30 to 5.67 on preparative isoelectricfocussing, followed by a single 
HPLC size exclusion chromatography. The yield of WGL.sup.+ antigen was 
thus higher (approximately 300 .mu.g from 1.3 kg of ticks). 
FIG. 5 shows SDS-polyacrylamide gel profiles of fraction GF 5/6, the 
starting material in this work, (lane 2) and of the purified WGL.sup.+ 
antigen (lanes 4 & 5) and LL.sup.+ antigen (lanes 6 & 7) together with 
appropriate molecular weight markers (lanes 1, 3 & 8). It is clear from 
these gels that the GF 5/6 fraction is very impure and contains a large 
number of components in addition to the WGL.sup.+ antigen which is in 
fact such a minor component that it can not be distinguished from the 
other components in the fraction. The WGL.sup.+ and LL.sup.+ antigens 
are highly purified. In lane 5 which is an overloaded sample of WGL.sup.+ 
antigen, a small amount of the contaminating material at lower molecular 
weight can just be seen. 
(g) Amino Acid Composition of WGL.sup.+, and LL.sup.+ Antigens 
Samples of the WGL.sup.+ and LL.sup.+ antigens isolated by the new 
purification procedure were analysed by amino acid analysis. The HPLC 
plots and calculated amino acid compositions derived from the HPLC 
printout by integration of the areas under each peak (Table 6) indicate 
that the antigens have different amino acid compositions. In addition the 
antigens clearly have different terminal sugar residues accounting for the 
different lectin binding characteristics. 
TABLE 6 
______________________________________ 
Amino Acid Compositions of Tick Antigens 
(mole %) 
WGL.sup.+ LL.sup.+ Antigen 
______________________________________ 
Asp 7.4 11.0 
Glu 6.8 10.3 
Ser 9.7 7.4 
Gly 7.4 10.5 
His 2.9 2.9 
Arg 5.0 5.2 
Thr 9.0 5.6 
Ala 9.1 6.8 
Pro 5.9 5.2 
Tyr 4.8 3.9 
Val 7.9 6.5 
Met 1.9 2.9 
Cys 1.4 0.5 
Ile 4.7 4.5 
Leu 6.6 8.8 
Phe 4.1 4.0 
Lys 5.4 3.8 
______________________________________ 
NOTES: 
Trp is destroyed in this assay. The results presented are obtained from 
samples taken after 24, 48 and 72 hours of acid hydrolysis. 
EXAMPLE 3 
Vaccinal Activities of WGL.sup.+ and LL.sup.+ Antigens 
Samples of WGL.sup.+ antigen (21 .mu.g) and LL.sup.+ antigen (400 .mu.g) 
were homogenised in Freunds Complete adjuvant and used to vaccinate cattle 
(1/10 of each preparation per animal per vaccination) as described in 
Australian Patent Application No. 45936/85. Vaccinated animals, together 
with control cattle were challenged with ticks and the numbers of engorged 
female ticks dropping from the experimental animals was monitored over a 
16 day period (Table 7). It is clear that cattle vaccinated with very 
small amounts of WGL.sup.+ antigen were strongly protected from 
infestation in that the number of ticks dropping from each animal per day 
was reduced, the weight of the surviving ticks was lower and a high 
proportion of the surviving ticks were visibly damaged as a result of gut 
damage allowing cattle blood components to pass into the haemolymph of the 
ticks (% Red column). In addition, the ticks which survived on the cattle 
vaccinated with the WGL.sup.+ antigen had a greatly reduced capacity to 
produce eggs compared to the control animals. 
TABLE 7 
______________________________________ 
Animal 
Wt. eggs/ Tick 
No. Wt. Ticks Antigen No. Tick Wt. 
% Red 
______________________________________ 
26 0.49 Controls 199 224 6 
29 0.52 237 231 3 
31 0.47 227 220 1 
28 269 223 9 
36 WGL.sup.- 186 228 3 
40 LL.sup.- 170 199 4 
27 272 224 2 
35 LL.sup.+ antigen 
338 262 0 
37 (130 .mu.g) 238 233 1 
30 0.16 25 152 86 
32 0.25 WGL.sup.+ antigen 
135 175 79 
34 0.22 (7 .mu.g) 38 152 70 
______________________________________ 
The LL.sup.+ antigen at a higher dose failed to give significant 
protection to the cattle despite the fact that the cattle had mounted a 
strong immune response to the vaccine as determined by ELISA [data not 
shown]. 
Both WGL.sup.+ and LL.sup.+ antigens appeared to be largely pure by SDS 
gel electrophoresis (FIG. 5) and both have similar molecular weights of 
approximately 89 kd in the gel system used compared to the BRL molecular 
weight standards used. 
The new purification procedure outlined above is an improvement over that 
used previously giving a yield of 33-300 .mu.g WGL.sup.+ antigen compared 
with approximately 3 .mu.g of "S2" antigen per 1.29 kg tick starting 
material. It is asserted that these two antigens (WGL.sup.+ and S2) are 
the same glycoprotein based on similar molecular weight, isoelectric 
point, lectin binding properties, amino acid composition and vaccinal 
efficacy. 
EXAMPLE 4 
Digestion of WGL.sup.+ antigen with endoproteinase lys-C, separation of 
oligopeptides, determination of the amino acid sequence of oligopeptides 
and design of oligonucleotide sequences suitable as hybridization probes 
to detect recombinant organisms containing DNA sequences coding for the 
WGL.sup.+ peptide. 
Approximately 40 .mu.g of WGL.sup.+ antigen purified as described in 
Example 2 was mixed with 100 .mu.l of 0.1M Tris-chloride buffer pH 8.3 
containing 20 mM dithiothreitol and 2% (.sup.w /v) SDS, then incubated at 
56.degree. C. for 30 min. The solution was then cooled to room temperature 
and sodium iodoacetate added to a final concentration of 0.14M. After 45 
min. in the dark, cold methanol was added in a ratio 9:1 methanol:sample 
(.sup.v /v). The sample was stored at -20.degree. C. overnight, 
centrifuged, the supernatant removed and the precipitate dried. 
The precipitate was then dissolved in 76 .mu.l of 0.1M Tris-chloride buffer 
containing 4M urea, pH 8.5, then 4 .mu.l of endo lys C (6 units per ml) 
was added. After 2 hrs at 37.degree. C., another 4 .mu.l of enzyme was 
added and the digestion was continued for a further 17 hrs. 
The digest was applied directly to an Aquapore RP-300 C-8 column in 0.1% 
trifluoroacetic acid and peptides were eluted in a linear gradient from 
0-60% .sup.v /v acetonitrile/water in 0.1% trifluoroacetic acid. If 
necessary, peptides were rechromatographed in the same solvent system 
using an Aquapore 318 column. Peptides were collected, concentrated to 
50-100 .mu.l by rotary dessication in a rotary evaporator. The amino acid 
sequences of the oligopeptides were determined using an Applied Biosystems 
amino acid sequencer. The following peptide sequences were obtained. The 
one letter and 3 letter codes used for amino acids are shown in Table 8. 
Fragment Number 
F1(SEQ ID NO:1) (K).sup.1 D P D P G K (20-mer oligonucleotide) 
F2(SEQ ID NO:2) (K).sup.1 W Y E D (G).sup.2 V L E A I (X).sup.3 T S I G K 
(50-mer oligonucleotide) 
F3(SEQ ID NO:3) (K).sup.1 (X).sup.4 Q A C E (H).sup.2 P I G E (W).sup.2 C M 
M Y P K (53-mer oligonucleotide) 
(C).sup.5 
F4(SEQ ID NO:4) (K).sup.1 E A G F V Q K (23-mer oligonucleotide) 
In addition, the following peptide sequences were deduced from mixed 
sequences which may assist in the characterization of the clones although 
there is a great deal of uncertainty in some of these sequences 
(especially F7). 
__________________________________________________________________________ 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
Oligonucleotides may be prepared using these amino acid sequences. 
For example the following could be used..sup.7 
20-mer (SEQ ID NO: 37) .sup.5' T T A C C T G G A T C T G G A T C C T 
T.sup.3' 
50-mer (SEQ ID NO: 38) TTA CCA ATG GAT GTA CAA ATA GCT TCA AGG ACA CCA 
TCT TCG TAC CAC TT 
##STR15## 
__________________________________________________________________________ 
NOTES: 
The following assumptions were made in interpreting the peptide 
sequences and in designing oligonucleotides probes: 
Numbers 1-6 refer to superscripts in the peptide sequence listed 
above. 
1. It was assumed that a lysine (K) preceded the first amino acid which 
was determined for each peptide based on the specificity of the endo 
lys-C. 
2. These amino acids were assumed to be correct although they were 
detected at lower molar ratios than expected. 
3. No amino acid could be confidently ascribed to the positions shown as 
X. 
4. This position contained a number of amino acids. For the design of 
oligonucleotides, the correct amino acid was assumed to be either D, A or 
but may be another. 
5. More than one amino acid was detected in some sequences. The 
uncertainty is denoted by brackets. 
6. These sequences were mixed (square brackets) and the relative molar 
abundance of the amino acids detected was approximately the same in each 
cycle. 
7. A number of approaches known in the art can be used to design 
oligonucleotides suitable for use as hybridization probes. For example 
inosine base can be incorporated in positions where a number of 
deoxyribonucleotides are used in the third positions of redundant 
codons. 
The reverse complementary sequences to those presented can also be used 
equally well as hybridization probes. In the examples shown the codon 
usage 
was based on the sequence for the mRNA coding for the brine shrimp 
elongation factor (12). 
TABLE 8 
______________________________________ 
amino acid three letter code 
one letter code 
______________________________________ 
alanine ala A 
arginine arg R 
asparagine asn N 
aspartic acid asp D 
cysteine cys C 
glutamic acid glu E 
glutamine gln Q 
glycine gly G 
histidine his H 
isoleucine ile I 
leucine leu L 
lysine lys K 
methionine met M 
phenylalanine phe F 
proline pro P 
serine ser S 
thrionine thr T 
tryptophan trp W 
tyrosine tyr Y 
valine val V 
______________________________________ 
EXAMPLE 5 
Approximately 40 .mu.g of WGL.sup.+ antigen was digested with endo lys-C 
as described in Example 4. The digest products were applied to an Aquapore 
RP-300 C-8 column in 0.1% heptafluorobutyric acid (HFBA) and peptides were 
eluted in a linear gradient from 0-60% acetonitrile/water in 0.1% HFBA. 
Selected fractions were then re-chromatographed on Aquapore RP-300 C-8 or 
C-18 columns using trifluoroacetic acid in place of HFBA. The most 
symmetrical fractions were analysed for the presence of amino acids by 
hydrolysis of one tenth of the sample in hydrochloric acid vapour, 
derivatization with O-phthalaldehyde followed by reverse phase separation 
on HPLC and detection by fluorescence. The remaining portions of the 
samples were dessicated to 50-100 .mu.l volumes in a rotary evaporator and 
the amino acid sequence was determined using an Applied Biosystems amino 
acid sequencer. 
The following peptide sequences were obtained. 
__________________________________________________________________________ 
FRAGMENT NUMBER 
__________________________________________________________________________ 
##STR16## 
F11 (SEQ ID NO: 13) (K) W Y E D R V L E A I R T S I G K 
##STR17## 
51-merF13 (SEQ ID NO: 15) (K) T R E C S Y G R C V E S N P S K 
##STR18## 
##STR19## 
##STR20## 
F17 (SEQ ID NO: 21) (K) L Q A C E H P I 
__________________________________________________________________________ 
NOTES: 
It was assumed that a lysine precedes each fragment (K). X indicates 
that no amino acid could be confidently ascribed to the position during 
the 
peptide sequencing. F10 and F15 were mixtures of two and three peptide 
fragments repectively (denoted by []). 
F10 is the sequence of the same mixture of two peptides as analysed for F9. 
It is surprising that these two oligopeptides co-purified on both 
occasions as the peptide fractionation procedure was different in the two 
examples. 
F11 and F2 are likely to be the same fragment as the only differences are 
that the two uncertain amino acids in the F2 sequence are both R in the 
F11 sequence. A larger amount of material was present in F11 so this 
sequence is likely to be correct. 
F17 and F3 appear to be sequences of the same peptide. F3 could be read 
further as more material was present but F17 contained less impurities so 
the first residue could be identified. 
From these amino acid sequences, oligonucleotides can be prepared which 
would be suitable for screening cDNA and genomic DNA banks to identify the 
gene coding for the WGL.sup.+ antigen. The following examples could be 
used (see note 7 in Example 4. In the following examples, the third 
position in the codons was chosen to minimise secondary structure, not on 
brine shrimp usage as used in Example 4). 
##STR21## 
In addition, degenerate shorter oligonucleotides could be synthesized. For 
example 64 fold degenerate 17-mer oligonucleotides could be designed using 
the sequence DFGNEF from the F12 sequence and from the sequences KAYECT 
and YECTCP from the F14 sequence. 16 fold degenerate 17-mer 
oligonucleotide mixtures could also be designed using the sequence CMMYPK 
from the amino acid sequence of F3 shown in Example 4. These short 
degenerate sequences may be useful to confirm that clones isolated using 
long oligonucleotides contain the desired DNA sequences coding for the 
WGL.sup.+ antigen. 
EXAMPLE 6 
Genetic Engineering of the WGL.sup.+ (S2) Antigen 
The major limitation to the development of commercial vaccines using the 
WGL.sup.+ antigen is the limited availability of WGL.sup.+ material 
which can be obtained from natural sources. Means by which this limitation 
can be overcome include the construction by genetic engineering techniques 
of bacteria, yeast or other readily cultivated cells including mammalian 
or insect cell lines which synthesize large amounts of all or part of the 
WGL.sup.+ antigen. There are several means by which this goal can be 
achieved but they basically fall into a small number of steps which, by 
means of example only, are set out below. 
In order to identify the recombinant organisms which contain the tick 
genetic information coding for the protein backbone of the WGL.sup.+ 
antigen, appropriate reagents must first be generated. These may be 
antibodies from animals vaccinated with the purified protective antigen or 
with partially purified preparations containing that antigen preferably 
following denaturation of the antigen with a suitable detergent such as 
SDS. Bacterial yeast or other cells which synthesize part or all of the 
WGL.sup.+ antigen must then be constructed and screened with the 
antiserum. 
Preferably the WGL.sup.+ antigen is isolated in sufficient quantities in 
order to determine the amino acid sequence of the protein portion of the 
antigen or of fragments of the antigen produced as a result of 
endoproteolytic enzyme digestion using enzymes such as trypsin, endo lys C 
or pepsin or by chemical cleavage of the peptide using reagents such as 
cyanogen bromide. Fragments produced as a result of these treatments are 
separated and isolated using methods known in the art such as 
fractionation by HPLC reverse phase liquid chromatography or HPLC on 
columns containing hydrophobic resins, ion exchange resins or size 
fractionation resins. Preferably reverse phase resins such as C1, C8 or 
C18 are used singly or consecutively depending on the characteristics of 
the fragments produced as a result of the enzymatic or chemical treatment 
chosen. 
Fragments of the WGL.sup.+ peptide produced as a result of these 
treatments are then analysed on a gas phase amino acid sequenator using 
methods known in the art. From the amino acid sequence and the known DNA 
sequences which encode each amino acid, oligonucleotide sequences can be 
prepared which are complementary to the DNA sequence coding for the 
antigen and these can be used in hybridisation experiments to identify 
recombinant organisms containing the DNA coding for the antigen. The DNA 
sequence of these reacting clones can then be determined to confirm that 
the sequence is the one of interest. The DNA sequence can then be used to 
design the best means by which the microorganisms can be engineered to 
manufacture large amounts of the polypeptide. 
(a) Construction of Gene Libraries 
Readily cultivated microorganisms which contain the genetic information 
coding for the WGL.sup.+ protective antigen can be constructed using 
synthesised DNA which is complementary to the RNA isolated from the 
appropriate developmental stage of ticks or using DNA fragments isolated 
from any stage of ticks, preferably eggs or larvae as they will not 
contain bovine blood. 
If antibody probes are the only reagents available for detection of the 
clones containing the WGL.sup.+ antigen, cDNA or genomic DNA libraries 
must be constructed in a phage, viral or plasmid system which would result 
in the expression of the tick antigen or parts thereof. Such vectors 
include lambda gt11, bacterial plasmids such as pUR290, pUR291, pUR282, 
pUK270, pUC8, pUC9 or eukaryotic viral vectors such as the baculovirus, 
pZipNeo, or SV40-based vectors. 
If oligonucleotide probes are available, clone libraries can be constructed 
using cDNA or genomic DNA fragments in a larger range of phage, plasmid 
and viral systems including, for example, lambda gt10, EMBL vectors, or 
plasmid vectors such as pBR327, pBR329 or pBR322. 
Preferably cDNA libraries are generated since smaller numbers of clones 
have to be screened and any problems with expression through introns are 
avoided. Ideally the developmental stage of ticks which synthesis maximum 
levels of the WGL.sup.+ antigen are first identified. If antibodies are 
the only means available to do this, in vitro translation of RNA isolated 
from ticks of various ages followed by immunoprecipitation of the 
translation products with antibodies and analysis by SDS gel 
electrophoresis and fluorography enables the identification of the most 
suitable stage of ticks for extraction of RNA for construction of cDNA 
banks. The apparently low abundance of the WGL.sup.+ protein makes this 
approach very difficult. If oligonucleotides are available, hybridisation 
to the RNA isolated from ticks of various ages should enable the 
identification of the RNA source containing the highest abundance of mRNA 
coding for the WGL.sup.+ antigen. 
RNA can be isolated from ticks and cDNA synthesized and cloned a number of 
methods known in the art can be used. The following methods are outlined 
by means of example only. 
(b) Ethanol Precipitation 
In the following methods, ethanol precipitation involves adding to the 
solution of nucleic acid, one tenth of the solution volume of 3M sodium 
acetate and three to four volumes of absolute ethanol. The mixture is then 
stored at -20.degree. C. for at least 2 hrs or at -70.degree. C. or in an 
ethanol/dry ice bath until the solution becomes viscous. The mixture is 
then centifuged usually at 12,000.times. g.sub.av for at least 10 minutes. 
The supernatant is carefully removed and the pellet containing the nucleic 
acid material (as well as other macro-molecules) is used in further 
manipulations. 
(c) Ethanol Precipitation from 2M Ammonium Acetate 
In high salt solutions (e.g. 2M ammonium acetate), the majority of 
unincorporated deoxynucleotide triphosphates (and other small molecular 
weight material) will remain in the supernatant after an ethanol 
precipitation. The procedure is as described above except an equal volume 
of 4M ammonium acetate is added to the solution instead of the sodium 
acetate followed by 3-4 volumes of ethanol before cooling as described 
above. 
(d) Phenol or Phenol/chloroform Extraction 
Phenol or phenol/chloroform extraction involves the addition to the nucleic 
acid solution of an equal volume of redistilled phenol or a 1:1 (.sup.v 
/v) mixture of phenol and chloroform equilibrated with 0.1M Tris pH 8. The 
contents of the tube are mixed and the phases separated by centrifugation. 
The upper (aqueous) phase is removed to a fresh tube and the phenol or 
phenol/chloroform is discarded. Usually the aqueous phase is re-extracted 
and then extracted with ether to remove remaining phenol. Optionally the 
phenol or phenol chloroform phase from the first extraction may be 
re-extracted by addition of TE, mixing and centrifugation. In this case, 
the two aqueous phases would be combined before ether extraction and 
further processing. 
(e) PEI Cellulose TLC 
To monitor incorporation of radioactive dATP into nucleic acids during the 
various reactions in this procedure, thin layer chromatography on PEI 
cellulose was performed in 0.75M phosphate buffer pH 3.5. An aliquot of 
material to be monitored is applied toward one end of a strip of PEI 
cellulose and, after the chromatogram is resolved, the strip is exposed to 
an X-ray film. Following development of the autoradiograph, the areas of 
the PEI cellulose strip containing radioactivity are cut, placed in vials 
and the radioactivity in each determined by Cherenkov counting in a 
scintillation counter. The proportion of the radioactive material at the 
origin of the chromatograph can be used to determine the success of the 
reaction. This procedure is referred to as PEI cellulose chromatography. 
(f) Extraction of DNA and RNA 
High molecular weight DNA and RNA are isolated from ticks picked from the 
host at different developmental stages. Ticks are homogenised at room 
temperature in an Omnimixer for 2-3 minutes in a buffer containing 
guanidine isothiocyanate (4.7M), Sarkosyl (7.4%), Tris (5 mM) and 
.beta.-mercaptoethanol (70 mM). The homogenate is centrifuged at 4.degree. 
C. at 14,000.times. g.sub.av for 10 minutes. Solid CsCl is added to the 
homogenate (1 g/2.5 ml) which is layered onto a CsCl cushion (2.5 g/ml) 
and centrifuged for 48 hours at 25,000 rpm in a SW28 rotor (Beckman). The 
upper layer is aspirated, the DNA band recovered and the RNA pellet 
recovered, precipitated with ethanol, washed several times with 70% 
ethanol and stored in TE at -70.degree. C. until used. 
Polyadenylated mRNA can be isolated by passage over oligo dT cellulose 
columns or poly U Sepharose columns using methods described by the 
manufacturers (Collaborative Research). 
(g) cDNA Synthesis 
Several methods can be used for the construction of cDNA banks in phage or 
plasmid vectors. The following method by means of example only is a 
modification of the "RNase H" method for construction of cDNA banks in 
lambda gt11. The method is outlined schematically in FIG. 6. 
(h) First Strand Synthesis 
2 .mu.g of poly A.sup.+ RNA is dissolved in TE. Water is added to give a 
final volume of 25 .mu.l. The solution is heated at 70.degree. C. for 3 
minutes then rapidly cooled on ice. To the cooled solution is added 5 
.mu.l 10.times.1st Strand Buffer, 5 .mu.l 0.1M DTT, 5 .mu.l Oligo-dT 
[Boehringer 100 ng/1 .mu.l], 1.25 .mu.l RNasin [Promega 40 U/.mu.l], 2 
.mu.l BSA (5 mg/ml), 5 .mu.l 10 mM d(GCT)TP, 0.5 .mu.l 10 mM dATP and 3 
.mu.l M-MLV Reverse transcriptase [BRL 200 U/.mu.l]. 
2.5 .mu.l of the mixture is transferred to tube A (analytical reaction for 
monitoring synthesis) and 0.2 .mu.l [.sup.32 P]dATP is added (0.2 .mu.Ci). 
To the remaining bulk reaction 0.5 .mu.l 50 mM dATP is added. The tubes are 
incubated for 30 minutes at 42.degree. C. 0.25 .mu.l 10 mM dATP is then 
added to tube A and the incubations continued for a further 30 minutes. A 
0.5 .mu.l sample is taken from tube A and ethanol precipitated for gel 
analysis. A further 0.2 .mu.l sample is taken from the tube to be 
monitored by TLC on PEI cellulose. 
If all of the 2 .mu.g of RNA added to the reaction was poly A-adenylated, 
it can be calculated that approximately 30% incorporation of [.sup.32 
P]dATP into nucleic acids is equivalent to 100% efficiency in first strand 
synthesis. Commonly RNA passaged over Oligo-dT cellulose once yields 6-10% 
incorporation. 
To prepare a sample to monitor the second strand reaction, 2.5 .mu.l of the 
bulk reaction is removed and precipitated with ethanol from 2M ammonium 
acetate. The sample is washed twice with 70% ethanol then resuspended in 
2.5 .mu.l 1.times.1st Strand Buffer in tube B. 
(i) Second Strand (RNase H) 
A solution of; 28 .mu.l of water, 10 .mu.l 10.times. RNase H Buffer; 1 
.mu.l 5 mg/.mu.l BSA, 1.25 .mu.l 10 mM d(GCT)TP, 0.5 .mu.l 10 mM dATP, 1.6 
.mu.l RNase H [BRL 20 U/.mu.l], 5 .mu.l DNA Polymerase 1 [holoenzyme 
(Biolabs) 100 U/.mu.l] and 2 .mu.l E. coli DNA ligase, is prepared. 
The solution is mixed, then 2.5 .mu.l is dispensed into Tube B with 0.2 
.mu.l [.sup.32 P]dATP, 1.8 .mu.l is dispensed into Tube A and the 
remainder is dispensed into the bulk reaction tube. 0.75 .mu.l of 10 mM 
dATP is added to the bulk reaction tube. The three tubes are incubated at 
15.degree. C. for 60 minutes, then at 22.degree. C. for a further 60 
minutes. 
A 0.2 .mu.l sample from tube B is chromatographed on PEI cellulose to 
monitor the reaction. A further sample from tube B is ethanol precipitated 
from 2M ammonium acetate for gel analysis. The tube A sample from the 
first strand synthesis and the tube B second strand synthesis sample are 
run on a 1.5% agarose gel to determine the size of the cDNA which has been 
synthesized. 
To prepare a sample to monitor the T.sub.4 polymerase reaction, 0.5 .mu.l 
is taken from the bulk reaction tube and placed in Tube C. 
The remaining contents of Tubes A and B are pooled with the bulk reaction. 
The contents of both the bulk reaction, and Tube C are extracted with 
phenol/chloroform (1:1), precipitated with ethanol from 2M ammonium 
acetate and the precipitates are washed twice with 70% ethanol. 
(j) EcoR1 Methylation 
A solution of 29.5 .mu.l of water, 4 .mu.l 0.1M DTT, 2 .mu.l 10.times. 
EcoR1 Methylase buffer, 4 .mu.l 1 mM S-adenosyl methonine [Biolabs] and 
0.5 .mu.1 EcoR1 Methylase [Biolabs 20 U/.mu.l] is prepared in a fresh 
tube. 2 .mu.l of the mix is dispensed into Tube C and the remainder into 
the bulk reaction tube. The two tubes are incubated at 37.degree. C. for 
30 minutes then at 70.degree. C. for a further 15 minutes then cooled in 
ice. 
In a fresh tube, the following buffer is prepared: 4 .mu.l 10.times. TA 
buffer, 2 .mu.l 5 mg/ml BSA, 1.4 .mu.l 0.1M DTT, 2 .mu.l T.sub.4 DNA 
polymerase [Biolabs 1 U/.mu.l] and 29.5 .mu.l of water. 2 .mu.l is added 
to tube C which is then incubated at 37.degree. C. for 10 minutes. 0.5 
.mu.l of a solution containing 10 mM d(GCTA)TP is added to the remainder 
of the solution and this is added to the bulk reaction tube which is then 
incubated at 37.degree. C. for 50 minutes, 70.degree. C. for 15 minutes 
then ice quenched. 
To Tube C 0.2 .mu.l of each of 50 .mu.M d(GTC)TP, [.sup.32 P]dATP [0.2 
.mu.Ci] and 5 .mu.M dATP are added and incubation is continued at 
37.degree. C. for a further 50 minutes, after which time 0.2 .mu.l of the 
sample is spotted and chromatographed on PEI cellulose. 
0.2 .mu.l of 0.2 mM dATP represents approximately three times the amount of 
dATP required to add 2 adenosine residues to the 5' ends of each molecule, 
assuming that there was a total of 2 .mu.g of dscDNA of average size of 1 
kb synthesized after 2nd strand synthesis. 
(k) Kinase 
There is some indication that the kinase step is not necessary and can 
probably be omitted. To the bulk reaction 20 .mu.l 5.times. Kinase buffer, 
0.2 .mu.l 0.1M ATP, and 0.5 .mu.l polynucleotide kinase [Biolabs 4 
U/.mu.l] is added. The mixture is incubated at 37.degree. C. for 60 
minutes. The reaction is extracted with an equal volume of a 
phenol/chloroform mixture (1:1), the aqueous phases are pooled, 
precipitated by ethanol from 2M ammonium acetate then washed twice with 
70% ethanol. 
(l) Linker Ligation 
To monitor the linker ligation reaction, samples are prepared for agarose 
and polyacrylamide gel analysis. 
______________________________________ 
Agarose gel: 
Samples from bulk reaction (.sup.32 P cDNA) 
______________________________________ 
Sample 1 cDNA before ligation to cold linkers 
Sample 2 cDNA after ligation to cold linkers 
Sample 3 cDNA after ligation to cold linkers and 
digestion with Eco R1 
______________________________________ 
Polyacrylamide gel: 
.sup.32 P linker samples 
______________________________________ 
Sample 4 Tube D .sup.32 P linkers + cDNA before 
digestion with Eco R1 
Sample 5 Tube D .sup.32 P linkers + cDNA after 
digestion with Eco R1 
Sample 6 Tube E .sup.32 P linkers alone before 
digestion with Eco R1 
Sample 7 Tube E .sup.32 P linkers alone after 
digestion with Eco R1 
______________________________________ 
The ligation mixture is prepared by adding to a fresh tube 9 .mu.l EcoR1 
linkers [Biolabs 200 ng/.mu.l], and 1.7 .mu.l of DNA ligase [IBI 3 
U/.mu.l]. 15 .mu.l of ligation mixture is dispensed into the bulk reaction 
tube mixed quickly, then a 0.25 .mu.l sample is removed and frozen on dry 
ice immediately for agarose gel analysis (Sample 1). 
A 1 .mu.l sample is taken from the bulk reaction tube and 0.2 .mu.l .sup.32 
P labelled EcoR1 linkers is added (Tube D: cDNA+linkers). 
A 1 .mu.l sample is taken from the remainder of the ligation mixture and 
0.2 .mu.l .sup.32 P labelled EcoR1 linkers are added (Tube E: linkers 
alone). 
The bulk reaction and tubes D and E are incubated at 25.degree. C. for 4 
hours. Samples of 0.25 .mu.l from the bulk reaction tube and 0.6 .mu.l 
from tubes D and E are removed for agarose or polyacrylamide gel analysis 
respectively (Samples 2, 4 and 6). 
The remainder of the bulk reaction and tubes D and E are heated for 5 hours 
at 70.degree. C. then cooled on ice. 
(m) EcoR1 Digestion 
To a fresh tube 11 .mu.l EcoR1 digestion buffer, 2 .mu.l EcoR1 [IBI 18 
U/.mu.l] and 82 .mu.l of water are added. 4 .mu.l of the mixture is 
dispensed into the bulk reaction tube. The three tubes are incubated at 
37.degree. C. for 60 minutes. A further 2 .mu.l aliquot of EcoR1 [36 U] is 
added to the bulk reaction tube and incubation is continued for a further 
60 minutes. The remaining samples in tubes D and E are electrophoresed on 
agarose and acrylamide gels together with the samples taken from tubes D 
and E above. Autoradiographs of those gels demonstrate whether the 
reactions have worked. 
A 1.4 .mu.l sample is removed from the bulk reaction tube (Sample 3). The 
remainder of the bulk reaction is extracted with phenol/chloroform. 
A 1% agarose gel is run loaded with 0.25 .mu.l each of samples 1, 2 and 3. 
Samples 4, 5, 6 and 7 are run on a 12% polyacrylamide gel. Both gels are 
autoradiographed to determine whether all reactions have succeeded. 
(n) Separation of Linkers from cDNA 
A 1.2.times.21 cm Sepharose 4B column is equilibrated with 0.1M TEAB. 150 
.mu.l samples of EcoR1 digested linkered cDNA are loaded on to the column 
and fractions collected in TEAB buffer (250-500 .mu.l). Fractions 
containing cDNA fragments with sizes greater than 600 bp as determined by 
mobility on agarose or polyacrylamide gels are pooled, evaporated to 
dryness in a rotary evaporator suspended in TE and ligated to EcoR1 
digested and phosphatased lambda gt11 or gt10, packaged in vitro and 
infected onto suitable host strains such as Y1090 or Y1089 in accordance 
with suppliers instructions (Promega or Integrated Sciences). 
(o) Screening Clones with Oligonucleotides 
From the amino acid sequence of the WGL.sup.+ protein, peptide fragments 
derived from chemical cleavage of the WGL.sup.+ protein or 
endoproteolytic digestion peptides derived from the WGL.sup.+ protein, 
oligonucleotides coding for specific portions of the DNA coding for the 
protein can be designed and used in hybridisation experiments using 
procedures known in the art. The DNA sequence of hybridising fragments 
isolated from the library can then be determined and used to design 
strategies for engineering the gene for expression of the WGL.sup.+ 
protein or portions thereof for incorporation into an effective vaccine. 
A cDNA library was constructed in lambda gt 11 using RNA isolated from 
young adult B. microplus which had been feeding on cattle for 
approximately 16 days. The phage were plated on E. coli strain RY1090 and 
grown at 37.degree. C. for 16 hours. Nitrocellulose filters were placed on 
the plates and triplicate filters were taken from each plate. The DNA on 
the filters was denatured and fixed by baking at 80.degree. C. under 
vacuum. The filters were incubated in prehybridization solution for 2-4 
hours and then in hybridization solution for 16 hours essentially as 
described (10). The hybridization solution contained oligonucleotides 
which had been labelled with .sup.32 P using polynucleotide kinase (10) 
and .psi. .sup.32 P-ATP (approximately 10.sup.5 cpm/ml of each 
oligonucleotide used). 
For each set of three filters, two were hybridized to the 63-mer 
oligonucleotide and the remaining replicate filter was hybridized to a 
mixture of 51-mer, 72-mer, 50-mer, and 53-mer oligonucleotides. Following 
washing and autoradiography, plaques which gave rise to signals on all 
three filters were identified, picked and purified to single plaques. 
EXAMPLE 7 
Analysis of DNA Sequence of Gene Coding for WGL.sup.+ Antigen 
The DNA isolated from one clone will be described in detail. This lambda 
gt11 clone contained three Eco R1 fragments of approximately 4 Kb, 1.5 Kb 
and 0.3 Kb. Southern hybridization (10) experiments showed that the 4 Kb 
fragment hybridized to the probes used. This fragment was therefore 
subcloned into a modified pUC 18 plasmid (giving pBTA 707) in host strain 
JM101 (recombinant host/plasmid referred to as BTA 1751 ATCC 67548). The 4 
Kb fragment was then sonicated and subcloned into M13 mp18 for DNA 
sequence analysis. 
M13 sub-clones were sequenced at random and the complete DNA sequence of 
the 4 kb inset compiled by assembly of the sequences of the sub-clones by 
use of an alignment computer program. 
FIGS. 6-6(2) show the DNA sequence (bases 1-2012 of SEQ ID NO:55) for the 4 
kb DNA fragment and the amino acid sequence (residues 11-688 of SEQ ID 
NO:56) which can be translated from one region of that DNA sequence into a 
protein sequence which is identified as the protein backbone of the 
WGL.sup.+ antigen. FIG. 8 shows that amino acid sequence using the one 
letter abbreviation code for amino acids (Table 8). 
The peptide fragments identified during the peptide sequence analysis of 
endo lys-C digest products from the WGL.sup.+ antigen isolated from ticks 
are identified in FIGS. 6-6(2) (SEQ ID NO:56) and 8 (residues 11-688 of 
SEQ ID NO:57) by underlines and are tabulated in a summary in Table 9. 
References to aa numbers correspond to the numbering of amino acid 
residues shown in FIG. 7 and SEQ ID NO:56. 
TABLE 9 
__________________________________________________________________________ 
##STR22## 
##STR23## 
##STR24## 
##STR25## 
##STR26## 
##STR27## 
##STR28## 
##STR29## 
##STR30## 
##STR31## 
##STR32## 
##STR33## 
##STR34## 
##STR35## 
__________________________________________________________________________ 
From the DNA sequence and the amino acid sequence deduced from that DNA 
sequence, it can be seen that the pre-pro-polypeptide of the WGL.sup.+ 
antigen consists of 650 amino acids (SEQ ID NO:56). 
The DNA sequence coding for peptide F12 (SEQ ID NO:42) can be identified at 
the region 90-152 bp (FIGS. 6-6(2) (SEQ ID NO:55)) of the DNA sequence and 
corresponds to amino acids 20-40 in the amino acid sequence (FIG. 8, 
residues 30-50 of SEQ ID NO:57) of the protein. The amino acid preceding 
the N-terminal glu residue identified in F12 is not a lysine (K) as would 
be expected if F12 was generated as a result of digestion by endo lys-C. 
Therefore it is assumed that the F12 peptide fragment was generated by the 
action of a proteinase other than endo lys-C. The 19 amino acid sequence 
preceding the F12 N-terminal glu residue begins with a methionine and has 
hydrophobicity properties which are very similar to leader sequences which 
precede other secreted and membrane-bound proteins in eukaryote cells (see 
9 for review). In addition, the majority of peptide leader sequences are 
cleaved at positions following A residues (9). It appears therefore that 
the F12 sequence is the N-terminus of the mature WGL.sup.+ polypeptide. 
This then indicates that the protein portion of the mature WGL.sup.+ 
polypeptide is 631 amino acids long and which would have a molecular 
weight of 69 729 daltons. 
Assuming that the consensus sequence for N-linked glycosylation is Asn X 
(Ser or Thr) in ticks as has been reported to be the case in other 
eukaryotic cells (10) 5 potential sites for N-linked glycosylation can be 
identified in the mature polypeptide sequence (FIGS. 6-6(2). Carbohydrate 
residues added to these residues or to other amino acids in the WGL.sup.+ 
antigen produced by ticks would account for the differences in the 
observed molecular weight for the native antigen compared with that 
predicted from the DNA sequence. 
By comparison of the amino acid sequence (Table 9) with the peptide 
sequences derived from the fractions from endo lys-C digestion, all of the 
peptides (F1-17) with the exception of F7 can be identified. In most 
cases, the amino acids which could not be confidently ascribed during the 
peptide sequence analysis can be shown to be correct following comparison 
with the sequence deduced from the DNA sequence. 
The amino acid sequence for peptide fragments F1, F11, F13 and F17 (SEQ ID 
NOS:1,173,15 and 21, respectively) all match precisely with the amino acid 
sequences deduced from the DNA sequence from the corresponding region of 
DNA (Table 9). 
Peptide F2 (SEQ ID NO:2) can be seen to be coded for by the DNA segment 
1104-1152 bp. Table 9 shows that the G and the X tentatively ascribed to 
positions 5 and 11 in the F2 peptide sequence are both N. N is very 
difficult to detect during gas phase sequencing and there was very little 
material in the sample. Otherwise the match is precise. F2 is the same 
peptide as F11 and all amino acids were ascribed correctly during the 
sequence analysis of the F11 peptide fragment. 
Peptides F3 (SEQ ID NO:3) and F17 show sequences of the same peptide 
obtained from two different endo lys-C digests of WGL.sup.+ (Examples 3 
and 4). The amino acid sequence for F17 matches precisely with the 
translated sequence from amino acids 405 to 412 of the WGL.sup.+ peptide. 
When sequencing F3, no amino acid could be ascribed to the first position 
(L from the DNA sequence) as there was a large amount of background but 
the rest of the amino acids match precisely with the amino acid sequence 
derived from the DNA sequence (amino acids 405-421 FIG. 8, residues 
415-431 of SEQ ID NO:57). 
F4 (SEQ ID NO:4) is found at amino acids 213-219 of the WGL.sup.+ protein 
(FIG. 8, residues 223-229 of SEQ ID NO:57). The sequence matches perfectly 
and the uncertain .sup.C /Q is shown from the DNA sequence to be C. 
Carboxy-methylated C migrates with a similar retention time to Q in the 
HPLC system used to separate the derivatized amino acids following the 
sequencing reactions. 
Very small amounts of material were sequencable in fragment F5 (SEQ ID 
NO:5) so there were several uncertainties. But it is clear that the 
sequence obtained corresponds to amino acids 200-210 in FIG. 8 (residues 
210-220 of SEQ ID NO:57). One of the two amino acids tentatively ascribed 
to each peptide is present in the amino acid sequence derived from the DNA 
sequence and those ascribed with confidence appear in the expected order. 
F6 (SEQ ID NO:6) sequence corresponds to amino acids 488-503 in the 
WGL.sup.+ protein sequence. The residues in the sequence derived for the 
F6 fragment differ from that derived from the DNA sequence. The F6 
sequence presented was derived from a mixed sequence in which the amino 
acids shown to be correct from the DNA sequence were in fact present. 
F7 (SEQ ID NO:7) has not been identified with confidence in the amino acid 
sequence derived from the DNA sequence. As with F6, the F7 sequence was 
derived from a mixed sequence and very little confidence can be placed in 
it. 
Small amounts of material were present in F8 (SEQ ID NO:8) sample so there 
were several uncertainties in the sequence. However, the F8 amino acid 
sequence appears to correspond to amino acids 444-450 in FIG. 8 (residues 
454-469 of SEQ ID NO:57). Again all uncertain residues can be identified 
in the translated DNA sequence. 
F9 (SEQ ID NOS:27 and 46) and F10 (SEQ ID NOS:27 and 29) were both mixtures 
of two amino acid sequences. It is apparent that one of those sequences 
corresponds to amino acids 51-63 in FIG. 8 (residues 61-72 of SEQ ID 
NO:57). In both cases, one of the two amino acids identified during the 
peptide sequence analysis can be ascribed to the amino acid sequence 
derived from the DNA sequence in the expected order. 
The remaining peptide sequence from F9 and F10 corresponds to amino acids 
514-531 in FIG. 8 (residues 524-541 of SEQ ID NO:57). The R recorded for 
position 11 in the F9 sequence is P from the DNA sequence which is in 
agreement with the sequence obtained for F10. The DNA sequence shows K at 
what would be position 10 of this peptide. The DNA sequence shown is that 
coding for one molecule of the WGL.sup.+ antigen and it is likely that 
different ticks have some variants of the sequence. This point will be 
expanded when discussing the F14 sequence. 
The fragment sequenced as F12 (SEQ ID NO:42) clearly corresponds to amino 
acids 20-41 in FIG. 8 (residues 30-51 of SEQ ID NO:57) and, as discussed 
previously is assumed to be the N-terminal fragment of the mature 
WGL.sup.+ peptide so the presumption that lysine preceded the first amino 
acids sequenced was incorrect in this case. The uncertain residue at 
position 6 of the peptide fragment is S from the DNA sequence. S is very 
difficult to detect during gas phase sequencing particularly as in this 
case, when the preceding amino acid is C and carboxy-methylated-C has a 
similar retention time to S in the HPLC system used to resolve the 
derivatized amino acids. Otherwise the F12 peptide sequence matches the 
sequence derived for WGL.sup.+ exactly. 
F14 (SEQ ID NO:16) is very interesting (amino acids 228-247). The peptide 
sequence showed RAF for amino acids 8-10 in the peptide, whereas the DNA 
sequence, when translated, shows SGS in these positions. Both sequences 
appear to be correct retrospectively so there is a clear discrepancy 
between the two sequences. The most likely explanation is that both 
sequences are correct for the molecule (in the case of the cDNA) and the 
mixture of molecules (in the case of F12) which have been sequenced. 
The tick population world wide is genetically diverse as is the case for 
all organisms which reproduce sexually. Each individual of a population 
differs subtly from the others in the population and these differences are 
a consequence of differences in the sequence of the DNA which each 
individual inherits from its parents. Thus for each gene coding for a 
particular protein, there are likely to be differences in the sequence 
among the population of individuals, referred to herein as homologues. In 
the particular example discussed here, the WGL.sup.+ protein which was 
digested in Example 4 to give rise to F12 was extracted from a large 
number of ticks (60,000-70,000). The peptide sequence determined for F12 
(SEQ ID NO:42) (and the rest of the peptide fragments sequenced) is that 
of the majority of the population of WGL.sup.+ molecules. Among that 
population of WGL.sup.+ molecules, it is likely that minor variance 
(homologues) will exist at a level too low to be detected during the 
peptide sequence analysis. The cDNA sequence shown in FIG. 7 (bases 1-2012 
of SEQ ID NO:55 and SEQ ID NO:56) and the amino acid sequence in FIG. 8 
are derived from one cDNA molecule from one individual in the population. 
This individual may have contained a DNA sequence coding for a minor 
variant of the WGL.sup.+ molecule. It is of course understood that other 
cDNA molecules may be derived from other individuals of the tick 
population world wide which will similarly vary in some small way from the 
sequence shown in FIGS. 6-6(2) but still code for a protein which is 
essentially the same as that for the WGL.sup.+ antigen molecule. These 
homologues are included within the scope of this invention. 
If the differences such as the one above are found in regions of the 
WGL.sup.+ molecule which are important epitopes for the protective immune 
response generated against the WGL.sup.+ molecule following vaccination, 
it is possible that the ticks with a WGL.sup.+ product which is a 
homologues of the sequence shown in FIG. 6-6(2) may survive feeding on 
vaccinated hosts. In this instance it is to be understood that cDNA can be 
synthesised or DNA isolated from these individuals as described above or 
by other methods known in the art. In hybridization experiments the 4 kb 
DNA fragment (or parts thereof) can be used as hybridization probes to 
identify clones containing DNA coding for the WGL.sup.+ protein from 
those variants which can then be used to construct bacteria or other 
micro-organisms which synthesize the variant WGL.sup.+ antigen to be 
incorporated into effective vaccine against the variant tick population. 
This principal extends to isolates of Boophilus microplus and to other 
species of ticks from anywhere in the world. 
The other difference in the sequence of F14 (SEQ ID NO:16) compared with 
the sequence for WGL.sup.+ polypeptide derived from the DNA sequence is 
that the residue at position 18 (S or H in F12 which was ascribed with low 
confidence) is T from the DNA sequence. 
F15 (SEQ ID NOS:17-19) was a mixture of at least three oligopeptides. Among 
those, one seems to be represented in the polypeptide at amino acids 
166-176. 
The F16 (SEQ ID NO:20) sequence can be seen in the WGL.sup.+ amino acid 
segment 274-281 (FIG. 8, residues 284-291 of SEQ ID NO:57). The two 
uncertain residues in the peptide sequences are X.dbd.T in the second 
position and X.dbd.C in the seventh position of F16, both of which were 
due to the very small amounts of material which were present in this 
peptide sample. 
In summary, it is clear that the DNA sequence shown in Table 9 codes for 
one homologue of the tick WGL.sup.+ polypeptide as 16 of the 17 endo 
lys-C peptide fragments shown which were generated from the antigen 
isolated from ticks can be coded for by that DNA sequence. Evidence has 
been obtained that homologues of that gene may exist in the tick 
population and these are included within the scope of this invention 
whether the homologues originate from Boophilus microplus or other species 
of ticks found world wide. 
The procedures outlined above refer to the WGL.sup.+ antigen derived from 
Boophilus microplus. It is clear that this antigen or the equivalent 
antigen isolated from other tick species may well be effective against 
other species of Boophilus such as B. annulatus, other tick species such 
as Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Ambylomma spp. 
Dermacentor spp, Ixodes spp and Hyalomma spp, and particular species 
thereof including Otobius megnini, Rhiphicephalus appendiculatus, 
Amblyomma variegatum, Haemaphysalis longicornis, Dermacentor andersoni, D. 
variabilis and Ixodes holocyclus each of which causes significant economic 
loss throughout the world either as a result of infestation or as vectors 
of diseases such as Babesia bovis, Babesia bigemina, Anaplasma marginale, 
Cowdria ruminatum, Theileria parva parva, T. parva lawrencii, T. annulata 
and T. hirci. The WGL.sup.+ gene product or the equivalent gene product 
from the related and other acarines would be expected to provide effective 
vaccines against the parasites as an extension of the work presented 
herein. 
Comments on Hybridization Probes Used 
The oligonucleotide probes which were chosen had several shortcomings which 
can be identified retrospectively. Some of these were due to incorrect 
choice of bases in the third codon positions and, of course to the 
uncertainties in the peptide sequence analysis. The oligonucleotide which 
was relied upon most heavily was the 63-mer as it was based on the most 
reliable amino acid sequence obtained at that time. When isolating the 
clones it was surprising that the hybridization signal with this probe was 
weaker than expected from theoretical considerations and at one stage 
there was doubt that the clone isolated coded for the WGL.sup.+ peptide. 
This uncertainty was alleviated to some extent by the use of the 
degenerate oligonucleotide sequences mentioned above as probes. These 
probes hybridized strongly to the DNA in the clone. The reason for the 
weaker than expected signal with the 63-mer can now be explained by the 
variation in the DNA sequence from that expected in this region. A large 
number of other clones were purified based on the hybridization signal 
obtained with one or two probes but these all turned out to be unrelated 
to the WGL.sup.+ gene by DNA sequence analysis. Therefore the strategy 
for isolating the clone by using triplicate filters and the use of the 
highly degenerate oligonucleotide sequences as hybridization probes to 
confirm the interest in the clone has been vindicated. 
EXAMPLE 8 
Construction of Recombinant Organisms Synthesizing WGL.sup.+ Antigen 
The major limitation to the development of a commercial vaccine based on 
the WGL.sup.+ antigen or homologues thereof is the limited amount of the 
antigen which can be obtained from ticks. The means by which this shortage 
can be overcome include the use of recombinant DNA techniques to engineer 
bacteria or eukaryote cells to synthesize large amounts of the antigen. 
The following by means of example only outline some approaches which could 
be taken. 
FIGS. 8A-8B show a restriction enzyme map of the gene coding for the 
WGL.sup.+ antigen isolated from B. microplus. In order to engineer 
bacteria which express the gene product at high levels, it would probably 
be desirable to remove the parts of the molecule which are hydrophobic. 
These include the hydrophobic leader sequence (amino acids 1-19) which is 
not found in the mature polypeptide, and the hydrophobic C-terminal 
sequence (amino acids 630-650) which is likely to be an anchor sequence 
involved in attaching the antigen to the outer surface of the tick cells. 
In FIGS. 8A-8B, cleavage sites for the restriction enzymes XmnI (116 
bases), PstI (1915 bases) and BamHI (1889 bases) are highlighted. DNA 
fragments produced by digestion of the WGL.sup.+ gene with XmnI in 
addition to BamHI or Pstl will contain the coding region for the majority 
of the gene without the N-terminal hydrophobic sequence or the C-terminal 
hydrophobic sequences. These 1773 bp and 1799 bp fragments can be 
subcloned into a number of plasmids including plasmids pBTA603 and pBTA224 
to yield recombinant plasmids which will direct the synthesis of fused 
proteins containing the majority of the WGL.sup.+ peptide. 
Plasmid pBTA603 has the PL promoter followed by a sequence from the 
N-terminus for the MS2 polymerase gene containing a multiple clone site vi 
s 
EQU ATG TCG AAG ACA ACA AAG AAG TTC AAC TCT TTA TCG ATG/GAT CCC 
Restriction endonuclease BamHl cuts the DNA where indicated (/) to give a 4 
base 5' single stranded overhang. When this is filled in with DNA 
polymerase 1, the sequence (bases 34-43 of SEQ ID NO:48) MS2 --TCG ATG GAT 
C is generated. When this is ligated to Xmnl cut WGL.sup.+ DNA (Xmnl cuts 
at the sequence (SEQ ID NO:49) GAANNNNTTC i.e. following base 120) the 
sequence (SEQ ID NO:50) MS2--TCG ATG GAT CAG TTC TGT--WGL.sup.+ is 
generated. The plasmid so constructed encodes a protein which contains 15N 
terminal amino acids from the MS2 polymerase and the cloning site 
sequences in place of the N-terminal 11 amino acids of the mature 
WGL.sup.+ sequence followed by the WGL.sup.+ amino acid sequence from 
amino acids 31 to 620 for the BamHl fragment or 31 to 628 for the Pstl 
fragment. When transformed into a suitable host such as N4830(10) which 
contains a mutation (cI.sup.ts) in the gene coding for the cI repressor, 
expression of the fused polypeptide is repressed at temperatures such as 
30.degree. C. but is active at temperatures such as 42.degree. C. This 
temperature dependence of expression is advantageous in instances where 
the fused product is deleterious to the cells. Cells are grown at 
30.degree. C. to the desired cell density and the temperature is then 
increased to 42.degree. C. to induce the synthesis of the fused protein. 
The expression vector pBTA224 was used to generate a strain capable of 
producing a .beta.-galactosidase-WGL.sup.+ fusion protein. pBTA224 was 
derived from pUR292 (EMBO J. 2, 1791-1794 (1983)) by eliminating the EcoR1 
site that lies outside of the .beta.-galactosidase-coding region. pBTA224 
DNA was cut with the restriction endonucleases Sacl and Pstl, and the 
resulting 4221 bp fragment was purified by agarose gel electrophoresis. 
Sacl cuts within the lac Z gene, 1181 bps from the 3' end. Pstl cuts 
pBTA224 at the 3' end of lacZ. A WGL.sup.+ gene fragment suitable for 
expression in this vector was prepared by first inserting an Xmnl 
restriction fragment of about 2 Kb (position 116 to past 3' end of 
WGL.sup.+ gene) into the vector M13um31 (obtained from International 
Biotechnologies, Inc.). By cutting the new construct with Sacl and Pstl, a 
fragment encoding most of the WGL.sup.+ and having Sacl and Pstl cohesive 
ends could be obtained. The sequences for the Sacl end are also shown in 
SEQ ID NOS:51 and 52. 
##STR36## 
The fusion protein expected to be produced after induction with IPTG 
consists of the first 651 amino acids of .beta. galactosidase, 599 amino 
acids of WGL.sup.+ and 19 amino acids that are encoded by other parts of 
the expression vector, such as the multiple cloning sites. The calculated 
molecular weight is 143,054 daltons. 
The plasmid described above has been designated pBTA708. A suitable E. coli 
host containing the lacI.sup.q gene is JM101. BTA1752 is JM101 transformed 
with pBTA708. 
Cell lysates prepared from IPTG induced and control cultures, were analysed 
by electrophoresis in SDS-polyacrylamide gels. One gel was stained with 
Coomassie brilliant blue and a band of about the expected size could be 
visualised (FIGS. 9A-9B). The band was absent in the non-induced control. 
A duplicate SDS-polyacrylamide gel was also run and the proteins in the 
gel were transferred to nitrocellulose paper. The nitrocellulose paper was 
incubated in BLOTTO (a solution of 5% powdered milk in Tris-saline) for 2 
hours, then in BLOTTO containing a 1/500 dilution of serum from a rabbit 
vaccinated with the fractions GF5 and 6 (see above) for 13 hrs at 
4.degree. C., then washed three times with BLOTTO, then incubated in a 
solution containing goat-anti-rabbit immunoglobulin conjugated to alkaline 
phosphatase (Promega Biotec). Following incubation for 1 hr, the 
nitrocellulose was removed, washed twice in BLOTTO and incubated in buffer 
containing Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl 
phosphate. A band appeared where the rabbit antibodies had bound to the 
.beta. galactosidase-WGL.sup.+ fused polypeptide synthesized by the 
bacteria (FIG. 10). The position of the band corresponds to the position 
of the band seen in the coomassie stained gel. 
EXAMPLE 9 
Fermentation Purification and Formulation of Vaccines Based on the 
WGL.sup.+ Antigen Produced by rDNA Techniques 
Strains expressing the WGL.sup.+ antigen or portions thereof are 
maintained as freeze-dried vials in the production culture collection. 
Cells from the storage vial are reconstituted and plated out on a 
selective medium, and the cells from this medium are used to prepare 
fermentor inocula. The inocula are used to seed fermentors containing a 
suitable growth medium and the fermentation proceeds under conditions 
appropriate for the production of the WGL.sup.+ proteins. At the 
completion of the fermentation the cells are harvested and the product is 
released from the cells and undergoes purification. The product is 
subjected to analyses and quality control, and is stored under conditions 
appropriate for good stability. The product is formulated for use by 
combination with other ingredients under conditions of strict hygiene. 
The strains produce the WGL.sup.+ fusion proteins in vivo as insoluble 
agglomerates termed inclusion bodies and can be produced and purified by 
the following procedure which is presented by means of example only. 
Overnight cultures of BTA1752 is diluted 1:50 into 2.times.1 liter fresh LB 
(10 g tryptone/5 g yeast extract/5 g NaCl per liter pH 7.5) in 2 liter 
baffled flasks and shaken at 30.degree. C. until the culture density 
reached OD 0.3-0.4. IPTG is added to a final concentration of 10 mM and 
incubation continued for a further 10 hours. The cells are harvested and 
resuspended in 20 ml of water per liter of original culture and broken by 
use of a French Press. The suspension is made 0.1 mM in 
phenylmethylsulfonyl fluoride (PMSF) and 5% Triton X-100 then centrifuged 
at 12,000.times. gav for 10 minutes. The supernatant is discarded and the 
pellet resuspended by ultrasound in 50 ml 1M NaCl/5% Triton X-100 and 
recentrifuged. This washing stage is repeated and the pellet finally 
resuspended using ultrasound in 2.5 ml 1M NaCl/5% Triton X-100 per liter 
original culture. 
Purified inclusion bodies are dissolved at 2 mg/ml in 8M urea/0.1M DTT/0.1M 
Tris HCl pH 8.0 under nitrogen at 37.degree. C. for 2 hrs. The solution is 
centrifuged at 20,000.times. gav for 20 min and the supernatant passed 
through a 0.1 .mu.m filter. The flow through is passed through a filter 
with a molecular weight cut-off of 30 kilodaltons and the retained 
material is applied to a DEAE resin which is poured into a column and 
washed with 0.1M tris buffer pH 8. The column is then resolved with a 
linear gradient of from 0-5M NaCl in 8M urea 0.1M Tris pH 8.0 and the 
fractions analysed by SDS-Polyacrylamide gel electrophores. Those 
containing the desired protein are pooled, concentrated and desalted on a 
XM30 filter. The partially purified protein is emulsified in an adjuvant 
such as Marcol 52:Montanide 888 (9:1) or Freunds complete or incomplete 
adjuvant and administered to animals. 
EXAMPLE 10 
Identification of DNA Sequences Coding for the WGL.sup.+ Antigen in 
Species of Tick Other than Boophilus microplus 
In various countries throughout the world, tick species other than 
Boophilus microplus are responsible for extensive productivity losses 
either due to the tick infestation or due to the other parasites which the 
ticks transmit or a combination of both. It would be highly desirable to 
develop vaccines against these tick species. This may be achieved by 
vaccinating animals with the WGL.sup.+ antigen derived from Boophilus 
microplus or the other immunogenic protective fractions described in this 
and Australian patent application No. 45936/85. It may also be possible to 
vaccinate animals with the WGL.sup.+ antigen produced by recombinant 
organisms described herein and elicit an immune response which protects 
against infestation of animals by other species of tick. 
As discussed above, the other species of tick probably contain a molecule 
which is functionally related to the Boophilus microplus WGL.sup.+ 
antigen but which differs in sequence from that shown in FIG. 8. If those 
differences occur in areas eliciting protective immune responses, then the 
Boophilus microplus WGL.sup.+ antigen may not be protective. However, the 
related gene product from the other species of tick is likely to be 
protective against those tick species, when incorporated into a vaccine. 
One means by which this proposal can be tested is to conduct a series of 
vaccination/challenge experiments using fractions derived from homogenates 
of other ticks and purify the WGL.sup.+ homologues from the other tick 
species. These can then be cleaved with proteinases, peptide fragments 
sequenced, oligonucleotides designed and used to identify recombinant 
organisms containing the genes in a similar way to that in which the 
Boophilus microplus WGL.sup.+ gene has been identified in the present 
work. 
A preferable approach is to construct cDNA or genomic DNA libraries from 
nucleic acids extracted from other tick species and to use the DNA 
fragment shown in FIGS. 6-6(2) (bases 1-2012 of SEQ ID NO:55) or portions 
thereof as hybridization probes to identify clones containing the 
homologous gene from the other tick species. Then, engineered recombinant 
microorganisms synthesizing the homologous gene product could be 
incorporated into an effective vaccine against the other species of ticks. 
In order to demonstrate that this latter approach is feasible and to 
generate information concerning the conditions under which the 
hybridization to the clone libraries should be carried out, preliminary 
"Southern blot" hybridization experiments can be conducted. Briefly by way 
of example only DNA isolated from a number of species of tick is purified 
and digested with restriction endonucleases. The DNA fragments so produced 
are size fractionated by electrophoresis on agarose gels, denatured and 
transferred to nylon or nitrocellulose filters by capillary action. The 
filter is, incubated in a prehybridization solution and then in a 
hybridization solution containing radioactively labelled DNA fragments 
derived from the WGL.sup.+ gene coding region. Following hybridization 
and washing of the filters, they are exposed to X-ray film and the 
resulting autoradiograph shows exposed areas which correspond to the DNA 
fragments from the various tick species which have hybridized to the 
WGL.sup.+ DNA fragments. There are many variations of protocols for 
carrying out this procedure which will be known to individuals skilled in 
the art and the following is detailed by means of example only. 
Eggs were obtained from female ticks of the species Rhiphicepalus 
appendiculatus, Amblyomma variegatim, Boophilus decoloratus and Boophilus 
microplus. They were incubated in a humidified incubator for 2-4 days then 
suspended in cold TE buffer and washed. They were then suspended in TE 
buffer containing 0.5% SDS in a loose fitting glass-homogeniser and gently 
homogenised to disrupt the eggs. Proteinase K was added to a final 
concentration of 50 .mu.g/ml and the mixture was incubated at 37.degree. 
C. for 1-2 h with gentle shaking. The viscous solution was gently 
extracted three times with phenol saturated with 0.1M Tris-HCl pH 8.0 and 
then twice with ether (centrifugation at 5,000.times. gav for 10 minutes 
was used to resolve the phases during the phenol extractions). Sodium 
acetate was added to 0.3M and 2 volumes of ethanol was slowly added with 
stirring. The DNA which came out of solution as a fibrous precipitate was 
removed with a pasteur pipette, washed in ethanol, and gently redissolved 
in TE. 
Aliquots (generally containing 10 .mu.g) of these DNA samples were digested 
with restriction endonucleases according to the manufacturers 
instructions. Aliquots of the digest products were fractionated by 
electrophoresis on a 1.6% agarose gel in SEB buffer (10). The DNA was 
depurinated with 2 volumes of 0.25M HCl for 15 minutes. The DNA was 
transferred by capillary action to a nylon membrane (Zetaprobe, Biorad). 
The filters were incubated in prehybridization in a solution (10) 
containing herring sperm DNA for 2-4 hours at 55.degree. C. Hybridization 
was carried out in the same solution containing heat denatured [.sup.32 P] 
labelled DNA fragments from the WGL.sup.+ gene (approximately 10.sup.5 
counts per minute/ml) for 20 hours at 68.degree. C. The filters were 
washed at 55.degree. C. for 30 minutes each in 2.times. SSC, 0.1% SDS then 
three times at 60.degree. C. for 15 minutes. After exposure to X-ray film 
for at least 24 hours the size of the hybridizing fragments could be 
determined by comparison with marker DNA fragments of known size. 
FIGS. 10A-10C show an autoradiogram of one such experiment. DNA was 
digested with restriction endonuclease Sau 3A. The WGL.sup.+ DNA clearly 
hybridizes to the DNA from all four species of ticks. In this experiment, 
the DNA was not intact so a smear is observed in all cases but 
hybridization is specific as no hybridization to control DNA on the same 
gel could be detected. 
EXAMPLE 11 
Isolation of Clones Coding for WGL.sup.+ Homologous from Other Tick 
Species 
The DNA from each of the species tested possesses sequences which are 
similar to and homologous with the DNA coding for the WGL.sup.+ antigen 
from Boophilus microplus. Clones containing those DNA sequences from other 
tick species can be isolated by constructing cDNA or genomic DNA libraries 
for the other tick species and hybridizing Boophilus microplus DNA 
fragments to those libraries, and purifying recombinant organisms 
containing the DNA sequences hybridizing to the homologous genes. 
More specifically, the genomic DNA isolated from the tick species listed 
was subjected to partial digestion with the restriction enzyme Sau 3A to 
give fragments with an average size of 15-20 Kb as judged by gel analysis. 
These were ligated into the Bam HI site of lambda EMBL 3 arms essentially 
as described by the suppliers (Promega Biotech). The libraries were plated 
on a restrictive host K62 and incubated overnight at 37.degree. C. The 
plaques were transferred to triplicate nitrocellulose filters, and the DNA 
denatured with 1.5M NaCl/0.5M NaOH, neutralised with 3M NaCl/0.5M Tris HCl 
pH 7.0. Then the filters were vacuum baked at 80.degree. C. for 2 hours 
and hybridized to Boophilus microplus DNA probes labelled with .sup.32 P. 
Following autoradiography, plaques which hybridized to the probes on both 
filters were identified, picked and purified to single plaques by repeated 
rounds of re hybridization. 
DNA was isolated from one plaque from a B. decoloratus genomic library and 
digested with restriction endonucleases HacIII and Apa 1. The fragments so 
produced were separated by electrophoresis on 1.6% agarose gels. One gel 
was stained with an ethidium bromide solution and the bands visualised 
under ultraviolet light (FIGS. 10A-10C). A replicate gel was transferred 
to nylon membrane and hybridized to Boophilus microplus DNA coding for the 
WGL.sup.+ antigen. FIGS. 10A-10C show that fragments from the Boophilus 
decoloratus, Amblyomma variegatum genomic clone hybridize to the WGL.sup.+ 
gene. 
The bands hybridising in the HaeIII digest are approximately 980, 630, and 
340 bp and in the Apa 1 digest 27,300 bp when compared with fragments of 
DNA from bacteriophage lambda digested with Hind III. 
The regions of the DNA in each plaque which codes for portions of the 
homologous gene for the WGL.sup.+ antigen from each species of tick are 
sequenced and engineered for expression in recombinant organisms 
essentially as described above for the Boophilus microplus WGL.sup.+ 
antigen. The same approach can also be taken to isolate cDNA clones from 
these and other tick species. 
The homologous WGL.sup.+ antigen proteins expressed by the microorganisms 
are then grown in fermenters, the expression of the recombinant antigen 
induced and the antigen is purified formulated with an adjuvant or carrier 
and used to vaccinate animals. 
It is understood that this procedure can be equally well applied to any 
species of tick to isolate clones coding for WGL.sup.+ related antigens 
and the WGL.sup.+ related proteins expressed by the so constructed 
genetically engineered microorganisms can be used as effective vaccines 
against a range of tick species which are responsible for productivity 
losses, morbidity and mortality to domestic animals and man. 
EXAMPLE 12 
RNA was extracted from ticks collected from different regions of the world 
and cDNA libraries were constructed using lambda vectors essentially as 
described in Example 6. Replicas of these cDNA libraries were hybridised 
with radioactively labelled restriction fragments derived from the DNA 
coding for the WGL+ antigen using hybridisation conditions designed to 
detect nucleic acid sequences having a minimum of 70% homology to the 
hybridising sequence. The resulting plaques that reacted with the DNA 
hybridisation probes were then purified to single plaques. The DNA 
sequences of the genes were determined using standard sequencing 
techniques. FIGS. 12-17 illustrate the DNA sequences and deduced amino 
acid sequences (SEQ ID NOS:58-69). 
The DNA sequence YBm017 (FIG. 12 (SEQ ID NO:58)), was derived from an 
Australian isolate of Boophilus microplus (Yeerongpilly, Queensland). The 
WGL+ antigen described in the preceding examples was also obtained from 
the same isolate. A comparison of the DNA sequences reveals: 
(i) The DNA sequences are not identical. There is approximately 95% 
homology at the DNA level but it is clear that the two sequences code for 
the same antigen. 
(ii) The translated amino acid sequence (SEQ ID NO:59) has 8 differences 
between the two antigens. For example, the WGL+ sequence encodes a 
phenylalanine at position 1551 while YBm017 encodes a cysteine at the 
corresponding position (nucleotide 1570 in FIG. 12 (SEQ ID NO:58)). In 
addition, the sequence serine glycine serine (encoded by nucleotides 735 
through 743 in the WGL+ sequence) is arginine alanine phenylalanine in the 
corresponding position of YBm017 (nucleotides 754 to 762 in FIG. 12 (SEQ 
ID NO:58)). 
A partial cDNA clone encoding a protein fragment with an amino acid 
sequence homologous to that of WGL+ is presented as YBm22M8 (FIG. 13 (SEQ 
ID NO:60)). This sequence extends from nucleotide position 276 to 
nucleotide position 1922 of the WGL+ sequence. There are 13 differences 
between the deduced amino acid sequences of YBm22M8 (SEQ ID NO:61) and 
WGL+ (SEQ ID NO:56). 
These observations further illustrate that the tick population, even within 
one isolate of ticks is genetically diverse and that homologues of the 
antigen are found within that population. 
A second form of the antigen consisting essentially of the sequence 
described for YBm22M8 but including the amino terminus of the original 
WGL+ clone has been expressed in recombinant bacteria and used to 
vaccinate cattle which were subsequently challenged with ticks. This 
recombinant antigen protects cattle as well as that encoded by the WGL+ 
antigen. 
The cDNA clone, Bm023 (FIG. 14 (SEQ ID NO:62)) was obtained from another 
Australian isolate of Boophilus microplus. The nucleotide sequence of this 
cDNA codes for a protein (SEQ ID NO:63) that has 13 amino acids that are 
different from those encoded by the WGL+ cDNA. This demonstrates that the 
major form of the WGL+ antigen is similar for the two populations of 
ticks. 
The VBm021 and MexBm86 cDNA molecules (FIGS. 15 (SEQ ID NO:64) and 16 (SEQ 
ID NO:66) respectively) were obtained from Boophilus microplus isolates 
form Venezuela and Mexico respectively. The VBm021 sequence is a partial 
cDNA clone in that the sequence does not extend to the start codon of the 
gene. The sequence begins at a position corresponding to amino acid 31 of 
the deduced WGL+ amino acid sequence (nucleotide position 123 in FIG. 7 
bases 1-2012 of SEQ ID NO:55). The MexBm86 cDNA sequence extends through 
the start codon and into the 5' untranslated region of the WGL+ sequence 
(FIG. 7 bases 1-2012 of SEQ ID NO:55). These sequences differ from the 
WGL+ deduced amino acid sequence by 28 (VBm021 SEQ ID NO:65) and 22 
(MexBm86 SEQ ID NO:66) amino acids. 
These results confirm that it is possible to isolate related genes from a 
diverse range of Boophilus microplus isolates using the WGL+ gene (or 
fragments derived from this gene) as hybridisation probes. The DNA 
sequences of the variants will enable the gene to be clearly identified as 
related to the WGL+ gene but the homology at the DNA sequence level may be 
no more than 50% over some regions. In addition the translated amino acid 
sequences of these genes clearly indicate that the genes code for proteins 
which are closely related to the WGL+ protein but may differ in amino acid 
sequence by as much as 30% over some stretches of the protein. 
The Ra442 sequence (FIG. 17, SEQ ID NO:68) was obtained from Rhipicephalus 
appendiculatus. Comparison of this cDNA with WGL+ demonstrates that the 
Ra442 sequence codes for a protein fragment (SEQ ID NO:69) which is 
homologous to the WGL+ sequence corresponding to nucleotides 1113 to 1553 
(FIG. 7 bases 1-2012 of SEQ ID NO:58). It contains structural elements 
which are characteristic of these molecules. The homology over this region 
is approximately 85% at the DNA level and approximately 70% at the amino 
acid level, with particular regions having higher homology than others. 
This is clearly a molecule which is closely related in structure (and 
presumably in function) to the WGL+ antigen from Boophilus microplus. 
The nucleotide sequences presented in both YBm017 (SEQ ID NO:58) and VBm021 
(SEQ ID NO:64) contain two nucleotides each that could not be determined 
unambiguously when reading the sequencing gels. These are represented 
using the IU ambiguity code and result in the translated amino acid, 
Xaa. These were not included when describing the number of amino acid 
differences between these clones and the WGL+ sequence. 
DEPOSITION OF MICROORGANISMS 
Strain BTA 1751 referred to herein has been deposited with the American 
Type Culture Collection of 12301 Parklawn Drive Rockville, Md. 20852 USA 
in accordance with the provisions of the Budapest Treaty on 26 Oct. 1987 
under accession number ATCC 67548. 
Strain BTA 1751 has also been deposited with the China Centre for Type 
Culture Collection under import licence IL-87044 and designated CTCC. 
INDUSTRIAL APPLICATION 
The current invention provides a means of vaccinating cattle against 
infestation with ticks such as Boophilus microplus, Boophilus annulatus; 
other species such as Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, 
Ambylomma spp, Dermacentor spp, Ixodes spp and Hyalomma spp; and 
particular examples thereof including Otobius megnini, Rhiphicephalus 
appendiculatus, Dermacentor andersoni, D. variabilis and Ixodes 
holocyclus. Further it provides a means of protecting cattle against 
diseases such as those caused by Babesia bovis, Cowdria ruminatum, 
Theleria parva parva, T. parva lawrencil, T. annulata and T. hirci. 
Further it provides diagnostic tools for the identification and 
quantification of tick antigens. 
REFERENCES 
1. Brown, S. J., Shapiro, S. Z. and Askenase, P. W. J. Immunol. 133, 1984, 
3319-3325. 
2. Ackerman, S., Floyd, M. and Sonenshine, D. E. J. Med. Entomol. 17, 1980, 
391-397. 
3. McGowan, M. J., Barker, M. J., Homer, J. T., McNew, R. W. and Holscher, 
K. M. 1971, J. Med. Entomol. 18, 1981, 328. 
4. Wikel, S. K., Am. J. Trop. Med. Hyg. 30, 1981, 284. 
5. Allen, J. R. and Humphries, S. J. Nature, 280, 1979, 481-493. 
6. Johnson, L. A. Y., Kemp, D. H. and Pearson, R. D. Int. J. Parasitol. 16, 
27-34, 1986. 
7. Kemp, D. H., Agbede, R. I. S., Johnston, L. A. Y. and Gough, J. M. Int. 
J. Parasitol. 16, 121-130, 1986. 
8. Agbede, R. I. S. and Kemp, D. H. Int. J. Parasitol. 16, 35-42, 1986. 
9. Briggs M. S. and Gierasch, L. M. (1986), Molecular Mechanisms of Protein 
Secretion: The Role of the Signal Sequence, pages 110-180 in Advances in 
Protein Chemistry, vol. 38, Academic Press. 
10. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982), Molecular 
cloning: A Laboratory Manual (Cold Spring Harbour Laboratory). 
11. Kornfeld, R. and Kornfeld, S., 1985, Ann. Rev. Biochem 54, 631-664 
12. Van Hemert, F. J., Amons, R., Pluijms, W. J. M., Van Ormondt, H. and 
Moeller, W. EMBO 3, 1109-1113 1984 
13. Willadsen, P., Int. J. Parasitol, 17, 671-677 (1987) 
14. Vretblad, P., Biochemica and Biophysica Acta, 434, 169-176 (1976). 
15. Sage, H. J. and Green, R. W., in Methods in Enzymology, 28, Guinsburg, 
V., ed., 332-339 (1972), London Academic Press. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 71 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LysAspProAspProGlyLys 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F2 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LysTrpTyrGluAspGlyValLeuGluAlaIleXaaThrSerIleGly 
151015 
Lys 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F3 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LysXaaGlnAlaCysGluHisProIleGlyGluTrpCysMetMetTyr 
151015 
ProLys 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F4 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note= "Xaa at position 7 
represents Cys or Gln" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LysGluAlaGlyPheValXaaLys 
15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F5 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 4 
(D) OTHER INFORMATION: /note= "Xaa at position 4 
represents Ser or Asp" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note= "Xaa at position 7 
represents Val or Cys" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 10 
(D) OTHER INFORMATION: /note= "Xaa at position 10 
represents Val or Ala" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note= "Xaa at position 11 
represents Ile or Cys" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
LysGlyProXaaGlyGlnXaaIleAsnXaaXaaLys 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F6 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note= "Xaa at position 3 
represents Gly or Asp" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
LysAlaXaaValSerThrAsnGluAsnGluGlnLeuGluGlnAlaAsp 
151015 
Lys 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F7 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 3 
(D) OTHER INFORMATION: /note= "Xaa at position 3 
represents Gly or Asp" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
LysSerXaaThrGlnXaaIleAspHisIleSerLys 
1510 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F8 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /note= "Xaa at position 2 
represents Asn or Asp" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 5 
(D) OTHER INFORMATION: /note= "Xaa at position 5 
represents Ala or Tyr" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note= "Xaa at position 6 
represents Ala or Tyr" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
LysXaaGlnGluXaaXaaTyr 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
LysCysProCysAspAsnMetTyrPheAsnAlaAlaGluGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AlaAsnGlnCysProProAspThrArgArgGlyGluIleGlyCysIle 
151015 
Glu 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
LysAlaProArgGlnAsnMetTyrPheAsnAlaAlaGluGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CysAsnCysAspCysProProAspThrArgProGlyGluIleGlyCys 
151015 
IleGlu 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F11 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
LysTrpTyrGluAspArgValLeuGluAlaIleArgThrSerIleGly 
151015 
Lys 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F12 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LysGluSerSerIleCysXaaAspPheGlyAsnGluPheCysArgAsn 
151015 
AlaGluCysGluValValPro 
20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F13 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
LysThrArgGluCysSerTyrGlyArgCysValGluSerAsnProSer 
151015 
Lys 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F14 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 19 
(D) OTHER INFORMATION: /note= "Xaa at position 19 
represents Ser or His" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
LysAlaTyrGluCysThrCysProArgAlaPheThrValAlaGluAsp 
151015 
GlyIleXaaCysLys 
20 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F15 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /note= "Xaa at position 8 
represents Ser or His" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
LysAspGluValAspAsnAlaXaaLeuValCysGlnAsnAla 
1510 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F15 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
LysAsnValLeuGlnSerAspGlyCysGlyProTyr 
1510 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F15 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 7 
(D) OTHER INFORMATION: /note= "Xaa at position 7 
represents Pro or Leu" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 11 
(D) OTHER INFORMATION: /note= "Xaa at position 11 
represents His or Ser" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
LysCysLeuAsnProArgXaaArgLeuLysXaa 
1510 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F16 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /note= "Xaa at position 2 
represents Ser, Ala, Cys or Gly" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
LysXaaXaaValLeuCysGluXaaPro 
15 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F17 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
LysLeuGlnAlaCysGluHisProIle 
15 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F3, F17 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
LysLeuGlnAlaCysGluHisProIleGlyGluTrpCysMetMetTyr 
151015 
ProLys 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F4 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
LysGluAlaGlyPheValCysLys 
15 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F5 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
LysGlyProAspGlyGlnCysIleAsnAlaCysLys 
1510 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F6 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
LysAlaGlyValSerCysAsnGluAsnGluGlnSerGluCysAlaAsp 
151015 
Lys 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F8 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
LysAspGlnGluAlaAlaTyrLys 
15 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
LysCysProArgAspAsnMetTyrPheAsnAlaAlaGluLys 
1510 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9, F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
LysAlaAsnCysGlnCysProProAspThrLysProGlyGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9, F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
LysAlaAsnCysGlnCysProProAspThrArgProGlyGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F12 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
AlaGluSerSerIleCysSerAspPheGlyAsnGluPheCysArgAsn 
151015 
AlaGluCysGluValValProGly 
20 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F14 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
LysAlaTyrGluCysThrCysProSerGlySerThrValAlaGluAsp 
151015 
GlyIleThrCysLys 
20 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F14 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
LysAlaTyrGluCysThrCysProArgAlaPheThrValAlaGluAsp 
151015 
GlyIleThrCysLys 
20 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F15 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
LysAsnLeuLeuGlnArgAspSerArgCysCysGln 
1510 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F16 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
LysGlyThrValLeuCysGluCysPro 
15 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
LysCysProCysAspAsnMetTyrPheAsnAlaAlaGluLys 
1510 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
LysAlaAsnArgGlnCysProProAspThrArgArgGlyGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
TTACCTGGATCTGGATCCTT20 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
TTACCAATGGATGTACAAATAGCTTCAAGGACACCATCTTCGTACCACTT50 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 53 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
TTTGGGTACATCATACACCATTCACCAATTGGGTGTTCACAAGCCTGADSCTT53 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
LysAlaProArgGlnAsnMetTyrPheAsnAlaAlaGluLys 
1510 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
LysCysAsnCysAspCysProProAspThrArgProGlyGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F12 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
LysGluSerSerIleCysXaaAspPheGlyAsnGluPheCysArgAsn 
151015 
AlaGluCysGluValValProLys 
20 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 72 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
TTTAGGTACAACCTCACATTCAGCATTCCTACAAAATTCATTACCGAAATCAAAACAAAT60 
ACTACTCTCCTT72 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
CTTCGACGGATTGGATTCGACGCATCTGCCATAGCTACATTCCCTCGTCTT51 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 63 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
CTTGCAATGGATTCCATCCTCGGCGACAGTGAAAGCTCTAGGGCAAGTGCACTCATAAGC60 
CTT63 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9 b 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
LysAlaAsnCysGlnCysProProAspThrArgArgGlyGluIleGly 
151015 
CysIleGlu 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F16 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
LysXaaXaaValLeuCysGluXaaPro 
15 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
ATGTCGAAGACAACAAAGAAGTTCAACTCTTTATCGATGGATCCC45 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
GAANNNNTTC10 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
TCGATGGATCAGTTCTGT18 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
CGGTACCCAGTTCTGT16 
(2) INFORMATION FOR SEQ ID NO:52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
ACAGAACTGGGTACCGAGCT20 
(2) INFORMATION FOR SEQ ID NO:53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
AACGAGCTCGGTACCCAGTCC21 
(2) INFORMATION FOR SEQ ID NO:54: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
GAACTGGGTACCGAGCTCGTT21 
(2) INFORMATION FOR SEQ ID NO:55: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2065 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 7 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 33..1985 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
CCGCGACAGCTGCGGTGGTTCGACGCAGTGAGATGCGTGGCATCGCTTTGTTC53 
MetArgGlyIleAlaLeuPhe 
15 
GTCGCCGCTGTTTCACTGATTGTAGAGGGCACAGCAGAATCATCCATT101 
ValAlaAlaValSerLeuIleValGluGlyThrAlaGluSerSerIle 
101520 
TGCTCTGACTTCGGGAACGAGTTCTGTCGCAACGCTGAATGTGAAGTG149 
CysSerAspPheGlyAsnGluPheCysArgAsnAlaGluCysGluVal 
253035 
GTGCCTGGTGCAGAGGATGATTTCGTGTGCAAATGTCCGCGAGATAAT197 
ValProGlyAlaGluAspAspPheValCysLysCysProArgAspAsn 
40455055 
ATGTACTTCAATGCTGCTGAAAAGCAATGCGAATATAAAGACACGTGC245 
MetTyrPheAsnAlaAlaGluLysGlnCysGluTyrLysAspThrCys 
606570 
AAGACAAGGGAGTGCAGCTATGGACGTTGCGTTGAAAGTAACCCGAGC293 
LysThrArgGluCysSerTyrGlyArgCysValGluSerAsnProSer 
758085 
AAGGCTAGCTGCGTCTGCGAAGCATCGGACGATCTAACGCTACAATGC341 
LysAlaSerCysValCysGluAlaSerAspAspLeuThrLeuGlnCys 
9095100 
AAAATTAAAAATGACTACGCAACTGACTGCCGAAATCGAGGTGGCACT389 
LysIleLysAsnAspTyrAlaThrAspCysArgAsnArgGlyGlyThr 
105110115 
GCTAAGTTGCGCACGGATGGGTTTATTGGCGCAACGTGTGACTGTGGT437 
AlaLysLeuArgThrAspGlyPheIleGlyAlaThrCysAspCysGly 
120125130135 
GAATGGGGTGCGATGAACATGACCACCCGGAACTGTGTCCCTACCACG485 
GluTrpGlyAlaMetAsnMetThrThrArgAsnCysValProThrThr 
140145150 
TGTCTTCGTCCCGACTTGACCTGCAAAGACCTCTGCGAGAAAAACCTG533 
CysLeuArgProAspLeuThrCysLysAspLeuCysGluLysAsnLeu 
155160165 
CTTCAAAGGGATTCTCGTTGTTGCCAGGGGTGGAACACAGCAAACTGT581 
LeuGlnArgAspSerArgCysCysGlnGlyTrpAsnThrAlaAsnCys 
170175180 
TCAGCCGCTCCTCCAGCTGACTCCTATTGCTCTCCTGGGAGCCCCAAA629 
SerAlaAlaProProAlaAspSerTyrCysSerProGlySerProLys 
185190195 
GGACCGGACGGACAGTGTATAAATGCTTGCAAGACGAAAGAAGCTGGG677 
GlyProAspGlyGlnCysIleAsnAlaCysLysThrLysGluAlaGly 
200205210215 
TTTGTCTGCAAGCATGGATGCAGGTCGACCGGCAAGGCGTACGAGTGC725 
PheValCysLysHisGlyCysArgSerThrGlyLysAlaTyrGluCys 
220225230 
ACGTGCCCGAGTGGCTCTACCGTCGCCGAAGATGGCATTACCTGCAAA773 
ThrCysProSerGlySerThrValAlaGluAspGlyIleThrCysLys 
235240245 
AGTATTTCGCACACAGTCAGCTGCACTGCTGAGCAAAAACAGACCTGC821 
SerIleSerHisThrValSerCysThrAlaGluGlnLysGlnThrCys 
250255260 
CGCCCAACCGAAGACTGTCGTGTGCACAAAGGAACTGTGTTGTGTGAG869 
ArgProThrGluAspCysArgValHisLysGlyThrValLeuCysGlu 
265270275 
TGCCCGTGGAATCAACATCTAGTGGGGGACACGTGCATAAGTGATTGC917 
CysProTrpAsnGlnHisLeuValGlyAspThrCysIleSerAspCys 
280285290295 
GTCGACAAGAAATGCCACGAAGAATTTATGGACTGTGGCGTATATATG965 
ValAspLysLysCysHisGluGluPheMetAspCysGlyValTyrMet 
300305310 
AATCGACAAAGCTGCTATTGTCCATGGAAATCAAGGAAGCCGGGCCCA1013 
AsnArgGlnSerCysTyrCysProTrpLysSerArgLysProGlyPro 
315320325 
AATGTCAACATCAATGAATGCCTACTGAATGAGTATTACTACACGGTG1061 
AsnValAsnIleAsnGluCysLeuLeuAsnGluTyrTyrTyrThrVal 
330335340 
TCATTCACCCCAAACATATCTTTTGATTCTGACCATTGCAAATGGTAT1109 
SerPheThrProAsnIleSerPheAspSerAspHisCysLysTrpTyr 
345350355 
GAGGATCGTGTTTTGGAAGCGATACGGACCAGTATCGGAAAAGAAGTT1157 
GluAspArgValLeuGluAlaIleArgThrSerIleGlyLysGluVal 
360365370375 
TTTAAGGTTGAGATACTTAACTGCACGCAGGACATTAAGGCAAGACTC1205 
PheLysValGluIleLeuAsnCysThrGlnAspIleLysAlaArgLeu 
380385390 
ATAGCAGAGAAACCACTGTCAAAACACGTGCTCAGGAAACTACAAGCA1253 
IleAlaGluLysProLeuSerLysHisValLeuArgLysLeuGlnAla 
395400405 
TGCGAGCATCCAATCGGCGAATGGTGCATGATGTATCCGAAGTTGCTG1301 
CysGluHisProIleGlyGluTrpCysMetMetTyrProLysLeuLeu 
410415420 
ATCAAGAAAAACTCTGCAACAGAAATCGAAGAAGAGAACCTTTGCGAC1349 
IleLysLysAsnSerAlaThrGluIleGluGluGluAsnLeuCysAsp 
425430435 
AGTCTGCTCAAGGATCAGGAAGCTGCCTACAAAGGTCAAAACAAATGC1397 
SerLeuLeuLysAspGlnGluAlaAlaTyrLysGlyGlnAsnLysCys 
440445450455 
GTCAAGGTCGACAACCTCTTCTGGTTCCAGTGCGCTGATGGTTACACA1445 
ValLysValAspAsnLeuPheTrpPheGlnCysAlaAspGlyTyrThr 
460465470 
ACAACTTACGAGATGACACGAGGTCGCCTACGCCGCTCCGTGTGTAAA1493 
ThrThrTyrGluMetThrArgGlyArgLeuArgArgSerValCysLys 
475480485 
GCTGGAGTTTCTTGCAACGAAAACGAGCAGTCGGAGTGTGCTGACAAA1541 
AlaGlyValSerCysAsnGluAsnGluGlnSerGluCysAlaAspLys 
490495500 
GGGCAAATATTTGTTTACGAAAACGGCAAAGCGAATTGCCAATGCCCA1589 
GlyGlnIlePheValTyrGluAsnGlyLysAlaAsnCysGlnCysPro 
505510515 
CCAGACACTAAACCTGGGGAGATTGGCTGCATTGAGCGTACCACATGC1637 
ProAspThrLysProGlyGluIleGlyCysIleGluArgThrThrCys 
520525530535 
AACCCTAAAGAAATACAAGAATGCCAAGACAAGAAGCTGGAGTGCGTT1685 
AsnProLysGluIleGlnGluCysGlnAspLysLysLeuGluCysVal 
540545550 
TACAAAAACCATAAAGCAGAATGCGAGTGTCCTGATGATCACGAGTGT1733 
TyrLysAsnHisLysAlaGluCysGluCysProAspAspHisGluCys 
555560565 
TACAGGGAGCCTGCCAAAGACTCTTGCAGTGAAGAGGATAATGGTAAA1781 
TyrArgGluProAlaLysAspSerCysSerGluGluAspAsnGlyLys 
570575580 
TGTCAAAGCAGTGGGCAGCGTTGTGTAATAGAAAACGGAAAGGCTGTT1829 
CysGlnSerSerGlyGlnArgCysValIleGluAsnGlyLysAlaVal 
585590595 
TGCAAGGAAAAGTCTGAAGCAACAACAGCTGCGACTACAACAACGAAA1877 
CysLysGluLysSerGluAlaThrThrAlaAlaThrThrThrThrLys 
600605610615 
GCGAAAGACAAGGATCCAGATCCTGGAAAGTCAAGTGCTGCAGCAGTA1925 
AlaLysAspLysAspProAspProGlyLysSerSerAlaAlaAlaVal 
620625630 
TCAGCTACTGGGCTCTTGTTACTGCTCGCAGCTACTTCAGTCACCGCA1973 
SerAlaThrGlyLeuLeuLeuLeuLeuAlaAlaThrSerValThrAla 
635640645 
GCATCGTTGTAAGGAAGATGTCCAACTTGAATACGGAACAGCTTGAATA2022 
AlaSerLeu 
650 
TGTATATATACATCACGCTTACATCGAACACCTAGCTTGGTTT2065 
(2) INFORMATION FOR SEQ ID NO:56: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 650 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
MetArgGlyIleAlaLeuPheValAlaAlaValSerLeuIleValGlu 
151015 
GlyThrAlaGluSerSerIleCysSerAspPheGlyAsnGluPheCys 
202530 
ArgAsnAlaGluCysGluValValProGlyAlaGluAspAspPheVal 
354045 
CysLysCysProArgAspAsnMetTyrPheAsnAlaAlaGluLysGln 
505560 
CysGluTyrLysAspThrCysLysThrArgGluCysSerTyrGlyArg 
65707580 
CysValGluSerAsnProSerLysAlaSerCysValCysGluAlaSer 
859095 
AspAspLeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAsp 
100105110 
CysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIle 
115120125 
GlyAlaThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThr 
130135140 
ArgAsnCysValProThrThrCysLeuArgProAspLeuThrCysLys 
145150155160 
AspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGln 
165170175 
GlyTrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyr 
180185190 
CysSerProGlySerProLysGlyProAspGlyGlnCysIleAsnAla 
195200205 
CysLysThrLysGluAlaGlyPheValCysLysHisGlyCysArgSer 
210215220 
ThrGlyLysAlaTyrGluCysThrCysProSerGlySerThrValAla 
225230235240 
GluAspGlyIleThrCysLysSerIleSerHisThrValSerCysThr 
245250255 
AlaGluGlnLysGlnThrCysArgProThrGluAspCysArgValHis 
260265270 
LysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGly 
275280285 
AspThrCysIleSerAspCysValAspLysLysCysHisGluGluPhe 
290295300 
MetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrp 
305310315320 
LysSerArgLysProGlyProAsnValAsnIleAsnGluCysLeuLeu 
325330335 
AsnGluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAsp 
340345350 
SerAspHisCysLysTrpTyrGluAspArgValLeuGluAlaIleArg 
355360365 
ThrSerIleGlyLysGluValPheLysValGluIleLeuAsnCysThr 
370375380 
GlnAspIleLysAlaArgLeuIleAlaGluLysProLeuSerLysHis 
385390395400 
ValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCys 
405410415 
MetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIle 
420425430 
GluGluGluAsnLeuCysAspSerLeuLeuLysAspGlnGluAlaAla 
435440445 
TyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPhe 
450455460 
GlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArg 
465470475480 
LeuArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGlu 
485490495 
GlnSerGluCysAlaAspLysGlyGlnIlePheValTyrGluAsnGly 
500505510 
LysAlaAsnCysGlnCysProProAspThrLysProGlyGluIleGly 
515520525 
CysIleGluArgThrThrCysAsnProLysGluIleGlnGluCysGln 
530535540 
AspLysLysLeuGluCysValTyrLysAsnHisLysAlaGluCysGlu 
545550555560 
CysProAspAspHisGluCysTyrArgGluProAlaLysAspSerCys 
565570575 
SerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysVal 
580585590 
IleGluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThr 
595600605 
AlaAlaThrThrThrThrLysAlaLysAspLysAspProAspProGly 
610615620 
LysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeu 
625630635640 
AlaAlaThrSerValThrAlaAlaSerLeu 
645650 
(2) INFORMATION FOR SEQ ID NO:57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 688 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
AlaThrAlaAlaValValArgArgSerGluMetArgGlyIleAlaLeu 
151015 
PheValAlaAlaValSerLeuIleValGluGlyThrAlaGluSerSer 
202530 
IleCysSerAspPheGlyAsnGluPheCysArgAsnAlaGluCysGlu 
354045 
ValValProGlyAlaGluAspAspPheValCysLysCysProArgAsp 
505560 
AsnMetTyrPheAsnAlaAlaGluLysGlnCysGluTyrLysAspThr 
65707580 
CysLysThrArgGluCysSerTyrGlyArgCysValGluSerAsnPro 
859095 
SerLysAlaSerCysValCysGluAlaSerAspAspLeuThrLeuGln 
100105110 
CysLysIleLysAsnAspTyrAlaThrAspCysArgAsnArgGlyGly 
115120125 
ThrAlaLysLeuArgThrAspGlyPheIleGlyAlaThrCysAspCys 
130135140 
GlyGluTrpGlyAlaMetAsnMetThrThrArgAsnCysValProThr 
145150155160 
ThrCysLeuArgProAspLeuThrCysLysAspLeuCysGluLysAsn 
165170175 
LeuLeuGlnArgAspSerArgCysCysGlnGlyTrpAsnThrAlaAsn 
180185190 
CysSerAlaAlaProProAlaAspSerTyrCysSerProGlySerPro 
195200205 
LysGlyProAspGlyGlnCysIleAsnAlaCysLysThrLysGluAla 
210215220 
GlyPheValCysLysHisGlyCysArgSerThrGlyLysAlaTyrGlu 
225230235240 
CysThrCysProSerGlySerThrValAlaGluAspGlyIleThrCys 
245250255 
LysSerIleSerHisThrValSerCysThrAlaGluGlnLysGlnThr 
260265270 
CysArgProThrGluAspCysArgValHisLysGlyThrValLeuCys 
275280285 
GluCysProTrpAsnGlnHisLeuValGlyAspThrCysIleSerAsp 
290295300 
CysValAspLysLysCysHisGluGluPheMetAspCysGlyValTyr 
305310315320 
MetAsnArgGlnSerCysTyrCysProTrpLysSerArgLysProGly 
325330335 
ProAsnValAsnIleAsnGluCysLeuLeuAsnGluTyrTyrTyrThr 
340345350 
ValSerPheThrProAsnIleSerPheAspSerAspHisCysLysTrp 
355360365 
TyrGluAspArgValLeuGluAlaIleArgThrSerIleGlyLysGlu 
370375380 
ValPheLysValGluIleLeuAsnCysThrGlnAspIleLysAlaArg 
385390395400 
LeuIleAlaGluLysProLeuSerLysHisValLeuArgLysLeuGln 
405410415 
AlaCysGluHisProIleGlyGluTrpCysMetMetTyrProLysLeu 
420425430 
LeuIleLysLysAsnSerAlaThrGluIleGluGluGluAsnLeuCys 
435440445 
AspSerLeuLeuLysAspGlnGluAlaAlaTyrLysGlyGlnAsnLys 
450455460 
CysValLysValAspAsnLeuPheTrpPheGlnCysAlaAspGlyTyr 
465470475480 
ThrThrThrTyrGluMetThrArgGlyArgLeuArgArgSerValCys 
485490495 
LysAlaGlyValSerCysAsnGluAsnGluGlnSerGluCysAlaAsp 
500505510 
LysGlyGlnIlePheValTyrGluAsnGlyLysAlaAsnCysGlnCys 
515520525 
ProProAspThrLysProGlyGluIleGlyCysIleGluArgThrThr 
530535540 
CysAsnProLysGluIleGlnGluCysGlnAspLysLysLeuGluCys 
545550555560 
ValTyrLysAsnHisLysAlaGluCysGluCysProAspAspHisGlu 
565570575 
CysTyrArgGluProAlaLysAspSerCysSerGluGluAspAsnGly 
580585590 
LysCysGlnSerSerGlyGlnArgCysValIleGluAsnGlyLysAla 
595600605 
ValCysLysGluLysSerGluAlaThrThrAlaAlaThrThrThrThr 
610615620 
LysAlaLysAspLysAspProAspProGlyLysSerSerAlaAlaAla 
625630635640 
ValSerAlaThrGlyLeuLeuLeuLeuLeuAlaAlaThrSerValThr 
645650655 
AlaAlaSerLeuXaaGlyArgCysProThrXaaIleArgAsnSerLeu 
660665670 
AsnMetTyrIleTyrIleThrLeuThrSerAsnThrXaaLeuGlyPhe 
675680685 
(2) INFORMATION FOR SEQ ID NO:58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2259 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 12 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 52..2004 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
GAATTCGCGGCCGCGAAAGTGCGACAGCTGCGGTGGTTCGACGCAGTCGAGATGCGT57 
MetArg 
GGCATCGCTTTGTTCGTCGCCGCTGTTTCACTGATTGTAGAGGGCACA105 
GlyIleAlaLeuPheValAlaAlaValSerLeuIleValGluGlyThr 
51015 
GCAGAATCATCCATTTGCTCTGACTTCGGGAACGAGTTCTGTCGCAAC153 
AlaGluSerSerIleCysSerAspPheGlyAsnGluPheCysArgAsn 
202530 
GCTGAATGTGAAGTGGTGCCTGGTGCAGAGGATGATTTCGTGTGCAAA201 
AlaGluCysGluValValProGlyAlaGluAspAspPheValCysLys 
35404550 
TGTCCGCGAGATAATATGTACTTCAATGCTGCTGAAAAGCAATGCGAA249 
CysProArgAspAsnMetTyrPheAsnAlaAlaGluLysGlnCysGlu 
556065 
TATAAAGACACGTGCAAAACAAGGGAGTGCAGCTATGGACGTTGCGTT297 
TyrLysAspThrCysLysThrArgGluCysSerTyrGlyArgCysVal 
707580 
GAAAGTAACCCGAGCAAGGCTAGCTGCGTCTGCGAAGCATCGGACGAT345 
GluSerAsnProSerLysAlaSerCysValCysGluAlaSerAspAsp 
859095 
CTAACGCTACAATGCAAAATTAAAAATGACTACGCAACTGACTGCCGA393 
LeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAspCysArg 
100105110 
AACCGAGGTGGCACTGCTAAGTTGCGCACGGATGGGTTTATTGGCGCA441 
AsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIleGlyAla 
115120125130 
ACGTGTGACTGTGGTGAATGGGGTGCGATGAACATGACCACCCGGAAC489 
ThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThrArgAsn 
135140145 
TGTGTCCCTACCACGTGTCTTCGTCCCGACTTGAGCTGCAAAGACCTC537 
CysValProThrThrCysLeuArgProAspLeuSerCysLysAspLeu 
150155160 
TGCGAGAAAAACCTGCTTCAAAGGGATTCTCGTTGTTGCCAGGGGTGG585 
CysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGlnGlyTrp 
165170175 
AACACAGCAAACTGTTCAGCCGCTCCTCCAGCTGACTCCTATTGCTCT633 
AsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyrCysSer 
180185190 
CCTGGGAGCCCCAAAGGACCGGACGGACAGTGTATAAATGCTTGCAAG681 
ProGlySerProLysGlyProAspGlyGlnCysIleAsnAlaCysLys 
195200205210 
ATGAAAGAAGCTGGGTTTGTCTGCAAGCATGGATGCAGGTCGACCGCC729 
MetLysGluAlaGlyPheValCysLysHisGlyCysArgSerThrAla 
215220225 
AAGGCGTACGAGTGCACGTGCCCACGTGCCTTTACCGTCGCGGAAGAT777 
LysAlaTyrGluCysThrCysProArgAlaPheThrValAlaGluAsp 
230235240 
GGCATTACCTGCAAAAGTATTTCGCACACAGTCAGCTGCACTGCTGAG825 
GlyIleThrCysLysSerIleSerHisThrValSerCysThrAlaGlu 
245250255 
CAAAAACAGACCTGCCGCCCAACCGAAGACTGTCGTGTGCACAAAGGA873 
GlnLysGlnThrCysArgProThrGluAspCysArgValHisLysGly 
260265270 
ACTGTGTTGTGTGAGTGCCCGTGGAATCAACATCTAGTGGGGGACACG921 
ThrValLeuCysGluCysProTrpAsnGlnHisLeuValGlyAspThr 
275280285290 
TGCATAAGTGATTGCGTCGACAAGAAATGCCACGAAGAATTTATGGAC969 
CysIleSerAspCysValAspLysLysCysHisGluGluPheMetAsp 
295300305 
TGTGGCGTATATATGAATCGACAAAGCTGCTATTGTCCATGGAAATCA1017 
CysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrpLysSer 
310315320 
AGGAAGCCGGGCCCAAATGTCAACATCAATGGATGCCTACTGAATGAG1065 
ArgLysProGlyProAsnValAsnIleAsnGlyCysLeuLeuAsnGlu 
325330335 
TATTACTACACGGTGTCATTCACCCCAAACATATCTTTTGATTCTGAC1113 
TyrTyrTyrThrValSerPheThrProAsnIleSerPheAspSerAsp 
340345350 
CATTGCAAATGGTATGAGGATCGTGTTTTGGAAGCGATACGGACCAGT1161 
HisCysLysTrpTyrGluAspArgValLeuGluAlaIleArgThrSer 
355360365370 
ATCGGAAAAGAAGTTTTTAAGGTTGAGATACTTAACTGCACGCAGGAC1209 
IleGlyLysGluValPheLysValGluIleLeuAsnCysThrGlnAsp 
375380385 
ATTAAGGCAAGACTCATAGCAGAGAAATTACTGTCAAAACACGTGCTC1257 
IleLysAlaArgLeuIleAlaGluLysLeuLeuSerLysHisValLeu 
390395400 
AGGAAACTACAAGCATGCGAGCATCCAATCGGCGAATGGTGCATGATG1305 
ArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCysMetMet 
405410415 
TATCCGAAGTTGCTGATCAAGAAAAACTCTGCAACAGAAATCGAAGAA1353 
TyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIleGluGlu 
420425430 
GAGAACCTTTGCGACAGTCTGCTCAAGGATCAGGAAGCTGCCTACAAA1401 
GluAsnLeuCysAspSerLeuLeuLysAspGlnGluAlaAlaTyrLys 
435440445450 
GGTCAAAACAAATGCGTCAAGGTCGACAACCTCTTCTGGTTCCAGTGC1449 
GlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPheGlnCys 
455460465 
GCTGATGGTTACACAACAACTTACGAGATGACACGAGGTCGCCTACGC1497 
AlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArgLeuArg 
470475480 
CGCTCCGTGTGTAAAGCTGGAGTTTCTTGCAACGAAAACGAGCAGTCG1545 
ArgSerValCysLysAlaGlyValSerCysAsnGluAsnGluGlnSer 
485490495 
GAGTGTGCTGACAAAGGGCAAATATGTGTTTACGAAAACGGCAAAGCG1593 
GluCysAlaAspLysGlyGlnIleCysValTyrGluAsnGlyLysAla 
500505510 
AATTGCCAATGCCCACCAGACACTAAACCTGGGGAGATTGGCTGCATT1641 
AsnCysGlnCysProProAspThrLysProGlyGluIleGlyCysIle 
515520525530 
GAGCGTACCACATGCAACCCTAAAGAGATACAAGAATGCCAAGACAAG1689 
GluArgThrThrCysAsnProLysGluIleGlnGluCysGlnAspLys 
535540545 
AAGCTGGAGTGCGTTTACAAAAACCATAAAGCAGAATSSAAGTGTCCT1737 
LysLeuGluCysValTyrLysAsnHisLysAlaGluXaaLysCysPro 
550555560 
GATGATCACGAGTGTTACAGGGAGCCTGCCAAAGACTCTTGCAGTGAA1785 
AspAspHisGluCysTyrArgGluProAlaLysAspSerCysSerGlu 
565570575 
GAGGATAATGGTAAATGTCAAAGCAGTGGGCAGCGTTGTGTAATAGAA1833 
GluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysValIleGlu 
580585590 
AACGGAAAGGCTGTTTGCAAGGAAAAGTCTGAAGCAACAACAGCTGCG1881 
AsnGlyLysAlaValCysLysGluLysSerGluAlaThrThrAlaAla 
595600605610 
ACTACAACAACGAAAGCGAAAGACAAGGATCCAGATCCTGGAAAGTCA1929 
ThrThrThrThrLysAlaLysAspLysAspProAspProGlyLysSer 
615620625 
AGTGCTGCAGCAGTATCAGCTACTGGGCTCTTGTTACTGCTCGCAGCT1977 
SerAlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeuAlaAla 
630635640 
ACTTCAGTCACCGCAGCATCGTTGTAAGGAAGATGTCCAACTTGAATACGGAAC2031 
ThrSerValThrAlaAlaSerLeu 
645650 
AGCTTGAATATGTATATATACATCACGCTTACATCGAACACCTAGCTTGGTTTTTGGAAT2091 
TTCAATATTGCGCATTGGTACTCACGGCAACATGAATGTATTACTTTAGAATGACAGGGA2151 
AGAGGGACGTGAAAGGAGTTTCCTTGTCTGAACATATCAAAGAAAATTTTCCCCTATCCG2211 
ACCGATGTCAAATAAAGATAGTTGGGTCTAAACAGCGGCCGCGAATTC2259 
(2) INFORMATION FOR SEQ ID NO:59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 650 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
MetArgGlyIleAlaLeuPheValAlaAlaValSerLeuIleValGlu 
151015 
GlyThrAlaGluSerSerIleCysSerAspPheGlyAsnGluPheCys 
202530 
ArgAsnAlaGluCysGluValValProGlyAlaGluAspAspPheVal 
354045 
CysLysCysProArgAspAsnMetTyrPheAsnAlaAlaGluLysGln 
505560 
CysGluTyrLysAspThrCysLysThrArgGluCysSerTyrGlyArg 
65707580 
CysValGluSerAsnProSerLysAlaSerCysValCysGluAlaSer 
859095 
AspAspLeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAsp 
100105110 
CysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIle 
115120125 
GlyAlaThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThr 
130135140 
ArgAsnCysValProThrThrCysLeuArgProAspLeuSerCysLys 
145150155160 
AspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGln 
165170175 
GlyTrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyr 
180185190 
CysSerProGlySerProLysGlyProAspGlyGlnCysIleAsnAla 
195200205 
CysLysMetLysGluAlaGlyPheValCysLysHisGlyCysArgSer 
210215220 
ThrAlaLysAlaTyrGluCysThrCysProArgAlaPheThrValAla 
225230235240 
GluAspGlyIleThrCysLysSerIleSerHisThrValSerCysThr 
245250255 
AlaGluGlnLysGlnThrCysArgProThrGluAspCysArgValHis 
260265270 
LysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGly 
275280285 
AspThrCysIleSerAspCysValAspLysLysCysHisGluGluPhe 
290295300 
MetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrp 
305310315320 
LysSerArgLysProGlyProAsnValAsnIleAsnGlyCysLeuLeu 
325330335 
AsnGluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAsp 
340345350 
SerAspHisCysLysTrpTyrGluAspArgValLeuGluAlaIleArg 
355360365 
ThrSerIleGlyLysGluValPheLysValGluIleLeuAsnCysThr 
370375380 
GlnAspIleLysAlaArgLeuIleAlaGluLysLeuLeuSerLysHis 
385390395400 
ValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCys 
405410415 
MetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIle 
420425430 
GluGluGluAsnLeuCysAspSerLeuLeuLysAspGlnGluAlaAla 
435440445 
TyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPhe 
450455460 
GlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArg 
465470475480 
LeuArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGlu 
485490495 
GlnSerGluCysAlaAspLysGlyGlnIleCysValTyrGluAsnGly 
500505510 
LysAlaAsnCysGlnCysProProAspThrLysProGlyGluIleGly 
515520525 
CysIleGluArgThrThrCysAsnProLysGluIleGlnGluCysGln 
530535540 
AspLysLysLeuGluCysValTyrLysAsnHisLysAlaGluXaaLys 
545550555560 
CysProAspAspHisGluCysTyrArgGluProAlaLysAspSerCys 
565570575 
SerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysVal 
580585590 
IleGluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThr 
595600605 
AlaAlaThrThrThrThrLysAlaLysAspLysAspProAspProGly 
610615620 
LysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeu 
625630635640 
AlaAlaThrSerValThrAlaAlaSerLeu 
645650 
(2) INFORMATION FOR SEQ ID NO:60: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1647 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 13 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1647 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
GTTGAAAGTAACCCGAGCAAGGCTAGCTGCGTCTGCGAACGATCGGAC48 
ValGluSerAsnProSerLysAlaSerCysValCysGluArgSerAsp 
151015 
GATCTAACGCTACAATGCAAAATTAAAAATGACTACGCAACTGACTGC96 
AspLeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAspCys 
202530 
CGAAATCGAGGTGGCACTGCTAAGTTGCGCACGGATGGGTTTATTGGC144 
ArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIleGly 
354045 
GCAACGTGTGACTGTGGTGAATGGGGTGCGATGAACATGACCACCCGG192 
AlaThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThrArg 
505560 
AACTGTGTCCCTACCACGTGTCTTCGTCCCGACTTGACCTGCAAAGAC240 
AsnCysValProThrThrCysLeuArgProAspLeuThrCysLysAsp 
65707580 
CTCTGCGAGAAAAACCTGCTTCAAAGGGATTCTCGTTGTTGCCAGGGG288 
LeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGlnGly 
859095 
TGGAACACAGCAAACTGTTCAGCCGCTCCTCCAGCTGACTCCTATTGC336 
TrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyrCys 
100105110 
TCTCCTGGGAGCCCCAAAGGACCGGACGGACAGTGTATAAATGCTTGC384 
SerProGlySerProLysGlyProAspGlyGlnCysIleAsnAlaCys 
115120125 
AAGATGAAAGAAGCTGGGTTTGTCTGCGAGCATGGATGCAGGTCGACC432 
LysMetLysGluAlaGlyPheValCysGluHisGlyCysArgSerThr 
130135140 
GCCAAGGCGTACGAGTGCACGTGCCCACGTGGCTTTACCGTCGCGGAA480 
AlaLysAlaTyrGluCysThrCysProArgGlyPheThrValAlaGlu 
145150155160 
GATGGCATTACCTGCAAAAGTATTTCGCACACAGTCAGCTGCACTGCT528 
AspGlyIleThrCysLysSerIleSerHisThrValSerCysThrAla 
165170175 
GAGCAAAAACAGACCTGCCGCCCAACCGAAGACTGTCGTGTGCACAAA576 
GluGlnLysGlnThrCysArgProThrGluAspCysArgValHisLys 
180185190 
GGAACTGTGTTGTGTGAGTGCCCGTGGAATCAACATCTAGTGGGGGAC624 
GlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGlyAsp 
195200205 
ACGTGCATAAGTGATTGCGTCGACAAGAAATGCCACGAAGAATTTATG672 
ThrCysIleSerAspCysValAspLysLysCysHisGluGluPheMet 
210215220 
GACTGTGGCGTATATATGAATCGACAAAGCTGCTATTGTCCATGGAAA720 
AspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrpLys 
225230235240 
TCAAGGAAGCCGGGCCCAAATGTCAACATCAATGGATGCCTACTGAAT768 
SerArgLysProGlyProAsnValAsnIleAsnGlyCysLeuLeuAsn 
245250255 
GAGTATTACTACACGGTGTCATTCACCCCAAACATATCTTTTGATTCT816 
GluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAspSer 
260265270 
GACCATTGCAAATGGTATGAGGATCGTGTTTTGGAAGCGATACGGACC864 
AspHisCysLysTrpTyrGluAspArgValLeuGluAlaIleArgThr 
275280285 
AGTATCGGAAAAGAAGTTTTTAAGGTTGAGATACTTAACTGCACGCAG912 
SerIleGlyLysGluValPheLysValGluIleLeuAsnCysThrGln 
290295300 
GACATTAAGGCAAGACTCATAGCAGAGAAACCACTGTCAAACCACGTG960 
AspIleLysAlaArgLeuIleAlaGluLysProLeuSerAsnHisVal 
305310315320 
CTCAGGAAACTACAAGCATGCGAGCATCCAATCGGCGAATGGTGCATG1008 
LeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCysMet 
325330335 
ATGTATCCGAAGTTGCTGATCAAGAAAAACTCTGCAACAGAAATCGAA1056 
MetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIleGlu 
340345350 
GAAGAGAACCTTTGCGACAGTCTGCTCAAGAATCAGGAAGCTGCCTAC1104 
GluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGluAlaAlaTyr 
355360365 
AAAGGTCAAAACAAATGCGTCAAGGTCGACAACCTCTTCTGGTTCCAG1152 
LysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPheGln 
370375380 
TGCGCTGATGGTTACACAACAACTTACGAGATGACACGAGGTCGCCTA1200 
CysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArgLeu 
385390395400 
CGCCGCTCCGTGTGTAAAGCTGGAGTTTCTTGCAACGAAAACGAGCAG1248 
ArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGluGln 
405410415 
TTGGAGTGTGCTGACAAAGGGCAAATATGTGTTTACGAAAACGGCAAA1296 
LeuGluCysAlaAspLysGlyGlnIleCysValTyrGluAsnGlyLys 
420425430 
GCGAATTGCCAATGCCCACCAGACACTAAACCTGGGGAGATTGGCTGC1344 
AlaAsnCysGlnCysProProAspThrLysProGlyGluIleGlyCys 
435440445 
ATTGAGCGTACCACATGCAACCCTAAAGAGATACAAGAATGCCAAGAC1392 
IleGluArgThrThrCysAsnProLysGluIleGlnGluCysGlnAsp 
450455460 
AAGAAGCTGGAGTGCGTTTACAAAAACCATAAAGCAGAATGCAAGTGT1440 
LysLysLeuGluCysValTyrLysAsnHisLysAlaGluCysLysCys 
465470475480 
CCTGATGATCACGAGTGTTCCAGGGAGCCTGCCAAAGACTCTTGCAGT1488 
ProAspAspHisGluCysSerArgGluProAlaLysAspSerCysSer 
485490495 
GAAGAGGATAATGGTAAATGTCAAAGCAGTGGGCAGCGTTGTGTAATA1536 
GluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysValIle 
500505510 
GAAAACGGAAAGGCTGTTTGCAAGGAAAAGTCTGAAGCAACAACAGCT1584 
GluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThrAla 
515520525 
GCGACTACAACAACGAAAGCGAAAGACAAGGATCCAGATCCTGGAAAG1632 
AlaThrThrThrThrLysAlaLysAspLysAspProAspProGlyLys 
530535540 
TCAAGTGCTGCAGCA1647 
SerSerAlaAlaAla 
545 
(2) INFORMATION FOR SEQ ID NO:61: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 549 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
ValGluSerAsnProSerLysAlaSerCysValCysGluArgSerAsp 
151015 
AspLeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAspCys 
202530 
ArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIleGly 
354045 
AlaThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThrArg 
505560 
AsnCysValProThrThrCysLeuArgProAspLeuThrCysLysAsp 
65707580 
LeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGlnGly 
859095 
TrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyrCys 
100105110 
SerProGlySerProLysGlyProAspGlyGlnCysIleAsnAlaCys 
115120125 
LysMetLysGluAlaGlyPheValCysGluHisGlyCysArgSerThr 
130135140 
AlaLysAlaTyrGluCysThrCysProArgGlyPheThrValAlaGlu 
145150155160 
AspGlyIleThrCysLysSerIleSerHisThrValSerCysThrAla 
165170175 
GluGlnLysGlnThrCysArgProThrGluAspCysArgValHisLys 
180185190 
GlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGlyAsp 
195200205 
ThrCysIleSerAspCysValAspLysLysCysHisGluGluPheMet 
210215220 
AspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrpLys 
225230235240 
SerArgLysProGlyProAsnValAsnIleAsnGlyCysLeuLeuAsn 
245250255 
GluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAspSer 
260265270 
AspHisCysLysTrpTyrGluAspArgValLeuGluAlaIleArgThr 
275280285 
SerIleGlyLysGluValPheLysValGluIleLeuAsnCysThrGln 
290295300 
AspIleLysAlaArgLeuIleAlaGluLysProLeuSerAsnHisVal 
305310315320 
LeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCysMet 
325330335 
MetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIleGlu 
340345350 
GluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGluAlaAlaTyr 
355360365 
LysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPheGln 
370375380 
CysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArgLeu 
385390395400 
ArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGluGln 
405410415 
LeuGluCysAlaAspLysGlyGlnIleCysValTyrGluAsnGlyLys 
420425430 
AlaAsnCysGlnCysProProAspThrLysProGlyGluIleGlyCys 
435440445 
IleGluArgThrThrCysAsnProLysGluIleGlnGluCysGlnAsp 
450455460 
LysLysLeuGluCysValTyrLysAsnHisLysAlaGluCysLysCys 
465470475480 
ProAspAspHisGluCysSerArgGluProAlaLysAspSerCysSer 
485490495 
GluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysValIle 
500505510 
GluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThrAla 
515520525 
AlaThrThrThrThrLysAlaLysAspLysAspProAspProGlyLys 
530535540 
SerSerAlaAlaAla 
545 
(2) INFORMATION FOR SEQ ID NO:62: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2308 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 14 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 58..2010 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
CCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCGCCGGCCGCCGAG57 
ATGCGTGGCATCGCTTTGTTCGTCGCCGCTGTTTCACTGATTGTAGAG105 
MetArgGlyIleAlaLeuPheValAlaAlaValSerLeuIleValGlu 
151015 
TGCACAGCAGAATCATCCATTTGCTCTGACTTCGGGAACGAGTTCTGT153 
CysThrAlaGluSerSerIleCysSerAspPheGlyAsnGluPheCys 
202530 
CGCAACGCTGAATGTGAAGTGGTGCCTGGTGCAGAGGATGATTTCGTG201 
ArgAsnAlaGluCysGluValValProGlyAlaGluAspAspPheVal 
354045 
TGCAAATGTCCGCGAGATAATATGTACTTCAATGCTGCTGAAAAGCAA249 
CysLysCysProArgAspAsnMetTyrPheAsnAlaAlaGluLysGln 
505560 
TGCGAATATAAAGACACGTGCAAGACAAGGGAGTGCAGCTATGGACGT297 
CysGluTyrLysAspThrCysLysThrArgGluCysSerTyrGlyArg 
65707580 
TGCGTTGAAAGTAACCCGAGCAAGGCTAGCTGCGTCTGCGAAGCATCG345 
CysValGluSerAsnProSerLysAlaSerCysValCysGluAlaSer 
859095 
GACGATCTAACGCTACAATGCAAAATTAAAAATGACTACGCAACTGAC393 
AspAspLeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAsp 
100105110 
TGCCGAAATCGAGGTGGCACTGCTAAGTTGCGCACGGATGGGTTTATT441 
CysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIle 
115120125 
GGCGCAACGTGTGACTGTGGTGAATGGGGTGCGATGAACATGACCACC489 
GlyAlaThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThr 
130135140 
CGGAACTGTGTCCCTACCACGTGTCTTCGTCCCGACTTGACCTGCAAA537 
ArgAsnCysValProThrThrCysLeuArgProAspLeuThrCysLys 
145150155160 
GACCTCTGCGAGAAAAACCTGCTTCAAAGGGATTCTCGTTGTTGCCAG585 
AspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGln 
165170175 
GGGTGGAACACAGCAAACTGTTCAGCCGCTCCTCCAGCTGACTCCTAT633 
GlyTrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyr 
180185190 
TGCTCTCCTGGGAGCCCCAAAGGACCGGACGGACAGTGTATAAATGCT681 
CysSerProGlySerProLysGlyProAspGlyGlnCysIleAsnAla 
195200205 
TGCAAGATGAAAGAAGCTGGGTTTGTCTGCGAGCATGGATGCAGGTCG729 
CysLysMetLysGluAlaGlyPheValCysGluHisGlyCysArgSer 
210215220 
ACCGCCAAGGCGTACGAGTGCACGTGCCCACGTGGCTTTACCGTCGCG777 
ThrAlaLysAlaTyrGluCysThrCysProArgGlyPheThrValAla 
225230235240 
GAAGATGGCATTACCTGCAAAAGTATTTCGCACACAGTCAGCTGCACT825 
GluAspGlyIleThrCysLysSerIleSerHisThrValSerCysThr 
245250255 
GCTGAGCAAAAACAGACCTGCCGCCCAACCGAAGACTGTCGTGTGCAC873 
AlaGluGlnLysGlnThrCysArgProThrGluAspCysArgValHis 
260265270 
AAAGGAACTGTGTTGTGTGAGTGCCCGTGGAATCAACATCTAGTGGGG921 
LysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGly 
275280285 
GACACGTGCATAAGTGATTGCGTCGACAAGAAATGCCACGAAGAATTT969 
AspThrCysIleSerAspCysValAspLysLysCysHisGluGluPhe 
290295300 
ATGGACTGTGGCGTATATATGAATCGACAAAGCTGCTATTGTCCATGG1017 
MetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrp 
305310315320 
AAATCAAGGAAGCCGGGCCCAAATGTCAACATCAATGGATGCCTACTG1065 
LysSerArgLysProGlyProAsnValAsnIleAsnGlyCysLeuLeu 
325330335 
AATGAGTATTACTACACGGTGTCATTCACCCCAAACATATCTTTTGAT1113 
AsnGluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAsp 
340345350 
TCTGACCATTGCAAATGGTATGAGGATCGTGTTTTGGAAGCGATACGG1161 
SerAspHisCysLysTrpTyrGluAspArgValLeuGluAlaIleArg 
355360365 
ACCAGTATCGGAAAAGAAGTTTTTAAGGTTGAGATACTTAACTGCACG1209 
ThrSerIleGlyLysGluValPheLysValGluIleLeuAsnCysThr 
370375380 
CAGGACATTAAGGCAAGACTCATAGCAGAGAAACCACTGTCAAACCAC1257 
GlnAspIleLysAlaArgLeuIleAlaGluLysProLeuSerAsnHis 
385390395400 
GTGCTCAGGAAACTACAAGCATGCGAGCATCCAATCGGCGAATGGTGC1305 
ValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCys 
405410415 
ATGATGTATCCGAAGTTGCTGATCAAGAAAAACTCTGCAACAGAAATC1353 
MetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIle 
420425430 
GAAGAAGAGAACCTTTGCGACAGTCTGCTCAAGAATCAGGAAGCTGCC1401 
GluGluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGluAlaAla 
435440445 
TACAAAGGTCAAAACAAATGCGTCAAGGTCGACAACCTCTTCTGGTTC1449 
TyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPhe 
450455460 
CAGTGCGCTGATGGTTACACAACAACTTACGAGATGACACGAGGTCGC1497 
GlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArg 
465470475480 
CTACGCCGCTCCGTGTGTAAAGCTGGAGTTTCTTGCAACGAAAACGAG1545 
LeuArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGlu 
485490495 
CAGTTGGAGTGTGCTGACAAAGGGCAAATATGTGTTTACGAAAACGGC1593 
GlnLeuGluCysAlaAspLysGlyGlnIleCysValTyrGluAsnGly 
500505510 
AAAGCGAATTGCCAATGCCCACCAGACACTAAACCTGGGGAGATTGGC1641 
LysAlaAsnCysGlnCysProProAspThrLysProGlyGluIleGly 
515520525 
TGCATTGAGCGTACCACATGCAACCCTAAAGAGATACAAGAATGCCAA1689 
CysIleGluArgThrThrCysAsnProLysGluIleGlnGluCysGln 
530535540 
GACAAGAAGCTGGAGTGCGTTTACAAAAACCATAAAGCAGAATGCAAG1737 
AspLysLysLeuGluCysValTyrLysAsnHisLysAlaGluCysLys 
545550555560 
TGTCCTGATGATCACGAGTGTTCCAGGGAGCCTGCCAAAGACTCTTGC1785 
CysProAspAspHisGluCysSerArgGluProAlaLysAspSerCys 
565570575 
AGTGAAGAGGATAATGGTAAATGTCAAAGCAGTGGGCAGCGTTGTGTA1833 
SerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysVal 
580585590 
ATAGAAAACGGAAAGGCTGTTTGCAAGGAAAAGTCTGAAGCAACAACA1881 
IleGluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThr 
595600605 
GCTGCGACTACAACAACGAAAGCGAAAGACAAGGATCCAGATCCTGGA1929 
AlaAlaThrThrThrThrLysAlaLysAspLysAspProAspProGly 
610615620 
AAGTCAAGTGCTGCAGCAGTATCAGCTACTGGGCTCTTGTTACTGCTC1977 
LysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeu 
625630635640 
GCAGCTACTTCAGTCACCGCAGCATCGTTGTAAGGAAGMTGTCCAACTNC2027 
AlaAlaThrSerValThrAlaAlaSerLeu 
645650 
AATACGGAACAGCTTGAATATGTATATATACATCACGCTTACATCGAACACCTAGCTTGG2087 
TTTTTGGAATTTCAATATTGCGCATTGGTACTCACNGCAACATGAATGTATTACTTTAGA2147 
ATGACAGGGAAGAGGGACGTGAAAGGAGTTTCCTTGTCTGAACATATCAAAGAAAATTTT2207 
CCCCTATCCGACCGATGTCAGCGGCCGCGAATTCCTGCAGCCCGGGGGATCCACTAGTTC2267 
TAGAGCGGGCGGCCGCGTTAACCACCGCGGTGGAGCTCCAG2308 
(2) INFORMATION FOR SEQ ID NO:63: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 650 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
MetArgGlyIleAlaLeuPheValAlaAlaValSerLeuIleValGlu 
151015 
CysThrAlaGluSerSerIleCysSerAspPheGlyAsnGluPheCys 
202530 
ArgAsnAlaGluCysGluValValProGlyAlaGluAspAspPheVal 
354045 
CysLysCysProArgAspAsnMetTyrPheAsnAlaAlaGluLysGln 
505560 
CysGluTyrLysAspThrCysLysThrArgGluCysSerTyrGlyArg 
65707580 
CysValGluSerAsnProSerLysAlaSerCysValCysGluAlaSer 
859095 
AspAspLeuThrLeuGlnCysLysIleLysAsnAspTyrAlaThrAsp 
100105110 
CysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIle 
115120125 
GlyAlaThrCysAspCysGlyGluTrpGlyAlaMetAsnMetThrThr 
130135140 
ArgAsnCysValProThrThrCysLeuArgProAspLeuThrCysLys 
145150155160 
AspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGln 
165170175 
GlyTrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyr 
180185190 
CysSerProGlySerProLysGlyProAspGlyGlnCysIleAsnAla 
195200205 
CysLysMetLysGluAlaGlyPheValCysGluHisGlyCysArgSer 
210215220 
ThrAlaLysAlaTyrGluCysThrCysProArgGlyPheThrValAla 
225230235240 
GluAspGlyIleThrCysLysSerIleSerHisThrValSerCysThr 
245250255 
AlaGluGlnLysGlnThrCysArgProThrGluAspCysArgValHis 
260265270 
LysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGly 
275280285 
AspThrCysIleSerAspCysValAspLysLysCysHisGluGluPhe 
290295300 
MetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrp 
305310315320 
LysSerArgLysProGlyProAsnValAsnIleAsnGlyCysLeuLeu 
325330335 
AsnGluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAsp 
340345350 
SerAspHisCysLysTrpTyrGluAspArgValLeuGluAlaIleArg 
355360365 
ThrSerIleGlyLysGluValPheLysValGluIleLeuAsnCysThr 
370375380 
GlnAspIleLysAlaArgLeuIleAlaGluLysProLeuSerAsnHis 
385390395400 
ValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCys 
405410415 
MetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIle 
420425430 
GluGluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGluAlaAla 
435440445 
TyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPhe 
450455460 
GlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArg 
465470475480 
LeuArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGlu 
485490495 
GlnLeuGluCysAlaAspLysGlyGlnIleCysValTyrGluAsnGly 
500505510 
LysAlaAsnCysGlnCysProProAspThrLysProGlyGluIleGly 
515520525 
CysIleGluArgThrThrCysAsnProLysGluIleGlnGluCysGln 
530535540 
AspLysLysLeuGluCysValTyrLysAsnHisLysAlaGluCysLys 
545550555560 
CysProAspAspHisGluCysSerArgGluProAlaLysAspSerCys 
565570575 
SerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysVal 
580585590 
IleGluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThr 
595600605 
AlaAlaThrThrThrThrLysAlaLysAspLysAspProAspProGly 
610615620 
LysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeu 
625630635640 
AlaAlaThrSerValThrAlaAlaSerLeu 
645650 
(2) INFORMATION FOR SEQ ID NO:64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2131 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 15 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1863 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
TTCTGTCGCAACGCTGAATGCGAAGAGGTGCCTGGTGCCGAGGATGAT48 
PheCysArgAsnAlaGluCysGluGluValProGlyAlaGluAspAsp 
151015 
TTCGTGTGCAAATGTCCGCGATATAATATGTACTTCAATGCTGCTGAA96 
PheValCysLysCysProArgTyrAsnMetTyrPheAsnAlaAlaGlu 
202530 
AAACAATGCGAATATAAAGATACGTGCAAGACAAGAGAGTGCAGCTAT144 
LysGlnCysGluTyrLysAspThrCysLysThrArgGluCysSerTyr 
354045 
GGCCGTTGCGTTCAAAGTAACCCGAGCAAGGCTAGCTGTGTCTGCGAA192 
GlyArgCysValGlnSerAsnProSerLysAlaSerCysValCysGlu 
505560 
GCATCTGACACTCTAACGCTACAATGCAACATTAACAATGACTACGCA240 
AlaSerAspThrLeuThrLeuGlnCysAsnIleAsnAsnAspTyrAla 
65707580 
ACTGACTGCCGAAACAGGGGTGGTACCGCTAAGTTGCGCACGGATGGG288 
ThrAspCysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGly 
859095 
TTTATTGGCGCAACGTGTGACTGTGGTGAATGGGGCGCAATGAACAAA336 
PheIleGlyAlaThrCysAspCysGlyGluTrpGlyAlaMetAsnLys 
100105110 
ACCACCCGGAACTGTGTCCCTACCACGTGTCTTCGTCCCGACTTGACC384 
ThrThrArgAsnCysValProThrThrCysLeuArgProAspLeuThr 
115120125 
TGCAAAGACCTCTGCGAGAAAAACCTGCTTCAAAGGGATTCTCGTTGT432 
CysLysAspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCys 
130135140 
TGCCAGGGGTGGAACACAGCAAACTGTTTAGCCGCTCCTCCAGCTGAC480 
CysGlnGlyTrpAsnThrAlaAsnCysLeuAlaAlaProProAlaAsp 
145150155160 
TCCTATTGCTCTCCTGGGAGCCCCAAAGGACCGGACGGACAGTGTAAA528 
SerTyrCysSerProGlySerProLysGlyProAspGlyGlnCysLys 
165170175 
AATGCTTGCAGGACGAAAGAAGCTGGGTTTGTCTGCAAGCATGGATGC576 
AsnAlaCysArgThrLysGluAlaGlyPheValCysLysHisGlyCys 
180185190 
AGGTCCACCGACAAGGCGTACGAGTGCACGTGCCCGAGTGGCTCTACC624 
ArgSerThrAspLysAlaTyrGluCysThrCysProSerGlySerThr 
195200205 
GTCGCCGAAGATGGCATTACCTGCAAAAGTATTTCGTACACAGTCAGC672 
ValAlaGluAspGlyIleThrCysLysSerIleSerTyrThrValSer 
210215220 
TGCACTGTTGAGCAAAAACAGACCTGCCGCCCAACCGAAGACTGTCGT720 
CysThrValGluGlnLysGlnThrCysArgProThrGluAspCysArg 
225230235240 
GTGCAGAAAGGAACTGTGTTGTGTGAGTGCCCGTGGAATCAACATCTA768 
ValGlnLysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeu 
245250255 
GTGGGGGACAAGTGCATAAGTGATTGCGTCGACAAGAAATGTCACGAA816 
ValGlyAspLysCysIleSerAspCysValAspLysLysCysHisGlu 
260265270 
GAATTTATGGACTGTGGCGTATATATGAATCGACAAAGCTGCTATTGT864 
GluPheMetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCys 
275280285 
CCATGGAAATCAAGGAAGCCGGGCCCAAATGTCAACATCAATGAATGC912 
ProTrpLysSerArgLysProGlyProAsnValAsnIleAsnGluCys 
290295300 
CTACTGAATGAGTATTACTACACGGTGTCATTCACCCCGAACATATCT960 
LeuLeuAsnGluTyrTyrTyrThrValSerPheThrProAsnIleSer 
305310315320 
TTTGATTCTGACCATTGCAAACGGTATGAGGATCGTGTTTTGGAAGCG1008 
PheAspSerAspHisCysLysArgTyrGluAspArgValLeuGluAla 
325330335 
ATACGGACCAGTATCGGAAAAGAAGTTTTTAAGGTTGAGATACTTAAC1056 
IleArgThrSerIleGlyLysGluValPheLysValGluIleLeuAsn 
340345350 
TGCACGCAGGACATTAAGGCAAGACTCATAGCAGAGAAACCACTGTCA1104 
CysThrGlnAspIleLysAlaArgLeuIleAlaGluLysProLeuSer 
355360365 
AAATACGTGCTCAGGAAACTACAAGCATGCGAGCATCCAATCGGCGAA1152 
LysTyrValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGlu 
370375380 
TGGTGCATGATGTATCCGAAGTTGCTGATCAAGAAAAACTCTGCAACA1200 
TrpCysMetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThr 
385390395400 
GAAATTGAAGAAGAGAACCTTTGCGACAGTCTGCTCAAGAATCAGGAA1248 
GluIleGluGluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGlu 
405410415 
GCTGCCTACAAAGGTCAAAACAAATGCGTCAAGGTCGACAACCTCTTC1296 
AlaAlaTyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPhe 
420425430 
TGGTTCCAGTGCGCTGATGGTTACACAACAACTTACGAGATGACACGA1344 
TrpPheGlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArg 
435440445 
GGTCGCCTACGCCGCTCCGTGTGTAAAGCTGGAGTTTCTTGCAACGAA1392 
GlyArgLeuArgArgSerValCysLysAlaGlyValSerCysAsnGlu 
450455460 
AACGAGCAGTTGGAGTGTGCTAACAAAGGTCAAATATGTGTCTACGAA1440 
AsnGluGlnLeuGluCysAlaAsnLysGlyGlnIleCysValTyrGlu 
465470475480 
AACGGCAAAGCGAATTGCCAATGCCCACCAGACACTAAACCAGGGGAG1488 
AsnGlyLysAlaAsnCysGlnCysProProAspThrLysProGlyGlu 
485490495 
ATTGGCTGCATTGAGCGTACCACATGCAACCCTAAAGAGATACAAGAA1536 
IleGlyCysIleGluArgThrThrCysAsnProLysGluIleGlnGlu 
500505510 
TGCCAAGACAAGAAGCTCGAGTGCGTTTACAAAAACCATAAAGCAGAA1584 
CysGlnAspLysLysLeuGluCysValTyrLysAsnHisLysAlaGlu 
515520525 
TSSAAGTGTCCTGATGATCACGAGTGTTCTAGGGAGCCTGCCAAAGAC1632 
XaaLysCysProAspAspHisGluCysSerArgGluProAlaLysAsp 
530535540 
TCTTGCAGTGAAGAAGATAATGGTAAATGTCAAAGCAGTGGGCAGCGT1680 
SerCysSerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArg 
545550555560 
TGTGTAATGGAAAACGGAAATGCTGTTTGCAAAGAGAAGTCTGATGCA1728 
CysValMetGluAsnGlyAsnAlaValCysLysGluLysSerAspAla 
565570575 
ACAACAGCTTCGACTACAACAACGAAAGCGAAAGACAAGGATCCAGAT1776 
ThrThrAlaSerThrThrThrThrLysAlaLysAspLysAspProAsp 
580585590 
CCTGAAAAGTCAAGTGCTGCAGCAGTATCAGCTACTGGGCTCTTGTTA1824 
ProGluLysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeu 
595600605 
CTGCTCGCAGCTACTTCAGTCACCGCAGCATCGTTGTAATGAAGAT1870 
LeuLeuAlaAlaThrSerValThrAlaAlaSerLeu 
610615620 
GTCCAACTTGAATACGGAACAGCTTGAAAATGTATATATACATCACGCTTACATCGAACA1930 
TCTAGCTTGGTCTTTGGAATTTAAATATTGCACATGGGTACTCACGGCAAAATGGACGTA1990 
TTATTTTAGAATGACAGGGAAGATGGACGTGAAAGGAGTTTCCTTGTCTGAAAATATCAA2050 
AGAAAAACTTTCCCTATCTGAATGATGTCAAATAAAGATAGTTGGGTCTAAACAAAAAAA2110 
AAAAAAAAAAAAAAGCGGCCG2131 
(2) INFORMATION FOR SEQ ID NO:65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 620 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
PheCysArgAsnAlaGluCysGluGluValProGlyAlaGluAspAsp 
151015 
PheValCysLysCysProArgTyrAsnMetTyrPheAsnAlaAlaGlu 
202530 
LysGlnCysGluTyrLysAspThrCysLysThrArgGluCysSerTyr 
354045 
GlyArgCysValGlnSerAsnProSerLysAlaSerCysValCysGlu 
505560 
AlaSerAspThrLeuThrLeuGlnCysAsnIleAsnAsnAspTyrAla 
65707580 
ThrAspCysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGly 
859095 
PheIleGlyAlaThrCysAspCysGlyGluTrpGlyAlaMetAsnLys 
100105110 
ThrThrArgAsnCysValProThrThrCysLeuArgProAspLeuThr 
115120125 
CysLysAspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCys 
130135140 
CysGlnGlyTrpAsnThrAlaAsnCysLeuAlaAlaProProAlaAsp 
145150155160 
SerTyrCysSerProGlySerProLysGlyProAspGlyGlnCysLys 
165170175 
AsnAlaCysArgThrLysGluAlaGlyPheValCysLysHisGlyCys 
180185190 
ArgSerThrAspLysAlaTyrGluCysThrCysProSerGlySerThr 
195200205 
ValAlaGluAspGlyIleThrCysLysSerIleSerTyrThrValSer 
210215220 
CysThrValGluGlnLysGlnThrCysArgProThrGluAspCysArg 
225230235240 
ValGlnLysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeu 
245250255 
ValGlyAspLysCysIleSerAspCysValAspLysLysCysHisGlu 
260265270 
GluPheMetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCys 
275280285 
ProTrpLysSerArgLysProGlyProAsnValAsnIleAsnGluCys 
290295300 
LeuLeuAsnGluTyrTyrTyrThrValSerPheThrProAsnIleSer 
305310315320 
PheAspSerAspHisCysLysArgTyrGluAspArgValLeuGluAla 
325330335 
IleArgThrSerIleGlyLysGluValPheLysValGluIleLeuAsn 
340345350 
CysThrGlnAspIleLysAlaArgLeuIleAlaGluLysProLeuSer 
355360365 
LysTyrValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGlu 
370375380 
TrpCysMetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThr 
385390395400 
GluIleGluGluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGlu 
405410415 
AlaAlaTyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPhe 
420425430 
TrpPheGlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArg 
435440445 
GlyArgLeuArgArgSerValCysLysAlaGlyValSerCysAsnGlu 
450455460 
AsnGluGlnLeuGluCysAlaAsnLysGlyGlnIleCysValTyrGlu 
465470475480 
AsnGlyLysAlaAsnCysGlnCysProProAspThrLysProGlyGlu 
485490495 
IleGlyCysIleGluArgThrThrCysAsnProLysGluIleGlnGlu 
500505510 
CysGlnAspLysLysLeuGluCysValTyrLysAsnHisLysAlaGlu 
515520525 
XaaLysCysProAspAspHisGluCysSerArgGluProAlaLysAsp 
530535540 
SerCysSerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArg 
545550555560 
CysValMetGluAsnGlyAsnAlaValCysLysGluLysSerAspAla 
565570575 
ThrThrAlaSerThrThrThrThrLysAlaLysAspLysAspProAsp 
580585590 
ProGluLysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeu 
595600605 
LeuLeuAlaAlaThrSerValThrAlaAlaSerLeu 
610615620 
(2) INFORMATION FOR SEQ ID NO:66: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2147 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 16 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 49..2001 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
CAGGATCCGTGGAAAGTGCGACAGCTGCGGTGGTTCGACGCAGTCGAGATGCGTGGC57 
MetArgGly 
1 
ATCGCTTTGTTCGTCGCCGCTGTTTCACTGATTGTAGAGTGCACAGCA105 
IleAlaLeuPheValAlaAlaValSerLeuIleValGluCysThrAla 
51015 
GAATCATCCATTTGCTCTGACTTCGGGAACGAGTTCTGTCGCAACGCT153 
GluSerSerIleCysSerAspPheGlyAsnGluPheCysArgAsnAla 
20253035 
GAATGTGAAGTGGTGCCTGGTGCAGAGGATGATTTCGTGTGCAAATGT201 
GluCysGluValValProGlyAlaGluAspAspPheValCysLysCys 
404550 
CCGCGAGATAATATGTACTTCAATGCTGCTGAAAAGCAATGCGAATAT249 
ProArgAspAsnMetTyrPheAsnAlaAlaGluLysGlnCysGluTyr 
556065 
AAAGATACGTGCAAGACAAGGGAGTGCAGCTATGGACGTTGCGTTGAA297 
LysAspThrCysLysThrArgGluCysSerTyrGlyArgCysValGlu 
707580 
AGTAACCCGAGCAAGGGTAGCTGCGTCTGCGAAGCATCGGACGATCTA345 
SerAsnProSerLysGlySerCysValCysGluAlaSerAspAspLeu 
859095 
ACGCTACAATGCAAAATTAAAAATGACTTCGCAACTGACTGCCGAAAC393 
ThrLeuGlnCysLysIleLysAsnAspPheAlaThrAspCysArgAsn 
100105110115 
CGAGGTGGCACTGCTAAGTTGCGCACGGATGGGTTTATTGGCCCAACG441 
ArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIleGlyProThr 
120125130 
TGTGACTGTGGTGAATGGGGTGCGATGAACAAGACCACACGGAACTGT489 
CysAspCysGlyGluTrpGlyAlaMetAsnLysThrThrArgAsnCys 
135140145 
GTCCCTACCACGTGTCTTCGTCCCGACTTGACCTGCAAAGACCTCTGC537 
ValProThrThrCysLeuArgProAspLeuThrCysLysAspLeuCys 
150155160 
GAGAAAAACCTGCTTCAAAGGGATTCTCGTTGTTGTCAGGGGTGGAAC585 
GluLysAsnLeuLeuGlnArgAspSerArgCysCysGlnGlyTrpAsn 
165170175 
ACAGCAAACTGTTCAGCCGCTCCTCCAGCTGACTCCTATTGCTCTCCT633 
ThrAlaAsnCysSerAlaAlaProProAlaAspSerTyrCysSerPro 
180185190195 
GGGAGCCCCAAAGGACCGGACGGACAGTGTAAAAATGCTTGCAGGACG681 
GlySerProLysGlyProAspGlyGlnCysLysAsnAlaCysArgThr 
200205210 
AAAGAAGCTGGGTTTGTCTGCAAGCATGGATGCAGGTCCACCGACAAG729 
LysGluAlaGlyPheValCysLysHisGlyCysArgSerThrAspLys 
215220225 
GCGTACGAGTGCACGTGCCCGAGTGGCTCTACCGTCGCCGAAGATGGC777 
AlaTyrGluCysThrCysProSerGlySerThrValAlaGluAspGly 
230235240 
ATTACCTGCAAAAGTATTTCGTACACAGTCAGCTGCACTGTTGAGCAA825 
IleThrCysLysSerIleSerTyrThrValSerCysThrValGluGln 
245250255 
AAACAGACCTGCCGCCCAACCGAAGACTGTCGTGTGCAGAAAGGAACT873 
LysGlnThrCysArgProThrGluAspCysArgValGlnLysGlyThr 
260265270275 
GTGTTGTGTGAGTGCCCGTGGAATCAACATCTAGTGGGGGACACGTGC921 
ValLeuCysGluCysProTrpAsnGlnHisLeuValGlyAspThrCys 
280285290 
ATAAGTGATTGCGTCGACAAGAAATGTCACGAAGAATTTATGGACTGT969 
IleSerAspCysValAspLysLysCysHisGluGluPheMetAspCys 
295300305 
GGCGTATATATGAATCGACAAAGCTGCTATTGTCCATGGAAATCAAGG1017 
GlyValTyrMetAsnArgGlnSerCysTyrCysProTrpLysSerArg 
310315320 
AAGCCGGGCCCAAATGTCAACATCAATGAATGCCTACTGAATGAGTAT1065 
LysProGlyProAsnValAsnIleAsnGluCysLeuLeuAsnGluTyr 
325330335 
TACTACACGGTGTCATTCACCCCGAACATATCTTTTGATTCTGACCAT1113 
TyrTyrThrValSerPheThrProAsnIleSerPheAspSerAspHis 
340345350355 
TGCAAACGGTATGAGGATCGTGTTTTGGAAGCGATACGGACCAGTATC1161 
CysLysArgTyrGluAspArgValLeuGluAlaIleArgThrSerIle 
360365370 
GGAAAAGAAGTTTTTAAGGTTGAGATACTTAACTGCACGCAGGACATT1209 
GlyLysGluValPheLysValGluIleLeuAsnCysThrGlnAspIle 
375380385 
AAGGCAAGACTCATAGCAGAGAAACCACTGTCAAAATACGTGCTCAGG1257 
LysAlaArgLeuIleAlaGluLysProLeuSerLysTyrValLeuArg 
390395400 
AAACTACAAGCATGCGAGCATCCAATCGGCGAATGGTGCATGATGTAT1305 
LysLeuGlnAlaCysGluHisProIleGlyGluTrpCysMetMetTyr 
405410415 
CCGAAGTTGCTGATCAAGAAAAACTCTGCAACAGAAATTGAAGAAGAG1353 
ProLysLeuLeuIleLysLysAsnSerAlaThrGluIleGluGluGlu 
420425430435 
AACCTTTGCGACAGTCTGCTCAAGAATCAGGAAGCTGCCTACAAAGGT1401 
AsnLeuCysAspSerLeuLeuLysAsnGlnGluAlaAlaTyrLysGly 
440445450 
CAAAACAAATGCGTCAAGGTCGACAACCTCTTCTGGTTCCAGTGCGCT1449 
GlnAsnLysCysValLysValAspAsnLeuPheTrpPheGlnCysAla 
455460465 
GATGGTTACACAACAACTTACGAGATGACACGAGGTCGCCTACGCCGC1497 
AspGlyTyrThrThrThrTyrGluMetThrArgGlyArgLeuArgArg 
470475480 
TCCGTGTGTAAAGCTGGAGTTTCTTGCAACGAAAACGAGCAGTTGGAG1545 
SerValCysLysAlaGlyValSerCysAsnGluAsnGluGlnLeuGlu 
485490495 
TGTGCTAACAAAGGTCAAATATGTGTCTACGAAAACGGCAAAGCGAAT1593 
CysAlaAsnLysGlyGlnIleCysValTyrGluAsnGlyLysAlaAsn 
500505510515 
TGCCAATGCCCACCAGACACTAAACCAGGGGAGATTGGCTGCATTGAG1641 
CysGlnCysProProAspThrLysProGlyGluIleGlyCysIleGlu 
520525530 
CGTACCACATGCAACCCTAAAGAGATACAAGAATGCCAAGACAAGAAG1689 
ArgThrThrCysAsnProLysGluIleGlnGluCysGlnAspLysLys 
535540545 
CTCGAGTGCGTTTACAAAAACCATAAAGCAGAATGCAAGTGTCCTGAT1737 
LeuGluCysValTyrLysAsnHisLysAlaGluCysLysCysProAsp 
550555560 
GATCACGAGTGTTCTAGGCAGCCTGCCAAAGACTCTTGCAGTGAAGAG1785 
AspHisGluCysSerArgGlnProAlaLysAspSerCysSerGluGlu 
565570575 
GATAATGGTAAATGTCAAAGCAGTGGGCAGCGTTGTGTAATGGAAAAC1833 
AspAsnGlyLysCysGlnSerSerGlyGlnArgCysValMetGluAsn 
580585590595 
GGAAAGGCTGTTTGCAAAGAGAAGTCTGAAGCAACAACAGCTGCGACT1881 
GlyLysAlaValCysLysGluLysSerGluAlaThrThrAlaAlaThr 
600605610 
ACAACAACGAAAGCGAAAGACAAGGATCCAGATCCTGGAAAGTCAAGT1929 
ThrThrThrLysAlaLysAspLysAspProAspProGlyLysSerSer 
615620625 
GCTGCAGCAGTATCAGCTACTGGGCTCTTGTTACTGCTCGCAGCTACT1977 
AlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeuAlaAlaThr 
630635640 
TCAGTCACCGTAGCATCGTTGTAATGAAGATGTCCAACTTGAATACGGAAC2028 
SerValThrValAlaSerLeu 
645650 
AGCTTGAAAATGTATATATACATCGCGCTTACATCGAACACCTAGCTTGGTTTTTGGGAT2088 
TTCAATATTGCGCATGGGTACTCACGTCAACATGGGATGTATTATTTGAGAATGACAAG2147 
(2) INFORMATION FOR SEQ ID NO:67: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 650 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
MetArgGlyIleAlaLeuPheValAlaAlaValSerLeuIleValGlu 
151015 
CysThrAlaGluSerSerIleCysSerAspPheGlyAsnGluPheCys 
202530 
ArgAsnAlaGluCysGluValValProGlyAlaGluAspAspPheVal 
354045 
CysLysCysProArgAspAsnMetTyrPheAsnAlaAlaGluLysGln 
505560 
CysGluTyrLysAspThrCysLysThrArgGluCysSerTyrGlyArg 
65707580 
CysValGluSerAsnProSerLysGlySerCysValCysGluAlaSer 
859095 
AspAspLeuThrLeuGlnCysLysIleLysAsnAspPheAlaThrAsp 
100105110 
CysArgAsnArgGlyGlyThrAlaLysLeuArgThrAspGlyPheIle 
115120125 
GlyProThrCysAspCysGlyGluTrpGlyAlaMetAsnLysThrThr 
130135140 
ArgAsnCysValProThrThrCysLeuArgProAspLeuThrCysLys 
145150155160 
AspLeuCysGluLysAsnLeuLeuGlnArgAspSerArgCysCysGln 
165170175 
GlyTrpAsnThrAlaAsnCysSerAlaAlaProProAlaAspSerTyr 
180185190 
CysSerProGlySerProLysGlyProAspGlyGlnCysLysAsnAla 
195200205 
CysArgThrLysGluAlaGlyPheValCysLysHisGlyCysArgSer 
210215220 
ThrAspLysAlaTyrGluCysThrCysProSerGlySerThrValAla 
225230235240 
GluAspGlyIleThrCysLysSerIleSerTyrThrValSerCysThr 
245250255 
ValGluGlnLysGlnThrCysArgProThrGluAspCysArgValGln 
260265270 
LysGlyThrValLeuCysGluCysProTrpAsnGlnHisLeuValGly 
275280285 
AspThrCysIleSerAspCysValAspLysLysCysHisGluGluPhe 
290295300 
MetAspCysGlyValTyrMetAsnArgGlnSerCysTyrCysProTrp 
305310315320 
LysSerArgLysProGlyProAsnValAsnIleAsnGluCysLeuLeu 
325330335 
AsnGluTyrTyrTyrThrValSerPheThrProAsnIleSerPheAsp 
340345350 
SerAspHisCysLysArgTyrGluAspArgValLeuGluAlaIleArg 
355360365 
ThrSerIleGlyLysGluValPheLysValGluIleLeuAsnCysThr 
370375380 
GlnAspIleLysAlaArgLeuIleAlaGluLysProLeuSerLysTyr 
385390395400 
ValLeuArgLysLeuGlnAlaCysGluHisProIleGlyGluTrpCys 
405410415 
MetMetTyrProLysLeuLeuIleLysLysAsnSerAlaThrGluIle 
420425430 
GluGluGluAsnLeuCysAspSerLeuLeuLysAsnGlnGluAlaAla 
435440445 
TyrLysGlyGlnAsnLysCysValLysValAspAsnLeuPheTrpPhe 
450455460 
GlnCysAlaAspGlyTyrThrThrThrTyrGluMetThrArgGlyArg 
465470475480 
LeuArgArgSerValCysLysAlaGlyValSerCysAsnGluAsnGlu 
485490495 
GlnLeuGluCysAlaAsnLysGlyGlnIleCysValTyrGluAsnGly 
500505510 
LysAlaAsnCysGlnCysProProAspThrLysProGlyGluIleGly 
515520525 
CysIleGluArgThrThrCysAsnProLysGluIleGlnGluCysGln 
530535540 
AspLysLysLeuGluCysValTyrLysAsnHisLysAlaGluCysLys 
545550555560 
CysProAspAspHisGluCysSerArgGlnProAlaLysAspSerCys 
565570575 
SerGluGluAspAsnGlyLysCysGlnSerSerGlyGlnArgCysVal 
580585590 
MetGluAsnGlyLysAlaValCysLysGluLysSerGluAlaThrThr 
595600605 
AlaAlaThrThrThrThrLysAlaLysAspLysAspProAspProGly 
610615620 
LysSerSerAlaAlaAlaValSerAlaThrGlyLeuLeuLeuLeuLeu 
625630635640 
AlaAlaThrSerValThrValAlaSerLeu 
645650 
(2) INFORMATION FOR SEQ ID NO:68: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 441 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: Figure 17 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..441 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
GCCCTTGTTTTGGACGCGATAAAGACCAGTATCGGAAGCGAAGTTTCT48 
AlaLeuValLeuAspAlaIleLysThrSerIleGlySerGluValSer 
151015 
AAACTTGAGATACTGAACTGCACGCAGGATATTAAGGCAAGGCTCATA96 
LysLeuGluIleLeuAsnCysThrGlnAspIleLysAlaArgLeuIle 
202530 
GTACCGAAACCGCTATCAAAGCACGTGCTCAAGAAGCTTCAAGCATGC144 
ValProLysProLeuSerLysHisValLeuLysLysLeuGlnAlaCys 
354045 
GAGCATCCCGTCGGGGACTTGTGTATGCTGTATCCGAAGTTGCCGATC192 
GluHisProValGlyAspLeuCysMetLeuTyrProLysLeuProIle 
505560 
AAGAAAAACTCTGCGACAGAAATTGAAGAAGAGAACCTTTGCGACAGC240 
LysLysAsnSerAlaThrGluIleGluGluGluAsnLeuCysAspSer 
65707580 
CTCCTCAAGCGTCAGGAAGCTGCCTACAAGGGTCAGAACAAATGCGTC288 
LeuLeuLysArgGlnGluAlaAlaTyrLysGlyGlnAsnLysCysVal 
859095 
AAGGTCGGTAACATTTTCTGGTTCCAGTGCGCTGATGGTTACAGATCA336 
LysValGlyAsnIlePheTrpPheGlnCysAlaAspGlyTyrArgSer 
100105110 
GTTTACGACATCACACAAGGTCGCCTACGCCGCTCCGTGTGCGAACGT384 
ValTyrAspIleThrGlnGlyArgLeuArgArgSerValCysGluArg 
115120125 
GGAATTTCTTGCAGTGATAATGAACAGTTGGAGTGTGCCAAGAAAGGA432 
GlyIleSerCysSerAspAsnGluGlnLeuGluCysAlaLysLysGly 
130135140 
CAAATATGT441 
GlnIleCys 
145 
(2) INFORMATION FOR SEQ ID NO:69: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 147 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
AlaLeuValLeuAspAlaIleLysThrSerIleGlySerGluValSer 
151015 
LysLeuGluIleLeuAsnCysThrGlnAspIleLysAlaArgLeuIle 
202530 
ValProLysProLeuSerLysHisValLeuLysLysLeuGlnAlaCys 
354045 
GluHisProValGlyAspLeuCysMetLeuTyrProLysLeuProIle 
505560 
LysLysAsnSerAlaThrGluIleGluGluGluAsnLeuCysAspSer 
65707580 
LeuLeuLysArgGlnGluAlaAlaTyrLysGlyGlnAsnLysCysVal 
859095 
LysValGlyAsnIlePheTrpPheGlnCysAlaAspGlyTyrArgSer 
100105110 
ValTyrAspIleThrGlnGlyArgLeuArgArgSerValCysGluArg 
115120125 
GlyIleSerCysSerAspAsnGluGlnLeuGluCysAlaLysLysGly 
130135140 
GlnIleCys 
145 
(2) INFORMATION FOR SEQ ID NO:70: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F9 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
LysAlaAsnArgGlnCysProProAspThrArgArgGlyLys 
1510 
(2) INFORMATION FOR SEQ ID NO:71: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: F10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
LysCysAsnCysAspCysProProAspThrArgProGlyLys 
1510 
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