Mareks' disease virus vaccine

The invention is concerned with the MD18 and MD20 polypeptides of Marek's Disease virus which can be used to vaccinate poultry against MD. The invention also relates to nucleic acid sequences encoding the MD18 or MD20 polypeptides. Said sequences can be used for the preparation of a subunit or vector vaccine.

BACKGROUND OF THE INVENTION 
The present invention is concerned with a nucleic acid sequence encoding a 
Marek's Disease virus polypeptide, a recombinant nucleic acid molecule 
comprising such a nucleic acid sequence, a vector virus comprising said 
nucleic acid sequence, a host cell transformed with such a nucleic acid 
sequence, a Marek's Disease virus polypeptide and antibodies reactive 
therewith, as well as a vaccine against Marek's Disease. 
Marek's Disease (MD) is a malignant, lymphoproliferative disorder of 
domestic fowl caused by a herpesvirus: Marek's Disease Virus (MDV). MD is 
ubiquitous, occurring in poultry-producing countries throughout the world. 
Chickens raised under intensive production systems will inevitably suffer 
losses from MD. MD affects chickens from about 6 weeks of age, occurring 
most frequently between ages of 12 and 24 weeks. 
Three forms of MD are recognized clinically, classical MD, acute MD and 
transient paralysis. 
Classical MD is characterized by peripheral nerve enlargement caused by 
lymphoid infiltration and demyelination, and paralysis is the dominant 
clinical sign. Mortality is variable but normally under 10-15 percent. 
In the acute form there are multiple and diffuse lymphomatous tumors in the 
visceral organs. Mortality from this form of MD is usually higher than 
from the classical form. An incidence of 10-30 percent is common in 
unvaccinated flocks and outbreaks involving up to 70% of the flock may be 
encountered. The pathological lesions in both classical and acute MD are 
essentially similar, involving the proliferation and infiltration of 
malignantly transformed T-lymphoblasts into normal tissues, peripheral 
nerves in the case of the classical form and visceral organs in the case 
of the acute form. 
Furthermore, the MDV has been shown to be responsible for encephalitis of 
young chickens characterized by sudden paralysis. 
Serological classification of MD related viruses yielded three serotypes: 
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Type I naturally occurring virulent strains of 
Marek's disease virus which are pathogenic 
and tumorigenic to chickens, and attenuated 
nonpathogenic strains derived therefrom 
Type II naturally occurring nonpathogenic strains 
of Marek's disease virus; and 
Type III herpesvirus of turkeys ("HVT"), which is 
nonpathogenic to chickens. 
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Serial passage of pathogenic strains of MDV serotype I was found to result 
in loss of pathogenicity and oncogenicity, but not of immunogenicity. 
Attenuated strains derived from HPRS-16 and CVI-988 strains have been 
applied as vaccines. SB-I and HN-I MDV strains (serotype 2) have also been 
shown to be useful in vaccination. HVT, first isolated from turkeys, is 
apathogenic in turkeys and domestic fowls, antigenically related to 
serotype 1 and 2 MD viruses and extensively used as a vaccine against MD. 
There are no methods of treatment of MD and control is based on management 
methods which isolate growing chickens from sources of infection, the use 
of genetically resistant stock, and vaccination. However, management 
procedures are normally not cost-effective and the progress has been 
disappointing with respect to the selection of poultry stock with 
increased genetically controlled resistance. Nowadays, control of MD is 
almost entirely based on vaccination. 
Current vaccines comprise chemically inactivated virus vaccines or modified 
live-virus vaccines. However, inactivated vaccines require additional 
immunizations, disadvantageously contain adjuvants, are expensive to 
produce and are laborious to administer. Further, some infectious virus 
particles may survive the inactivation process and may cause disease after 
administration to the animal. 
In general, attenuated live virus vaccines are preferred because they evoke 
an immune response often based on both humoral and cellular reactions. Up 
to now, such vaccines based on MDV serotype I strains could only be 
prepared by serial passage of virulent strains in tissue culture. However, 
because of this treatment uncontrolled mutations are introduced into the 
viral genome, resulting in a population of virus particles heterogeneous 
with regard to virulence and immunizing properties. Overattenuation during 
passage in cell culture can also be a problem with these vaccines. One 
must achieve a delicate balance between ensuring that the vaccine is not 
virulent while making certain that it is still protective. In addition it 
is well known that such traditional attenuated live virus vaccines can 
revert to virulence resulting in disease outbreaks in inoculated animals 
and the possible spread of the pathogen to other animals. The occurrence 
of very virulent field strains of MD virus against which live HVT vaccines 
provided poor protection have now been isolated and are responsible for 
excessive losses in various parts of the world. Bivalent vaccines 
consisting of serotype 2 and serotype 3 strains are reasonably effective 
against very virulent field isolates in some cases. Multivalent vaccines 
containing serotype antigens should be even more effective at eliciting 
immunity against these very virulent strains. 
Improved vaccines might be constructed based on recombinant DNA technology. 
These vaccines would only contain the necessary and relevant MDV 
immunogenic material which is capable of eliciting a protective immune 
response against the MDV pathogens, or the genetic information encoding 
said material, and would not display above mentioned disadvantages of the 
live or inactivated vaccines. 
SUMMARY OF THE INVENTION 
According to the present invention a nucleic acid sequence encoding a MDV 
polypeptide is provided which can be applied for the preparation of a 
vaccine for the immunization of poultry against MD. 
"Nucleic acid sequence" as used herein refers to a polymeric form of 
nucleotides of any length, both to ribonucleic acid sequences and to 
deoxyribonucleic acid sequences. In principle, this term refers to the 
primary structure of the molecule. Thus, this term includes double and 
single stranded DNA, as well as double and single stranded RNA, and 
modifications thereof. 
In general, the term "polypeptide" refers to a molecular chain of amino 
acids with a biological activity and does not refer to a specific length 
of the product. If required the polypeptide can be modified in vivo or in 
vitro, for example by glycosylation, amidation, carboxylation or 
phosphorylation; thus inter alia, peptides, oligopeptides and proteins are 
included. 
According to the invention a nucleic acid sequence containing a gene 
encoding the MDV polypeptide MD18 or MD20, respectively, have been 
isolated and characterized and were found to be recognized by the immune 
system of the host. The genes encoding said polypeptides were identified 
by screening bacteriophage expression libraries made in the lambda gt11 
vector, with polyvalent sera from chickens infected with a virulent MD 
virus strain. 
The gene encoding the MD18 polypeptide maps to the unique long (U.sub.L) 
region of the MDV genome and encodes a polypeptide of about 663 amino 
acids in length. The amino acid sequence of the polypeptide encoded by the 
MD18 gene is shown in SEQ ID NO: 2. 
The gene encoding the MD20 polypeptide also maps to the U.sub.L region of 
the MDV genome and encodes a polypeptide of about 1074 amino acids in 
length. The amino acid sequence of the polypeptide encoded by the MD20 
gene is shown in SEQ ID NO: 4. 
More particularly, this invention provides a nucleic acid sequence that 
encodes the MD18 polypeptide or MD20 polypeptide having an amino acid 
sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, respectively. 
Also included within the scope of the present invention are nucleic acid 
sequences encoding a functional equivalent of said MD18 or MD20 
polypeptide having corresponding immunological characteristics. 
It will be understood that for the particular MD18 or MD20 polypeptide 
embraced herein, derived from the serotype 1 GA strain, natural variations 
can exist between individual viruses or strains of MDV of Type 1. These 
variations may be demonstrated by (an) amino acid difference(s) in the 
overall sequence or by deletions, substitutions, insertions, inversions or 
additions of (an) amino acid(s) in said sequence. Amino acid substitutions 
can be expected which can be expected probably do not essentially alter 
biological and immunological activities have been described. Amino acid 
replacements between related amino acids or replacements which have 
occurred frequently in evolution are, for example Ser/Ala, Ser/Gly, 
Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence 
and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, 
suppl. 3). Based on this information Lipman and Pearson developed a method 
for rapid and sensitive protein comparison (Science 227, 1435-1441, 1985) 
and determining the similarity between homologous polypeptides. Nucleic 
acid sequences encoding such functional equivalents are included within 
the scope of this invention. Moreover, the potential exists to use 
recombinant DNA technology for the preparation of nucleic acid sequences 
encoding these various functional equivalents. 
Preferably, nucleic acid sequences according to the invention may be 
derived from available isolates of MDV of Type 1, strains such as GA, JM, 
HPRS-16, Conn A, RB-IB CVI-988 or Md 11, the GA strain being the most 
preferred strain. 
In addition nucleic acid sequences encoding the MD18 polypeptide or MD20 
polypeptide or variations thereof as mentioned above may also be derived 
from MDV strains belonging to Type 2 or Type 3, e.g. HN, HPRS-24, SB-1 or 
FC126. 
DETAILED DESCRIPTION OF THE INVENTION 
The information provided in SEQ ID NO: 1-4 allows a person skilled in the 
art to isolate and identify the nucleic acid sequences encoding the 
variant functional equivalent polypeptides mentioned above having 
corresponding immunological characteristics with the MD18 or MD20 
polypeptide disclosed herein. The generally known blotting and 
hybridization techniques can be used for that purpose (Experiments in 
Molecular Biology, ed. R. J. Slater, Clifton, U.S.A., 1986; Singer-Sam, J. 
et al., Proc. Natl. Acad. Sci. 80, 802-806, 1983; Maniatis, T. et al., in 
Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory 
Press, U.S.A., 1989). For example, restriction enzyme digested DNA 
fragments derived from a specific MDV strain is electrophoresed and 
transferred, or "blotted" thereafter onto a piece of nitrocellulose 
filter. It is now possible to identify the nucleic sequences encoding the 
functional equivalent polypeptides on the filter by hybridization to a 
defined labelled DNA or "probe" back translated from the amino acid 
sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, under specific conditions 
of salt concentration and temperature that allow hybridization of the 
probe to any homologous DNA sequences present on the filter. After washing 
the filter, hybridized material may be detected by autoradiography. From 
an agarose gel with starting DNA that was not blotted, DNA can now be 
obtained that encodes a polypeptide functionally equivalent to a 
polypeptide disclosed in SEQ ID NO: 2 or 4. 
In another way, DNA obtained from a specific MDV strain may be cloned into 
a .lambda.gt11 phage and expressed into a bacterial host. Recombinant 
phages can then be screened with polyclonal serum raised against the 
purified MD18 or MD20 polypeptide, determining the corresponding 
immunological characteristic of the variant polypeptide. The above 
mentioned procedure is outlined in detail in Example 1. The production of 
the polyclonal serum elicited against MD18 or MD20 is described below. 
As is well known in the art, the degeneracy of the genetic code permits 
substitution of bases in a codon resulting in another codon but still 
coding for the same amino acid, e.g. the codon for the amino acid glutamic 
acid is both GAT and GAA. Consequently, it is clear that for the 
expression of a polypeptide with the amino acid sequence shown in SEQ ID 
NO: 2 or SEQ ID NO: 4 use can be made of a derivate nucleic acid sequence 
(functional equivalent) with such an alternative codon composition 
different from the nucleic acid sequence shown in said SEQ ID's. 
A preferred nucleic acid sequence according to the invention is 
characterized in that said sequence contains the deoxyribonucleic acid 
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. 
Furthermore, also fragments of the nucleic acid sequences encoding the MD18 
or MD20 polypeptide or functional equivalents thereof as mentioned above 
are included in the present invention. 
The term "fragment" as used herein means a DNA or amino acid sequence 
comprising a subsequence of one of the nucleic acid sequences or 
polypeptides of the invention. Said fragment is or encodes a polypeptide 
having one or more immunoreactive and/or antigenic determinants of a MDV 
polypeptide, i.e. has one or more epitopes which are capable of eliciting 
an immune response in a chicken and/or is capable of specifically binding 
to a complementary antibody. Methods for determining usable polypeptide 
fragments are outlined below. Fragments can inter alia be produced by 
enzymatic cleavage of precursor molecules, using restriction endonucleases 
for the DNA and proteases for the polypeptides. Other methods include 
chemical synthesis of the fragments or the expression of polypeptide 
fragments by DNA fragments introduced in a suitable host cell environment. 
All modifications resulting in such functional equivalents of the MD18 or 
MD20 polypeptide are included within the scope of the present invention 
for as long as the immunological characteristics of the MD18 or MD20 
polypeptide remain unaffected in essence. 
A nucleic acid sequence according to the present invention can be ligated 
to various replication effecting DNA sequences with which it is not 
associated or linked in nature, optionally containing portions of DNA 
encoding fusion protein sequences such as .beta.-galactosidase, resulting 
in a so-called recombinant nucleic acid molecule which can be used for the 
transformation of a suitable host. Such hybrid DNA molecules are 
preferably derived from, for example plasmids, or from nucleic acid 
sequences present in bacteriophages, cosmids or viruses. Specific vectors 
which can be used to clone nucleic acid sequences according to the 
invention are known in the art and include plasmid vectors such as pBR322, 
the various pUC, pGEM and Bluescript plasmids, bacteriophages, e.g. 
.lambda.gt-Wes-.lambda. B, Charon 28 and the M13 derived phages or viral 
vectors such as SV40, adenovirus or polyoma virus (see also Rodriquez, R. 
L. and D. T. Denhardt, ed., Vectors: A survey of molecular cloning vectors 
and their uses, Butterworths, 1988; Lenstra, J. A. et al., Arch. Virol. 
110, 1-24, 1990). The methods to be used for the construction of a 
recombinant nucleic acid molecule according to the invention are known to 
those of ordinary skill in the art and are inter alia set forth in 
Maniatis, T. et al. (ibid, 1989). For example, the insertion of the 
nucleic acid sequence according to the invention into a cloning vector can 
easily be achieved by ligation with an enzyme such as T4 DNA ligase when 
both the genes and the desired cloning vehicle have been cut with the same 
restriction enzyme(s) as complementary DNA termini are thereby produced. 
Alternatively, it may be necessary to modify the restriction sites that are 
produced into blunt ends either by digesting the single-stranded DNA or by 
filling in the recessive termini with an appropriate DNA polymerase. 
Subsequently, blunt end ligation with an enzyme such as T4 DNA ligase may 
be carried out. 
If desired, any restriction site may be produced by ligating linkers onto 
the DNA termini. Such linkers may comprise specific oligonucleotide 
sequences that encode restriction site sequences. The restriction enzyme 
cleaved vector and nucleic acid sequence may also be modified by 
homopolymeric tailing. 
"Transformation", as used herein, refers to the introduction of a 
heterologous nucleic acid sequence into a host cell, irrespective of the 
method used, for example, by direct uptake or transduction. The 
heterologous nucleic acid sequence may be maintained through autonomous 
replication or alternatively may be integrated into the host genome. If 
desired, the recombinant DNA molecules are provided with appropriate 
control sequences compatible with the designated host which can regulate 
the expression of the inserted nucleic acid sequence. 
The recombinant nucleic acid molecule according to the invention preferably 
contains one or more marker activities that may be used to select for 
desired transformants, such as ampicillin and tetracycline resistance in 
pBR322, ampicillin resistance and .beta.-galactosidase activity in pUC8. 
A suitable host cell is a cell which can be transformed by a nucleic acid 
sequence encoding a polypeptide or by a vector virus or a recombinant 
nucleic acid molecule comprising such a nucleic acid sequence and which 
can if desired be used to express said polypeptide encoded by said nucleic 
acid sequence. The host cell can be of procaryotic origin, e.g. bacteria 
such as Escherichia coli, Bacillus subtilis and Pseudomonas species; or of 
eucaryotic origin such as yeast, e.g. Saccharomyces cerevisiae or higher 
eucaryotic cells such as insect, plant or mammalian cells, including HeLa 
cells and Chinese hamster ovary (CHO) cells. Insect cells include the Sf9 
or Sf21 cell line of Spodoptera frugiperda (Luckow et al., Bio-technology 
6, 47-55, 1989). Information with respect to the cloning and expression of 
the nucleic acid sequence of the present invention in eucaryotic cloning 
systems can be found in Esser, K. et al. (Plasmids of Eukaryotes, 
Springer-Verlag, 1986). 
In general, prokaryotes are preferred for cloning and manipulation of DNA 
sequences and for constructing the vectors useful in the invention. For 
example E. coli K12 is particularly useful. Other microbial strains which 
may be used include E. coli strains such as DH5.alpha., JM101 or HB101. 
For expression, nucleic acid sequences of the present invention are 
operably linked to expression control sequences. Such control sequences 
may comprise promoters, enhancers, operators, inducers, ribosome binding 
sites etc. 
When the host cells are bacteria, illustrative useful expression control 
sequences include the Trp promoter and operator (Goeddel, et al., Nucl. 
Acids Res. 8, 4057, 1980); the lac promoter and operator (Chang, et al., 
Nature 275, 615, 1978); the outer membrane protein (OMP) promoter 
(Nakamura, K. and Inouge, M., EMBO J. 1, 771-775, 1982); the bacteriophage 
.lambda. promoters and operators (Remaut, E. et al., Nucl. Acids Res. 11, 
4677-4688, 1983); the .alpha.-amylase (B. subtilis) promoter and operator, 
termination sequence and other expression enhancement and control 
sequences compatible with the selected host cell. When the host cell is 
yeast, illustrative useful expression control sequences include, e.g., 
.alpha.-mating factor. For insect cells the polyhedrin or p10 promoters of 
baculoviruses can be used (Smith, G. E. et al., Mol. Cell. Biol. 3, 
2156-65, 1983). When the host cell is of mammalian origin illustrative 
useful expression control sequences include, e.g., the SV-40 promoter 
(Berman, P. W. et al., Science 222, 524-527, 1983) or, e.g. the 
metallothionein promoter (Brinster, R. L., Nature 296, 39-42, 1982) or a 
heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA 82, 
4949-53, 1985). Alternatively, also expression control sequences present 
in MDV, in particular those regulating the expression of MD18 or MD20 may 
be applied. For maximizing gene expression, see also Roberts and Lauer 
(Methods in Enzymology 68, 473, 1979). 
The present invention also comprises a polypeptide displaying MDV 
immunological characteristics containing at least part of the MD18 or MD20 
amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, respectively, 
or derivatives thereof, essentially free from the whole virus or other 
proteins with which it is ordinarily associated. 
It will be understood that derivatives of said amino acid sequences 
displaying the same immunological properties in essence, i.e. 
immunological equivalents, are also within the scope of the present 
invention. 
Immunological equivalents of the MD18 or MD20 polypeptide disclosed herein 
are the corresponding polypeptides present in viruses of other strains of 
MD Type 1 or in viruses of strains belonging to Type 2 or 3. Said 
equivalents can be produced through the expression of the genes encoding 
said equivalents, the genes Leing identified and isolated making use of 
the information provided herein as described above. 
In addition a polypeptide comprising a fragment of the MD18 or MD20 
polypeptide or functional equivalent thereof, which can be used for 
immunization of poultry against MD is included in the present invention. 
Various methods are known for detecting such usable polypeptide fragments 
within a known amino acid sequence. 
Suitable immunochemically active polypeptide fragments of a polypeptide 
according to the invention containing (an) epitope(s) can be found by 
means of the method described in Patent Application WO 86/06487, Geysen, 
H. M. et al. (Prod. Natl. Acad. Sci. 81, 3998-4002, 1984), Geysen, H. M. 
et al. (J. Immunol. Meth. 102, 259-274, 1987) based on the so-called 
pepscan method, wherein a series of partially overlapping polypeptides 
corresponding with partial sequences of the complete polypeptide under 
consideration, are synthesized and their reactivity with antibodies is 
investigated. 
In addition, a number of regions of the polypeptide, with the stated amino 
acid sequence, can be designated epitopes on the basis of theoretical 
considerations and structural agreement with epitopes which are now known. 
The determination of these regions was based on a combination of the 
hydrophilicity criteria according to Hopp and Woods (Proc. Natl. Acad. 
Sci. 78, 3824-3828, 1981) and the secondary structure aspects according to 
Chou and Fasman (Advances in Enzymology 47, 45-148, 1987). 
T-cell epitopes which may be necessary can likewise be derived on 
theoretical grounds with the aid of Berzofsky's amphiphilicity criterion 
(Science 235, 1059-62, 1987). 
Such epitopes can also be generated experimentally by limited exposure of 
the polypeptide to the proteolytic activity of Cathepsin D. Cleavage of a 
protein antigen by this enzyme specifically recognizes amino acid sequence 
patterns which are also found within the NH.sub.2 -terminal residues of 
peptides recognized by the major histocompability complex on the surface 
of antigen-presenting cells (v. Noort, J. M. and v.d. Drift, A. C. M., J. 
Biol. Chem. 264, 14159, 1989). 
In another embodiment of the invention a polypeptide having an amino acid 
sequence encoded by a nucleic acid sequence mentioned above is used. 
Immunization of poultry against MDV infection can, for example be achieved 
by administering to the animals a polypeptide according to the invention 
in an immunologically relevant context as a so-called subunit vaccine. The 
subunit vaccine according to the invention may comprise a polypeptide in a 
pure form, optionally in the presence of a pharmaceutically acceptable 
carrier. The polypeptide can optionally be covalently bonded to a 
non-related protein, which, for example can be of advantage in the 
purification of the fusion product. Examples are .beta.-galactosidase, 
protein A, prochymosine, blood clotting factor Xa, etc. 
In some cases the ability to raise neutralizing antibodies against these 
polypeptides per se may be low. Small fragments are preferably conjugated 
to carrier molecules in order to increase their immunogenicity. Suitable 
carriers for this purpose are macromolecules, such as natural polymers 
(proteins like keyhole limpet hemocyanin, albumin, toxins), synthetic 
polymers like polyamino acids (polylysine, polyalanine), or micelles of 
amphiphilic compounds like saponins. Alternatively these fragments may be 
provided as polymers thereof, preferably linear polymers. 
Polypeptides to be used in such subunit vaccines can be prepared by methods 
known in the art, e.g. by isolating said polypeptides from MDV, by 
recombinant DNA techniques or by chemical synthesis. 
If required the polypeptides according to the invention to be used in a 
vaccine can be modified in vitro or in vivo, for example by glycosylation, 
amidation, carboxylation or phosphorylation. 
An alternative to subunit vaccines are live vector vaccines. A nucleic acid 
sequence according to the invention is introduced by recombinant DNA 
techniques into a microorganism (e.g. a bacterium or virus) in such a way 
that the recombinant microorganism is still able to replicate thereby 
expressing a polypeptide coded by the inserted nucleic acid sequence. 
For example the technique of in vivo homologous recombination can be used 
to introduce a heterologous nucleic acid sequence, e.g. a nucleic acid 
sequence according to the invention into the genome of the vector 
microorganism. 
First, a DNA fragment corresponding with an insertion region of the vector 
genome, i.e. a region which can be used for the incorporation of a 
heterologous sequence without disrupting essential functions of the vector 
such as those necessary for infection or replication, is inserted into a 
cloning vector according to standard recDNA techniques. Insertion regions 
have been reported for a large number of microorganisms (e.g. EP 80,806, 
Ep 110,385, EP 83,286, EP 3-14,569, WO 88/02022 and WO 88/07088). 
Second, if desired, a deletion can be introduced into the insertion region 
present in the recombinant DNA molecule obtained from the first step. This 
can be achieved for example by appropriate exonuclease III digestion or 
restriction enzyme treatment of the recombinant DNA molecule from the 
first step. 
Third, the heterologous nucleic acid sequence is inserted into the 
insertion region present in the recombinant DNA molecule of the first step 
or in place of the DNA deleted from said recombinant DNA molecule. The 
insertion region DNA sequence should be of appropriate length as to allow 
homologous recombination with the vector genome to occur. Thereafter, 
suitable cells can be transformed with vector genomic DNA in the presence 
of the recombinant DNA molecule containing the insertion flanked by 
appropriate vector DNA sequences whereby recombination occurs between the 
corresponding regions in the recombinant DNA molecule and the vector 
genome. Recombinant vector progeny can now be produced in cell culture and 
can be selected for example genotypically or phenotypically, e.g. by 
hybridization, detecting enzyme activity encoded by a gene co-integrated 
along with the heterologous nucleic acid sequence, or detecting the 
antigenic heterologous polypeptide expressed by the recombinant vector 
immunologically. 
Next, this recombinant microorganism can be administered to the host animal 
for immunization whereafter it is maintained and optionally replicates in 
the body of the inoculated animal, expressing in vivo a polypeptide coded 
for by the nucleic acid sequence according to the invention inserted in a 
vector organism and resulting in the stimulation of the immune system of 
the inoculated animal. 
Suitable vectors for the incorporation of a nucleic acid sequence according 
to the invention can be derived from viruses such as (avian) pox viruses, 
e.g. vaccinia virus or fowl pox virus (EP 314,569 and WO 88/02022), herpes 
viruses such as HVT (WO 88/07088), adeno virus or influenza virus, or 
bacteria such as E. coli or specific Salmonella species. With recombinant 
microorganisms of this type, the polypeptide synthesized in the host cell 
can be exposed as a surface antigen. In this context fusion of the said 
polypeptide with OMP proteins, or pilus proteins of for example E. coli or 
with synthetic provision of signal and anchor sequences which are 
recognized by the organism are conceivable. It is also possible that the 
said immunogenic polypeptide, if desired as part of a larger whole, is 
released inside the animal to be immunized. In all of these cases it is 
also possible that one or more immunogenic products will be expressed, 
generating protection against various pathogens and/or against various 
antigens of a given pathogen. 
A vaccine according to the invention can be prepared by culturing a host 
cell infected with a vector virus comprising a nucleic acid sequence 
according to the invention, whereafter virus containing cells and/or 
vector viruses grown in the cells can be collected, optionally in a pure 
form, and formed to a vaccine optionally in a lyophilized form. 
Above mentioned host cells comprising a nucleic acid sequence according to 
the invention can also be cultured under conditions which are favourable 
for the expression of a polypeptide coded by said nucleic acid sequence. 
Vaccines may be prepared using samples of the crude culture, host cell 
lysates or host cell extracts, although in another embodiment more 
purified polypeptides according to the invention are formed to a vaccine, 
depending on its intended use. In order to purify the polypeptides 
produced, host cells containing a nucleic acid sequence according to the 
invention are cultured in an adequate volume and the polypeptides produced 
are isolated from such cells or from the medium if the protein is 
secreted. Polypeptides secreted into the medium can be isolated and 
purified by standard techniques, e.g. salt fractionation, centrifugation, 
ultrafiltration, chromatography, gel filtration or immuno affinity 
chromatography, whereas intracellular polypeptides can be isolated by 
first collecting said cells, disrupting the cells, for example by 
sonication or by other mechanically disruptive means such as French press 
followed by separation of the polypeptides from the other intracellular 
components and forming the polypeptides to a vaccine. Cell disruption 
could also be accomplished by chemical (e.g. EDTA treatment) or enzymatic 
means such as lysozyme digestion. 
Antibodies or antiserum directed against a polypeptide according to the 
invention have potential uses in passive immunotherapy, diagnostic 
immunoassays and generation of anti-idiotype antibodies. 
The MDV polypeptides MD18 or MD20 as described above can be used to produce 
antibodies, both polyclonal, monospecific and monoclonal. If polyclonal 
antibodies are desired, techniques for producing and processing polyclonal 
sera are known in the art (e.g. Mayer and Walter, eds, Immunochemical 
Methods in Cell and Molecular Biology, Academic Press, London, 1987). In 
short, a selected mammal, e.g. rabbit is given (multiple) injections with 
one of the above mentioned immunogens, about 20 .mu.g to about 80 .mu.g of 
protein per immunization. Immunizations are given with an acceptable 
adjuvant, generally equal volumes of immunogen and adjuvant. Acceptable 
adjuvants include Freund's complete, Freund's incomplete, alum-precipitate 
or water-in-oil emulsions, with Freund's complete adjuvant being preferred 
for the initial immunization. Freund's incomplete adjuvant is preferred 
for all booster immunizations. The initial immunization consists of the 
administration of about 1 ml of emulsion at multiple subcutaneous sites on 
the backs of the rabbits. Booster immunizations utilizing an equal volume 
of immunogen are given at about one month intervals and are continued 
until adequate levels of antibodies are present in an individual rabbits 
serum. Blood is collected and serum isolated by methods known in the art. 
Monospecific antibodies to each of the immunogens are prepared by 
immunizing rabbits as described above with the purified proteins and 
thereafter affinity purified from polyspecific antisera by a modification 
of the method of Hall et al. (Nature 311, 379-387 1984). Monospecific 
antibody as used herein is defined as a single antibody species or 
multiple antibody species with homogeneous binding characteristics for the 
relevant antigen. Homogeneous binding as used herein refers to the ability 
of the antibody species to bind to a specific antigen or epitope. 
Monoclonal antibodies reactive against each of the MDV immunogens can be 
prepared by immunizing inbred mice, preferably Balb/c with the appropriate 
protein. The mice are immunized intraperitoneally with about 100 ng to 
about 10 .mu.g immunogen per 0.5 ml in an equal volume of a suitable 
adjuvant. Such acceptable adjuvants include Freund's complete, Freund's 
incomplete, alum-precipitate and water-in-oil emulsions. The mice are 
given intravenous booster immunizations of an equal amount of the 
immunogen without adjuvant at about days 14, 21 and 63 post primary 
immunization. At about day three after the final booster immunization, 
individual mice are serologically tested for anti-immunogen antibodies. 
Spleen cells from antibody producing mice are isolated and fused with 
murine myeloma cells, such as SP-2/0 or the like, by techniques known in 
the art (Kohler and Milstein, Nature 256; 495-497, 1975). Hybridoma cells 
are selected by growth in appropriate cell culture medium such as 
Dulbecco's modified Eagle's medium (DMEM) containing hypoxanthine, 
thymidine and aminopterin in an antibody producing hybridomas are cloned, 
preferably using the soft agar technique of MacPherson (Soft Agar 
Techniques, Tissue Culture Methods and Applications, Kruse and Paterson, 
eds., Academic Press, 276, 1973), Discrete colonies are transferred into 
individual wells of culture plates for cultivation in an appropriate 
culture medium. Antibody producing cells are identified by screening with 
the appropriate immunogen. Immunogen positive hybridoma cells are 
maintained by techniques known in the art. Specific monoclonal antibodies 
are produced by cultivating the hybridomas in vitro or preparing ascites 
fluid in mice following hybridoma injection by procedures known in the 
art. 
Anti-idiotype antibodies are immunoglobulins which carry an "internal 
image" of the antigen of the pathogen against which protection is desired 
and can be used as an immunogen in a vaccine (Dreesman et al., J. Infect. 
Disease 151, 761, 1985). Techniques for raising anti-idiotype antibodies 
are known in the art (MacNamara et al., Science 226, 1325, 1984). 
The vaccine according to the invention can be administered in a 
conventional active immunization scheme: single or repeated administration 
in a manner compatible with the dosage formulation and in such amount as 
will be prophylactically and/or therapeutically effective and immunogenic. 
The administration of the vaccine can be done, e.g. intradermally, 
subcutaneously, intramusculary, intravenously or intranasally. 
Additionally, the vaccine may also contain an aqueous medium or a 
water-containing suspension, often mixed with other constituents, e.g. in 
order to increase the activity and/or shelf life. These constituents may 
be salts, pH buffers, stabilizers (such as skimmed milk or casein 
hydrolysate), emulsifiers, adjuvants to improve the immune response (e.g. 
oils, muramyl dipeptide, aluminium-hydroxide, tocol derivatives, saponin, 
polyanions and amphipatic substances) and preservatives. 
It is clear that a vaccine according to the invention may also contain 
immunogens related to other pathogens of poultry or may contain nucleic 
acid sequences encoding these immunogens, like antigens of Infectious 
Bronchitis Virus, Newcastle Disease virus Infectious Bursal Disease virus 
of Marek's Disease Virus different from those disclosed herein, to produce 
a multivalent vaccine. 
The invention also relates to an "immunochemical reagent", which reagent 
comprises at least one of the polypeptides according to the invention or 
an antigenic fragment thereof. 
The term "immunochemical reagent" signifies that the polypeptides according 
to the invention have been bound to a suitable support or have been 
provided with a labelling substance. 
The supports which can be used are, for example, the inner wall of a 
microtest well or a cuvette, a tube or capillary, a membrane, filter, test 
strip or the surface of a particle such as, for example, a latex particle, 
an erythrocyte, a dye sol, a metal sol or metal compound as sol particle. 
Labelling substances which can be used are, inter alia, a radioactive 
isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal 
compound as sol particle. 
A nucleic acid sequence according to the invention can also be used to 
design specific probes for hybridization experiments for the detection of 
MDV related nucleic acids in any kind of tissue. 
The present invention also provides a test kit comprising said nucleic acid 
sequence useful for the diagnosis of MDV infection. 
The invention also relates to a test kit to be used in an immunoassay, this 
test kit containing at least one immunochemical reagent according to the 
invention. 
The immunochemical reaction which takes place using this test kit is 
preferably a sandwich reaction, an agglutination reaction, a competition 
reaction or an inhibition reaction. 
For carrying out a sandwich reaction, the test kit can consist, for 
example, of a polypeptide according to the invention bonded to a solid 
support, for example the inner wall of a microtest well, and either a 
labelled polypeptide according to the invention or a labelled 
anti-antibody.

EXAMPLE 1 
Screening of Bacterial Expression Libraries with Convalescent Chicken Serum 
The vector used for establishment of the library has been .lambda.gt11 
(Young, R. A. and Davis, R. W. Proc. Natl. Acad. Sci. 80, 1194-1198, 
1983). The genome of this expression vector contains a functional LacZ 
gene encoding the enzyme .beta.-galactosidase with a unique EcoRI 
restriction site near the carboxy terminus. Insertion of DNA fragments 
from the MDV genome at this site, potentially results in the expression of 
a protein consisting of a MDV specific polypeptide fused to a major part 
of the .beta.-galactosidase. Libraries representing a complex range of DNA 
fragments can be screened at high density for recombinant clones producing 
a fusion protein which is recognized by an antibody probe such as the 
serum from MDV infected birds. The library for these experiments was made 
using the DNA of a pool of BamHI plasmid clones which represented about 
90% of the viral genome of MDV strain GA (Fukuchi et al., J. Virol. 51, 
102, 1984). DNA from these plasmids was digested with BamHI, fragmented by 
sonication and size-selected by centrifugation on sucrose gradients. 
Fragments with a size between 0.5 and 4.0 kb were isolated, tailed with 
dG-residues and inserted by means of a synthetic adaptor into the unique 
EcoRI-site of the .lambda.gt10 vector (Le Bouc et al., FEBS lett. 196, 
108, 1986; Huynh et al., in: Cloning Techniques, A Practical Approach, ed. 
Glover, D., 49-78, 1985) resulting in a library with an effective size of 
5.times.10.sup.4 pfu. Phages were amplified and purified on 
CsCl-gradients, DNA was extracted and inserts were recovered by 
restriction with EcoRI. Finally, these inserts were ligated into the 
EcoRI-site of the vector and recombinant phages were screened with chicken 
serum against MDV. This convalescent serum was obtained by infecting 
single-comb white leghorns (SPAFAS) with a virulent passage of the GA 
strain from MDV obtained from Dr. Calnek (Cornell University, NY, USA). 
Serum was collected over a 12 week period and samples were tested 
individually by indirect fluorescence for binding to MDV plaques in tissue 
cultures of chicken embryo fibroblasts (CEF). 
Six of the sera were selected based on titer and specificity. A mixture of 
these samples was used in a 1:100 dilution to screen the .lambda.gt11 
library on nitro-cellulose filters according to Young, R. A. et al. (Proc. 
Natl. Acad. Sci. U.S.A. 82, 2583, 1985). The second antibody for 
incubation of the filters was an alkaline phosphatase conjugated 
rabbit-anti-chicken serum (Sigma, St. Louis, USA) and positive signals 
were developed by the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl 
phosphate colour reaction (McGadey, Histochemie 23, 180, 1970). Series of 
candidates were selected and recombinants were plaque-purified until 
homogeneity. From four of these, the DNA insert in the .lambda.gt11 
recombinant was recovered as a EcoRI fragment and transferred to the 
plasmid vector pUC8 (Vieira, J. and Messing, J., Gene 19. 259, 1982) or 
pGEM3Z (Promega, Madison, USA). The resulting constructs were designated 
as pMD18, pMD20 and pMD21. 
EXAMPLE 2 
Cloning of the MD18 Gene and Structural Analysis of the Antigen MD18 
The gene encoding the complete amino acid sequence of antigen MD18 was 
isolated by screening a .lambda.EMBL3 library representing the genome of 
MDV strain GA. The DNA used for establishment of this library was prepared 
by infecting chicken embryo fibroblasts (CEF) with a tissue culture 
adapted passage of MD strain GA provided by Dr. Nonoyama, Showa Univ., St. 
Petersburg, U.S.A. Cultures were incubated until 90% cytophatic effect 
(CPE) had developed and total DNA was prepared by proteinase K digestion 
and phenol/chloroform extraction. DNA was partially digested with Sau 3A 
(Promega, Madison, USA) and the size fraction of about 20 kb was isolated 
after separation in a 0.8% agarose gel. DNA fragments were ligated with 
BamHI/EcoRI digested .lambda.EMBL3 DNA (Promega., Madison, USA), packaged 
in vitro and plated on E.coli strain LE392. Screening of this library 
(Maniatis, T. et al., 1989, ibid) with the insert from pMD18 resulted 
among others in the isolation of .lambda.GA12 containing a 21 kb DNA 
mapped by restriction analysis to a region of the MDV viral genome about 
halfway the U.sub.L structural element. Within this 21 kb of DNA, the 
position of the sequence hybridizing with pMD18 was defined to a 3.8 kb 
BamHI fragment which was subcloned as such in both orientations using the 
vector pGEM3Z and resulting in the plasmids pMD41 and pMD42 respectively. 
Nucleotide sequence analysis in both orientations of the DNA was performed 
on progressively deleted subclones generated with the enzyme exonuclease 
III as described by Henikoff, S. (Gene 28, 357, 1984). After assemblage of 
all sequence data and translation of the sequence in the region of 
interest, a primary structure was deduced for the antigen originally 
identified in the immuno-screening by means of the convalescent chicken 
serum. The complete amino acid sequence of this antigen designated MD18, 
is presented in SEQ ID NO: 2. 
EXAMPLE 3 
Cloning of the MD20 Gene and Structural Analysis of the Antigen MD20 
Analysis directly on the insert of pMD20 and pMD21 revealed a partially 
overlapping nucleotide sequence suggesting that both candidates were 
representing the same antigen. 
The gene encoding the complete amino acid sequence of this antigen was 
isolated by screening a .lambda.EMBL3 library representing the genome of 
MDV strain GA. The DNA used for establishment of this library was prepared 
by infecting chicken embryo fibroblasts (CEF) with a tissue culture 
adapted passage of MD strain GA provided by Dr. Nonoyama, Showa Univ., St. 
Petersburg, U.S.A. Cultures were incubated until 90% cytophatic effect 
(CPE) had developed and total DNA was prepared by proteinase K digestion 
and phenol/chloroform extraction. DNA was partially digested with Sau 3A 
(Promega, Madison, USA) and the size fraction of about 20 kb was isolated 
after separation in a 0.8% agarose gel. DNA fragments were ligated with 
BamHI/EcoRI digested .lambda.EMBL3 DNA (Promega., Madison, USA), packaged 
in vitro and plated on E.coli strain LE392. Screening of this library 
(Maniatis, T. et al., 1982, ibid) with the insert from pMD21 resulted in 
the isolation of clone .lambda.GA09. This clone contained a 17 kb DNA 
insert that is located near the junction of the U.sub.L and IR.sub. L in 
the MDV viral genome. Referring to the restriction map as published by 
Fukuchi et al. (J. Virol. 51, 102, 1984), the 17 kb insert included the 
region in between the S and I.sub.2 BamHI fragments. Restriction mapping 
on the DNA from .lambda.GA09 and hybridization with the insert from pMD21 
identified the position of the gene in a 7.5 kb SalI fragment which was 
subcloned as such in pGEM3Z to result in pMD26. Nucleotide sequence 
analysis was performed on subclones generated by both the exonuclease III 
treatment (Henikoff, S., Gene 28, 257, 1984) and the use of convenient 
restriction sites. The final sequence obtained after assembling all data 
from the reactions done in both orientations was translated into the 
complete amino acid sequence of the antigen denominated MD20 and is shown 
in SEQ ID NO: 4. 
EXAMPLE 4 
Insertion of the Genes Encoding MDV Antigens MD=18 and MD20 into the Viral 
Genome of Herpes Virus of Turkey (HVT) 
Based on the genome structure of HVT as published by Igarashi, T. et al. 
(Virology 157, 351, 1987) a region in the unique-short sequence element 
(Us) of the virus was selected for the insertion of foreign genes. The 
corresponding DNA fragment was screened from a .lambda.EMBL3 library 
constructed by partially digesting total DNA from HVT infected CEF 
following a procedure used previously for MDV strain GA. The insert of one 
of the .lambda.-isolates, characterized by the absence of any BamHI 
restriction site, was denominated .lambda.HVT04 and analyzed in detail by 
physical mapping (FIG. 1). The sequence present in the 17.5 kb inserted 
fragment represented a major part of the Us region including part of the 
inverted repeat structure (Igarashi, T. et al., 1987, ibid). One of the 
1.2 kb XhoI restriction fragments from .lambda.HVT04 was subcloned in 
pGEM3Z digested with Sal I resulting in plasmid pMD07 which contained a 
unique BglII site available for insertion of DNA fragments. The gene 
encoding antigen MD18 or MD20 was assembled from pMD41 and pMD 26 
respectively by removal of the excess of nucleotide sequences preceding 
the ATG-initiator and the creation of convenient restriction sites such as 
Sal I or XhoI flanking the coding region. For pMD41 this resulted in 
pMD46, and pMD26 gave pMD47, both restriction maps being presented in FIG. 
2. 
A strong promoter which could direct the expression of foreign genes after 
their insertion into the genome of the HVT virus was selected from the LTR 
sequence of Rous Sarcoma Virus (RSV). The promoter has been mapped on a 
580 bp NdeI/HindIII restriction fragment from pRSVcat (Gorman et al., 
Proc. Natl. Acad. Sci. 79, 6777, 1982) and was inserted between the 
HindIII and PstI sites of pGEM3Z (Promega) by means of double stranded 
synthetic linkers on both sides of the fragment. The connection between 
the HindIII site from the vector pGEM3Z and the NdeI site of the RSV 
fragment carrying the LTR-promoter was made with a 30 bp linker containing 
cohesive ends compatible with HindIII on one and NdeI on the other site. 
However, after ligation both restriction sites are not restored due to 
deliberate, modifications in the outer nucleotides of the six base pair 
recognition sequence. In addition to the removal of these two sites, a new 
restriction site (BamHI) present within the linker itself was created at 
the corresponding position. A second 20 bp linker was synthesized which 
connected the HindIII site from the LTR fragment to the PstI site from 
pGEM3Z, in this case without destruction of the recognition sequence on 
either of the ends and adding the three convenient unique restriction 
sites BglII, XhoI and EcoRV, to those already present in the polylinker of 
pGEM3Z, e.g. PstI, SalI, XhoI and BamHI. The resulting derivative of 
pGEM3Z, designated pVEC01, therefore contains a 650 bp restriction 
fragment carrying the LTR promoter sequence immediately followed by seven 
restriction sites available for the insertion of foreign genes. The 650 bp 
fragment is flanked on either end by a BamHI restriction site and has been 
transferred as such to the unique BglII site present in the 1.2 kb HVT 
insert from pMD07. The cohesive ends generated by these two restriction 
enzymes are compatible but ligation does not restore either of the 
original recognition sequences for BglII or BamHI. One of the resulting 
constructs, carrying the LTR in the orientation towards the TRs, was 
designated pVEC04 and checked by restriction mapping (FIG. 3). The 
structure of this universal HVT recombination vector allows the insertion 
of foreign genes immediately downstream of the LTR promoter and subsequent 
integration of the complete expression cassette into the HVT genome by in 
vivo recombination. The positions of the different restriction sites 
downstream of the LTR in particular those for the enzymes BglII, XhoI and 
EcoRV are designed in such a way that even multiple gene insertion can be 
envisaged. A 2.5 kb SalI/XhoI restriction fragment derived from pMD46 
carrying the MD18 gene, was inserted into the unique BglII site of pVEC04 
downstream of the LTR promoter, resulting in pMD48. A 3.6 kb SalI 
restriction fragment derived from pMD47 carrying the MD20 gene was 
inserted into the unique XhoI site of pVEC04 downstream of the LTR 
promoter, resulting in pMD49. 
DNA of the plasmids pMD48 or pMD49 was introduced together with total DNA 
prepared from HVT infected cells into CEF by a method based on the calcium 
phosphate DNA precipitation according to Graham, F. and v.d. Eb, A., 
(Virology 52, 456, 1973) with modifications described by Morgan et al. 
(Avian Diseases 34, 345, 1990). Two microgram of plasmid DNA from the 
constructs were mixed with 15 .mu.g of DNA from HVT infected cells in a 
final volume of 560 .mu.l H.sub.2 O and added to 750 .mu.l of HBSP (20 mM 
KCl, 560 mM NaCl, 24 mM glucose, 3 mM Na.sub.2 HPO.sub.4, 100 mM HEPES, pH 
7.0). Precipitates were formed by gradually adding 190 .mu.l of 1M 
CaCl.sub.2 solution and incubating the mixtures at room-temperature for 30 
minutes. In the meantime, 15 ml of a suspension of secondary CEF from 10 
day old embryos in medium 6/B8, for which the composition is based on 
Glasgow's modification of Eagle's Minimal Essential Medium supplemented 
with 2% of foetal calf serum, were seeded in .phi. 10 cm dishes at a 
density of 5.times.10.sup.5 cells per ml. Calcium phosphate precipitated 
DNA was gently added to the cell suspension and dishes were incubated at 
37.degree. C. in a humified incubator containing 5% CO.sub.2 in air. After 
5 hours, medium was removed and 10 ml of solution containing equal volumes 
of HBSP and 30% glycerol was layered onto the cells. After a one to two 
minute incubation, the solution was removed, cells were washed with medium 
6/B8 and dishes were incubated with fresh medium for 3 to 5 days until 
viral CPE developed. Detection of HVT recombinants expressing the MD18 or 
MD20 polypeptides was done by immunofluorescence staining using specific 
mono- or polyvalent sera against these MDV antigens. 
BRIEF DESCRIPTION OF THE DRAWINGS 
FIG. 1 
Restriction enzyme map of a DNA fragment essentially corresponding to the 
Us region of the HVT genome. The relative position of the insertion region 
consisting of four open reading frames and non-coding sequences in between 
is indicated. 
FIG. 2 
A. Restriction enzyme map of pMD46, containing the gene encoding 
MD18-antigen flanked by SalI and XhoI restriction sites. Vector plasmid 
was derived from pSP72 (Promega, Wisconsin, U.S.A.) by modification of the 
ClaI restriction site in the polylinker into SalI. 
B. Restriction enzyme map of pMD47, containing the gene encoding 
MD20-antigen flanked by SalI restriction sites. Vector plasmid was derived 
from pSP72 (Promega, Wis., U.S.A.) by modification of the ClaI restriction 
site in the polylinker into SalI. 
FIG. 3 
Restriction enzyme map of pVEC04 showing the LTR-promoter inserted into the 
unique BglII site of the 1,2 kb XhoI HVT fragment from pMD07. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2015 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
( A) ORGANISM: Marek's disease herpesvirus 
(B) STRAIN: GA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 14..2005 
(D) OTHER INFORMATION: /label=pMD18 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATTTTTATCTGAAATGAATCCGGCCGACCATCCATCGGTGTATGTAGCG49 
Met AsnProAlaAspHisProSerValTyrValAla 
1510 
GGCTATCTGGCATTATATGGGGCGGATGAAAGTGATGAATTGAATATC97 
GlyTyrLeuAlaLeuTyrGl yAlaAspGluSerAspGluLeuAsnIle 
152025 
GACCGCAAAGATATTCGCGCCGCGATTCCGACACCAGCTCCTTTACCA145 
AspArgLysAspIleArgAlaAlaIle ProThrProAlaProLeuPro 
303540 
ATAAATATAGATCACAGAAGAGATTGCACAGTCGGAGCAGTTCTTGCG193 
IleAsnIleAspHisArgArgAspCysThrValGly AlaValLeuAla 
45505560 
CTAATAGATGATGAACATGGATTATTTTTCCTGGGAAAGATAAATTGT241 
LeuIleAspAspGluHisGlyLeuPhePheLeuG lyLysIleAsnCys 
657075 
CCTGTGATGGTACGTACACTAGAGACAGCCGCCAGTCAAGAAATATTC289 
ProValMetValArgThrLeuGluThrAlaAlaSe rGlnGluIlePhe 
808590 
AGCGAACTTGATAATCTTAAACCAGATGATAAATTGCTATATATAATT337 
SerGluLeuAspAsnLeuLysProAspAspLysLeuLeu TyrIleIle 
95100105 
ACAAATTATCTTCCATCGGTATCGCTGTCCTCACGACGCCTAGCACCG385 
ThrAsnTyrLeuProSerValSerLeuSerSerArgArgLeuAla Pro 
110115120 
GGGGAAACGGCAGATGAGACTTTTTTGGCACATGTTGCTTTGTGTTTA433 
GlyGluThrAlaAspGluThrPheLeuAlaHisValAlaLeuCysLeu 
125 130135140 
TTGGGGAAGCGAATTGGAACTATTGTTACATATGATCTCACCCCGGAA481 
LeuGlyLysArgIleGlyThrIleValThrTyrAspLeuThrProGlu 
145150155 
GAGGCTATAGAGCCGTTCAGAAAGCTTTCTCCAAATTCTAAAGCGACC529 
GluAlaIleGluProPheArgLysLeuSerProAsnSerLysAlaThr 
160165170 
TTGCTATCACAGGGCAAGGAAACTGAACGGCTCTTAGGTGAGATGGTG577 
LeuLeuSerGlnGlyLysGluThrGluArgLeuLeuGlyGluMetVal 
175 180185 
TGGTATCCGAGCAAAAATGCAATAACCAAAGCGTTATTAGGAACGGCG625 
TrpTyrProSerLysAsnAlaIleThrLysAlaLeuLeuGlyThrAla 
190 195200 
GTTAATAATATGTTACTGCGAGATAGATGGCAAATTATCTCCGAACGA673 
ValAsnAsnMetLeuLeuArgAspArgTrpGlnIleIleSerGluArg 
205210 215220 
AGACGCATGGCTGGTATAACTGGACAAAAGTATTTGCAAGCATCATCT721 
ArgArgMetAlaGlyIleThrGlyGlnLysTyrLeuGlnAlaSerSer 
225 230235 
TTTACGGCATTGACCGATTCAATGACGTCAAATAACGTGTCAGTCACC769 
PheThrAlaLeuThrAspSerMetThrSerAsnAsnValSerValThr 
240 245250 
CACCCAATTTGTGAAAACGCAAACCCGGGTAACATACAAAAGGATGAG817 
HisProIleCysGluAsnAlaAsnProGlyAsnIleGlnLysAspGlu 
255260 265 
GAAATGCAAGTGTGTATCAGTCCAGCACAAACGAGTGAAACGTTAAAT865 
GluMetGlnValCysIleSerProAlaGlnThrSerGluThrLeuAsn 
270275 280 
GCTGGAGTGCTGTCTGGATGCAACGATTTCCATAGACTTCCCCACTCC913 
AlaGlyValLeuSerGlyCysAsnAspPheHisArgLeuProHisSer 
285290295 300 
GACCCTGCATCAACGAGCGATCAAACCAATTTGCAATCGCTAATAGAA961 
AspProAlaSerThrSerAspGlnThrAsnLeuGlnSerLeuIleGlu 
305310 315 
CCGTCCATGAACACTCAATCTTCTCGCCCACCCGGAGACGATTTTATT1009 
ProSerMetAsnThrGlnSerSerArgProProGlyAspAspPheIle 
320325330 
TGGGTCCCGATTAAAAGCTATAATCAGCTAGTATCGAGAAATGCTTCT1057 
TrpValProIleLysSerTyrAsnGlnLeuValSerArgAsnAlaSer 
335340345 
CAGCC AACGAATATTCCCGATATTGCAATTACATCGAATCAGCCTCCG1105 
GlnProThrAsnIleProAspIleAlaIleThrSerAsnGlnProPro 
350355360 
TTTATTCCCCCGGCG TTAATGAATACATCGATATCAGGTCAACACTCC1153 
PheIleProProAlaLeuMetAsnThrSerIleSerGlyGlnHisSer 
365370375380 
ATCCCAAGTGGA TATGCCCAATATGGGTACCCTACACCCGTAGGTACC1201 
IleProSerGlyTyrAlaGlnTyrGlyTyrProThrProValGlyThr 
385390395 
CATAACTCTCTGC TTCCATTGGGACCTGTAAATCAAATGGGCGGATTT1249 
HisAsnSerLeuLeuProLeuGlyProValAsnGlnMetGlyGlyPhe 
400405410 
CAATATGGACCTCAGGT GTACCCCTTGTCATATGGACAATCGCCTTTA1297 
GlnTyrGlyProGlnValTyrProLeuSerTyrGlyGlnSerProLeu 
415420425 
GAAGCAAAACTGACAGCCTTACTT GAATGCATGACAAAGGAAAAGAGA1345 
GluAlaLysLeuThrAlaLeuLeuGluCysMetThrLysGluLysArg 
430435440 
CCAGTGGATGAGGAGCACAGAGGCGACGATATG CATACTACTAGGGAA1393 
ProValAspGluGluHisArgGlyAspAspMetHisThrThrArgGlu 
445450455460 
GAACGAGGACGACGTGGACGTAAGCGACCAT ACGAATTTGACAGATCT1441 
GluArgGlyArgArgGlyArgLysArgProTyrGluPheAspArgSer 
465470475 
ATCGAGTCTGATCTTTATTATCCCGGTGAATT CCGTCGGTCTAATTTT1489 
IleGluSerAspLeuTyrTyrProGlyGluPheArgArgSerAsnPhe 
480485490 
TCTCCTCCTCAAGCCAGTAGTATGAAATATGAAGAA ACTACTGGGGGT1537 
SerProProGlnAlaSerSerMetLysTyrGluGluThrThrGlyGly 
495500505 
CGGCATGATCTGAGTCAAACAGGACCCGTATTAAATAGTCTA ATGGGA1585 
ArgHisAspLeuSerGlnThrGlyProValLeuAsnSerLeuMetGly 
510515520 
GCTGTGACTTCCCTACAAAAAGAAGTCGAACGGCTAAATGGAGGAAAT163 3 
AlaValThrSerLeuGlnLysGluValGluArgLeuAsnGlyGlyAsn 
525530535540 
TTACCGATATCAAATGCACAAAGTTCATATGGAGTGCCCAATGGGATG 1681 
LeuProIleSerAsnAlaGlnSerSerTyrGlyValProAsnGlyMet 
545550555 
CATGCCCCAGTTTATTACTCATACCCTCCTCCGGGAACACATCCCACA1 729 
HisAlaProValTyrTyrSerTyrProProProGlyThrHisProThr 
560565570 
GTTTCATGGCCCATGGGAGTCGAACGCCCTATGCCTTCCACGGAAGGA1777 
ValSerTrpProMetGlyValGluArgProMetProSerThrGluGly 
575580585 
AAAACTTCTACCAATTCCACGGTCATTCCTGTGCCAGTTTCAGATCCG1825 
LysThrS erThrAsnSerThrValIleProValProValSerAspPro 
590595600 
GAGGCTGGTCGAAATGTACCAATAACTGCGACCATCTCTCAGGAGCGT1873 
GluAlaGlyArgAsnVa lProIleThrAlaThrIleSerGlnGluArg 
605610615620 
TCCGACGGAATTCAGAAGGAAAGCATCGAGCAATCACGGGATACCATG1921 
SerAspGlyIleGln LysGluSerIleGluGlnSerArgAspThrMet 
625630635 
AATGCTAGCGCCGTAGCTGGTATACACCGCACCAGTGATGCCGGCGTC1969 
AsnAlaSerAlaVal AlaGlyIleHisArgThrSerAspAlaGlyVal 
640645650 
GATGTATTTATTAATCAAATGATGGCGCATCAATAATACAGGGAGC2015 
AspValPheIleAsnGlnM etMetAlaHisGln 
655660 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 663 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetAsnProAlaAspHisProSerValT yrValAlaGlyTyrLeuAla 
151015 
LeuTyrGlyAlaAspGluSerAspGluLeuAsnIleAspArgLysAsp 
2025 30 
IleArgAlaAlaIleProThrProAlaProLeuProIleAsnIleAsp 
354045 
HisArgArgAspCysThrValGlyAlaValLeuAlaLeuIleAspAsp 
505560 
GluHisGlyLeuPhePheLeuGlyLysIleAsnCysProValMetVal 
65707580 
ArgThrLeuGluThrAl aAlaSerGlnGluIlePheSerGluLeuAsp 
859095 
AsnLeuLysProAspAspLysLeuLeuTyrIleIleThrAsnTyrLeu 
100 105110 
ProSerValSerLeuSerSerArgArgLeuAlaProGlyGluThrAla 
115120125 
AspGluThrPheLeuAlaHisValAlaLeuCysLeuLeuG lyLysArg 
130135140 
IleGlyThrIleValThrTyrAspLeuThrProGluGluAlaIleGlu 
145150155160 
ProPhe ArgLysLeuSerProAsnSerLysAlaThrLeuLeuSerGln 
165170175 
GlyLysGluThrGluArgLeuLeuGlyGluMetValTrpTyrProSer 
180 185190 
LysAsnAlaIleThrLysAlaLeuLeuGlyThrAlaValAsnAsnMet 
195200205 
LeuLeuArgAspArgTrpGlnIleIleSe rGluArgArgArgMetAla 
210215220 
GlyIleThrGlyGlnLysTyrLeuGlnAlaSerSerPheThrAlaLeu 
225230235 240 
ThrAspSerMetThrSerAsnAsnValSerValThrHisProIleCys 
245250255 
GluAsnAlaAsnProGlyAsnIleGlnLysAspGluGluMetGlnVal 
260265270 
CysIleSerProAlaGlnThrSerGluThrLeuAsnAlaGlyValLeu 
275280285 
SerGlyCysAsnAspPhe HisArgLeuProHisSerAspProAlaSer 
290295300 
ThrSerAspGlnThrAsnLeuGlnSerLeuIleGluProSerMetAsn 
305310315 320 
ThrGlnSerSerArgProProGlyAspAspPheIleTrpValProIle 
325330335 
LysSerTyrAsnGlnLeuValSerArgAsnAlaSerGl nProThrAsn 
340345350 
IleProAspIleAlaIleThrSerAsnGlnProProPheIleProPro 
355360365 
AlaLeu MetAsnThrSerIleSerGlyGlnHisSerIleProSerGly 
370375380 
TyrAlaGlnTyrGlyTyrProThrProValGlyThrHisAsnSerLeu 
385390 395400 
LeuProLeuGlyProValAsnGlnMetGlyGlyPheGlnTyrGlyPro 
405410415 
GlnValTyrProLeuSerTyrGlyGl nSerProLeuGluAlaLysLeu 
420425430 
ThrAlaLeuLeuGluCysMetThrLysGluLysArgProValAspGlu 
435440 445 
GluHisArgGlyAspAspMetHisThrThrArgGluGluArgGlyArg 
450455460 
ArgGlyArgLysArgProTyrGluPheAspArgSerIleGluSerAsp 
465 470475480 
LeuTyrTyrProGlyGluPheArgArgSerAsnPheSerProProGln 
485490495 
AlaSerSerMetLys TyrGluGluThrThrGlyGlyArgHisAspLeu 
500505510 
SerGlnThrGlyProValLeuAsnSerLeuMetGlyAlaValThrSer 
515520 525 
LeuGlnLysGluValGluArgLeuAsnGlyGlyAsnLeuProIleSer 
530535540 
AsnAlaGlnSerSerTyrGlyValProAsnGlyMetHisAlaProVa l 
545550555560 
TyrTyrSerTyrProProProGlyThrHisProThrValSerTrpPro 
565570575 
Met GlyValGluArgProMetProSerThrGluGlyLysThrSerThr 
580585590 
AsnSerThrValIleProValProValSerAspProGluAlaGlyArg 
595 600605 
AsnValProIleThrAlaThrIleSerGlnGluArgSerAspGlyIle 
610615620 
GlnLysGluSerIleGluGlnSerArgAspThrMet AsnAlaSerAla 
625630635640 
ValAlaGlyIleHisArgThrSerAspAlaGlyValAspValPheIle 
645650 655 
AsnGlnMetMetAlaHisGln 
660 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3265 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Marek's disease herpesvirus 
(B) STRAIN: GA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 41..3265 
(D) OTHER INFORMATION: /label=pMD20 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAGTCTTCTGTAGTCGTATGTTCTTGAGCTGGTTTGCATTATGGCGAGATTTTCG55 
MetAlaArgPheSer 
15 
TCTATATCCGATACGCTCGAAAGTGATGACTCGGGAATTAAGGTCTTA103 
SerIleSerAspThrLe uGluSerAspAspSerGlyIleLysValLeu 
101520 
TTTGCCGTAGATGGTTGTGCCGTGTCGTTTTCCCTGGCCCTTCTTACA151 
PheAlaValAspGlyCy sAlaValSerPheSerLeuAlaLeuLeuThr 
253035 
GGTCAGATACCCTCTACTAACTCCGTTTATGTTATCGGCTATTGGGAT199 
GlyGlnIleProSerThrAs nSerValTyrValIleGlyTyrTrpAsp 
404550 
CCAAGCGACCGATTTTCAAGCATACCCTTTCTCGACGGGGATCCTAAT247 
ProSerAspArgPheSerSerIlePr oPheLeuAspGlyAspProAsn 
556065 
ACTAATGAGAGAATATCTACCACCGTTTGTAATTTAGAGGATGTTCCC295 
ThrAsnGluArgIleSerThrThrValCysAsnLe uGluAspValPro 
70758085 
AGCCCTCTAAGAGTAGAATTTTGTCTTCTGAACCAAATGGCATCAGGT343 
SerProLeuArgValGluPheCysLeuLeuAs nGlnMetAlaSerGly 
9095100 
ATGGGCGGTGCTGATTTAAAACTGAGAACACGTGCAATATTCGTATGC391 
MetGlyGlyAlaAspLeuLysLeuArgThrAr gAlaIlePheValCys 
105110115 
CGATTTACATCATGGTCCGAAATGAACGCTATCGCAAATTCAATAATT439 
ArgPheThrSerTrpSerGluMetAsnAlaIleAl aAsnSerIleIle 
120125130 
TATGGAACGCCAATTCAAGCCGGTGTTTTACAAGCAACAATATCTGAA487 
TyrGlyThrProIleGlnAlaGlyValLeuGlnAlaThrIl eSerGlu 
135140145 
ACTGAAACGTTCATGTTACATGATGAATTCAACCTTGCTCTTCACGTC535 
ThrGluThrPheMetLeuHisAspGluPheAsnLeuAlaLeuHisVal 
150155160165 
TTTCTCAATGGGTTATCTCTGAAGGGTCGTAACAAAAAAGATGTTTGT583 
PheLeuAsnGlyLeuSerLeuLysGlyArgAsnLysLysAspValC ys 
170175180 
ATGTCATTGAATCACAATTATATATCGAGCGTATCTGAGAATTTCCCA631 
MetSerLeuAsnHisAsnTyrIleSerSerValSerGluAsnPheP ro 
185190195 
AGGGGTAAACGAGGTCTGACTGGACTCTATTTACAACACGAACAAAAG679 
ArgGlyLysArgGlyLeuThrGlyLeuTyrLeuGlnHisGluGlnLys 
200205210 
GTCACAGCAGCATATCGGCGTATATATGGTGGATCTACTACAACTGCT727 
ValThrAlaAlaTyrArgArgIleTyrGlyGlySerThrThrThrAla 
215 220225 
TTTTGGTACGTGTCCAAATTCGGACCAGATGAAAAAAGTCTTGTTTTG775 
PheTrpTyrValSerLysPheGlyProAspGluLysSerLeuValLeu 
230 235240245 
GCCCTACGTTATTACCTTTTGCAGGCACAGGAAGAAGTTACTGGTATT823 
AlaLeuArgTyrTyrLeuLeuGlnAlaGlnGluGluValThrGlyIle 
250255260 
GCAACAGGCTATGATCTGCAAGCCATAAAAGATATATGCAAAACATAC871 
AlaThrGlyTyrAspLeuGlnAlaIleLysAspIleCysLysThrTyr 
265 270275 
GCAGTGTCGGTAAATCCCAATCCCACGGGATTTTTGGCTGCCGATTTA919 
AlaValSerValAsnProAsnProThrGlyPheLeuAlaAlaAspLeu 
280 285290 
ACGTCATTTAGTAGATTATCACGTTTTTGTTGTTTAAGTTACTATTCC967 
ThrSerPheSerArgLeuSerArgPheCysCysLeuSerTyrTyrSer 
295300 305 
AAAGGCTCTGTGGCCATAGCATTTCCATCATATGTGGAACGCAGGATT1015 
LysGlySerValAlaIleAlaPheProSerTyrValGluArgArgIle 
310315 320325 
ATGGCCGATATCGCAGAAGTGGATGCATTGAGAGAATATATAGAAAGA1063 
MetAlaAspIleAlaGluValAspAlaLeuArgGluTyrIleGluArg 
330 335340 
GACAGACCCAGTTTGAAGATTTCGGATTTGGAATTCGTTAAATATATA1111 
AspArgProSerLeuLysIleSerAspLeuGluPheValLysTyrIle 
345350 355 
TATTTAGCTTATTTTGAATGTTATAACCGCGAACAGTTAAAACGACAT1159 
TyrLeuAlaTyrPheGluCysTyrAsnArgGluGlnLeuLysArgHis 
360365 370 
TTGAAAGATGTGACAGTAAGTTTGCCCGATGAAGACATTTACAAGAAG1207 
LeuLysAspValThrValSerLeuProAspGluAspIleTyrLysLys 
375380385 
TCTTCACTAGGCAAGTGTGCAGTAGAAAATTTTTTTACACATGTGAGA1255 
SerSerLeuGlyLysCysAlaValGluAsnPhePheThrHisValArg 
390395400 405 
TCTAGATTGAACGTGAATGACCACATAGCCCATAATGTATTGCCCGAA1303 
SerArgLeuAsnValAsnAspHisIleAlaHisAsnValLeuProGlu 
410415 420 
CAAGTAGAAATGGGAAATAAGCTAGTCCGAAAGTTTGGACGTGCCAGA1351 
GlnValGluMetGlyAsnLysLeuValArgLysPheGlyArgAlaArg 
425430435 
ATGTATCTGTCAACTACGATGACTAACGAGTCGCACTTCACTGGAATA1399 
MetTyrLeuSerThrThrMetThrAsnGluSerHisPheThrGlyIle 
440445450 
TGTGA ATGTGCATCTGTGATTTTAAAGCGACTGGACACTCTAGAAATG1447 
CysGluCysAlaSerValIleLeuLysArgLeuAspThrLeuGluMet 
455460465 
AAATTGCAAAAGTA TGGTTGGCCGTCTGATCGTGTGGATGGTTCCAAT1495 
LysLeuGlnLysTyrGlyTrpProSerAspArgValAspGlySerAsn 
470475480485 
CTAATGGCCGA TAATCAGAACAACTCTACTTTAATACCGTATGATAAA1543 
LeuMetAlaAspAsnGlnAsnAsnSerThrLeuIleProTyrAspLys 
490495500 
TCTAGGTCTTC TGGAATGATACTCGAGTGTTCGAACACTCATTCTCGA1591 
SerArgSerSerGlyMetIleLeuGluCysSerAsnThrHisSerArg 
505510515 
GGGGGGCCGATGAT AGTTAAAAGGTTATTAGCTTTAGTATCTGCCGAT1639 
GlyGlyProMetIleValLysArgLeuLeuAlaLeuValSerAlaAsp 
520525530 
TCTCGCGCAGGGGGAATCGG CCCAGCTAACATGCTCATGGGGATTGAC1687 
SerArgAlaGlyGlyIleGlyProAlaAsnMetLeuMetGlyIleAsp 
535540545 
TCTGCAATAGATGGACCCCTTCCAGTTTA CCGTGTGGGCATGTCAAAG1735 
SerAlaIleAspGlyProLeuProValTyrArgValGlyMetSerLys 
550555560565 
GGCAGACAGGCTTTTACGGTGCTTAT GACCGAATGTTGGGAAAGGACC1783 
GlyArgGlnAlaPheThrValLeuMetThrGluCysTrpGluArgThr 
570575580 
ATTCCATCTCCGGGAAGTGCGAAAGC GCATTTGATCAAGCTTAACAAC1831 
IleProSerProGlySerAlaLysAlaHisLeuIleLysLeuAsnAsn 
585590595 
TCTTACGGTACTTCGACAGAAGACTTGAT TTCACGAGACTTATTCCTA1879 
SerTyrGlyThrSerThrGluAspLeuIleSerArgAspLeuPheLeu 
600605610 
ACTTCTGAAATCGAACAGCTTATCGGAAGCACAGT AGAATTGCCGGAG1927 
ThrSerGluIleGluGlnLeuIleGlySerThrValGluLeuProGlu 
615620625 
ATTACATGTGGCTCTGCCGATGAACAGCAATATATAAACCGCAA TGAA1975 
IleThrCysGlySerAlaAspGluGlnGlnTyrIleAsnArgAsnGlu 
630635640645 
GTCTTTAATGGGAATCTTGCGATAGGAAATATAGTTTTAGA TGTGGAT2023 
ValPheAsnGlyAsnLeuAlaIleGlyAsnIleValLeuAspValAsp 
650655660 
ATACATTTAAGAAACCCCATACCTCTTAGACTTATGCATGC AGCGATA2071 
IleHisLeuArgAsnProIleProLeuArgLeuMetHisAlaAlaIle 
665670675 
CGAGGTTTTAGAAGTGGTATACTCAGAGCTTTGGCCTTATTGCT ACCA2119 
ArgGlyPheArgSerGlyIleLeuArgAlaLeuAlaLeuLeuLeuPro 
680685690 
AAGGCAAATATCGACCATGGCTCATACCCGTGTTACTTTTATAAGAGT 2167 
LysAlaAsnIleAspHisGlySerTyrProCysTyrPheTyrLysSer 
695700705 
TCGTGCAAGAAATCTAGAGTAATGGGGGGAGCGCCTTGGATGCTCCAT2215 
SerC ysLysLysSerArgValMetGlyGlyAlaProTrpMetLeuHis 
710715720725 
GATGCAGAACTTGCCCCAGATTATTCGATGTTTGAAAATGCGGAGTTT2263 
A spAlaGluLeuAlaProAspTyrSerMetPheGluAsnAlaGluPhe 
730735740 
GATTTAGAAATGGGCATAGATGACCCTTTACTCATAGACCAAATAGAT2311 
A spLeuGluMetGlyIleAspAspProLeuLeuIleAspGlnIleAsp 
745750755 
GAATCTCTTACTAGATGGAGCTCAGAATCATCAAGGAGTGTCGATTTG2359 
GluS erLeuThrArgTrpSerSerGluSerSerArgSerValAspLeu 
760765770 
GATCCAGATAAGCCATGCGGTTGCCATGATAAAATCGGATTGAGGGTT2407 
AspProAspL ysProCysGlyCysHisAspLysIleGlyLeuArgVal 
775780785 
TGCATTCCAGTACCCTCTCCATATTTACTTGTGGGTAGCAAGACATTG2455 
CysIleProValProSerP roTyrLeuLeuValGlySerLysThrLeu 
790795800805 
GCCGGATTGTCTCGAATCATTCAACAAGCCGTCCTCTTAGAGCGCAAT2503 
AlaGlyLeuSerArgI leIleGlnGlnAlaValLeuLeuGluArgAsn 
810815820 
TTTGTAGAAACTATAGGGCCATATCTGAAAAATTATGAGATAATTGAT2551 
PheValGluThrIleG lyProTyrLeuLysAsnTyrGluIleIleAsp 
825830835 
AGTGGCGTATATGGTCATGGGCGTAGCTTACGTCTGCCGTTTTTTGGC2599 
SerGlyValTyrGlyHisG lyArgSerLeuArgLeuProPhePheGly 
840845850 
AAAATTGATGAAAACGGTATCGTGTCTAGAAGACTTGTACCGTTTTTC2647 
LysIleAspGluAsnGlyIleValS erArgArgLeuValProPhePhe 
855860865 
GTGATACCAGATGATTGTGCTGACATGGAGAAGTTTATTGTGGCCCAT2695 
ValIleProAspAspCysAlaAspMetGluLysP heIleValAlaHis 
870875880885 
TTCGAACCTAAAAACTTCCATTTTCACAGCTCTATCCCGCTAGAAAAG2743 
PheGluProLysAsnPheHisPheHisSerS erIleProLeuGluLys 
890895900 
GCCGCCATAATTCTGAAAGATATAGGTGGCGAATATGCAGGTTTCTTC2791 
AlaAlaIleIleLeuLysAspIleGlyGlyG luTyrAlaGlyPhePhe 
905910915 
GAAAGAAAAATTACAGTAAATAGAGATATATTTTTCGGGACTCGATTA2839 
GluArgLysIleThrValAsnArgAspIlePheP heGlyThrArgLeu 
920925930 
TCTTTATCAATAGCTCTCAGGGAAAGGGGGGTAGATATAAATGATTGT2887 
SerLeuSerIleAlaLeuArgGluArgGlyValAspIleA snAspCys 
935940945 
GCTGCCATTACAACATTTGTAACAGATCACATTTTAGATGATATTATA2935 
AlaAlaIleThrThrPheValThrAspHisIleLeuAspAspIleIle 
950955960965 
ACATACGTATATGAGCATATACCAGATCACGCAATCGAATATCAAAAT2983 
ThrTyrValTyrGluHisIleProAspHisAlaIleGluTyrGlnA sn 
970975980 
CTTTCTGTCTCGTGTTGTGTTGTCAAATCGGATTGGATCCTGCTGCAG3031 
LeuSerValSerCysCysValValLysSerAspTrpIleLeuLeuG ln 
985990995 
CTAATCCCCAATAAAACAATAGGATATCGTCACGGGTTTACATGTGTG3079 
LeuIleProAsnLysThrIleGlyTyrArgHisGlyPheThrCysVal 
100010051010 
AGATTTAAGCATGCAAGAGCAAGGCGAGCGAGTGCACGTTCTTATTTG3127 
ArgPheLysHisAlaArgAlaArgArgAlaSerAlaArgSerTyrLeu 
1015 10201025 
GCTCTGAACGTCGATGCGCATGGTAGGTTGTGCGTATGTGTAATTCAA3175 
AlaLeuAsnValAspAlaHisGlyArgLeuCysValCysValIleGln 
10301 03510401045 
CAGTGTTTTGCGGCCAAGTGCGGAAATAATAAACTTCGCACACTTTTC3223 
GlnCysPheAlaAlaLysCysGlyAsnAsnLysLeuArgThrLeuPhe 
1 05010551060 
ACGGTAGATATTGACTCGAAATGTCGATTAGAACATCAATAG3265 
ThrValAspIleAspSerLysCysArgLeuGluHisGln 
1065 1070 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1074 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetAlaArgPheSerSerIleSerAspThrLeuGluSerAspAspSer 
1 51015 
GlyIleLysValLeuPheAlaValAspGlyCysAlaValSerPheSer 
202530 
LeuAlaLeuLeuThrGlyGlnIle ProSerThrAsnSerValTyrVal 
354045 
IleGlyTyrTrpAspProSerAspArgPheSerSerIleProPheLeu 
505560 
AspGlyAspProAsnThrAsnGluArgIleSerThrThrValCysAsn 
65707580 
LeuGluAspValProSerProLeuArgValGluPheCysLeuLeuAsn 
859095 
GlnMetAlaSerGlyMetGlyGlyAlaAspLeuLysLeuArgThrArg 
100105110 
AlaIlePheVal CysArgPheThrSerTrpSerGluMetAsnAlaIle 
115120125 
AlaAsnSerIleIleTyrGlyThrProIleGlnAlaGlyValLeuGln 
130135 140 
AlaThrIleSerGluThrGluThrPheMetLeuHisAspGluPheAsn 
145150155160 
LeuAlaLeuHisValPheLeuAsnGlyLeuSerLeuLys GlyArgAsn 
165170175 
LysLysAspValCysMetSerLeuAsnHisAsnTyrIleSerSerVal 
180185190 
S erGluAsnPheProArgGlyLysArgGlyLeuThrGlyLeuTyrLeu 
195200205 
GlnHisGluGlnLysValThrAlaAlaTyrArgArgIleTyrGlyGly 
210 215220 
SerThrThrThrAlaPheTrpTyrValSerLysPheGlyProAspGlu 
225230235240 
LysSerLeuValLeuAlaLeuArgTyr TyrLeuLeuGlnAlaGlnGlu 
245250255 
GluValThrGlyIleAlaThrGlyTyrAspLeuGlnAlaIleLysAsp 
260265 270 
IleCysLysThrTyrAlaValSerValAsnProAsnProThrGlyPhe 
275280285 
LeuAlaAlaAspLeuThrSerPheSerArgLeuSerArgPheCysCys 
290295300 
LeuSerTyrTyrSerLysGlySerValAlaIleAlaPheProSerTyr 
305310315320 
ValGluArgArgIleM etAlaAspIleAlaGluValAspAlaLeuArg 
325330335 
GluTyrIleGluArgAspArgProSerLeuLysIleSerAspLeuGlu 
340 345350 
PheValLysTyrIleTyrLeuAlaTyrPheGluCysTyrAsnArgGlu 
355360365 
GlnLeuLysArgHisLeuLysAspValThrValSerLeu ProAspGlu 
370375380 
AspIleTyrLysLysSerSerLeuGlyLysCysAlaValGluAsnPhe 
385390395400 
PheT hrHisValArgSerArgLeuAsnValAsnAspHisIleAlaHis 
405410415 
AsnValLeuProGluGlnValGluMetGlyAsnLysLeuValArgLys 
4 20425430 
PheGlyArgAlaArgMetTyrLeuSerThrThrMetThrAsnGluSer 
435440445 
HisPheThrGlyIleCysGluCysAla SerValIleLeuLysArgLeu 
450455460 
AspThrLeuGluMetLysLeuGlnLysTyrGlyTrpProSerAspArg 
465470475 480 
ValAspGlySerAsnLeuMetAlaAspAsnGlnAsnAsnSerThrLeu 
485490495 
IleProTyrAspLysSerArgSerSerGlyMetIleLeuGluCysSer 
500505510 
AsnThrHisSerArgGlyGlyProMetIleValLysArgLeuLeuAla 
515520525 
LeuValSerAlaAspS erArgAlaGlyGlyIleGlyProAlaAsnMet 
530535540 
LeuMetGlyIleAspSerAlaIleAspGlyProLeuProValTyrArg 
5455505 55560 
ValGlyMetSerLysGlyArgGlnAlaPheThrValLeuMetThrGlu 
565570575 
CysTrpGluArgThrIleProSerProGlySerAla LysAlaHisLeu 
580585590 
IleLysLeuAsnAsnSerTyrGlyThrSerThrGluAspLeuIleSer 
595600605 
ArgAs pLeuPheLeuThrSerGluIleGluGlnLeuIleGlySerThr 
610615620 
ValGluLeuProGluIleThrCysGlySerAlaAspGluGlnGlnTyr 
625630 635640 
IleAsnArgAsnGluValPheAsnGlyAsnLeuAlaIleGlyAsnIle 
645650655 
ValLeuAspValAspIleHisLeuA rgAsnProIleProLeuArgLeu 
660665670 
MetHisAlaAlaIleArgGlyPheArgSerGlyIleLeuArgAlaLeu 
675680 685 
AlaLeuLeuLeuProLysAlaAsnIleAspHisGlySerTyrProCys 
690695700 
TyrPheTyrLysSerSerCysLysLysSerArgValMetGlyGlyAla 
705 710715720 
ProTrpMetLeuHisAspAlaGluLeuAlaProAspTyrSerMetPhe 
725730735 
GluAsnAlaGluPh eAspLeuGluMetGlyIleAspAspProLeuLeu 
740745750 
IleAspGlnIleAspGluSerLeuThrArgTrpSerSerGluSerSer 
75576 0765 
ArgSerValAspLeuAspProAspLysProCysGlyCysHisAspLys 
770775780 
IleGlyLeuArgValCysIleProValProSerProTyrLeuLeuV al 
785790795800 
GlySerLysThrLeuAlaGlyLeuSerArgIleIleGlnGlnAlaVal 
805810815 
Leu LeuGluArgAsnPheValGluThrIleGlyProTyrLeuLysAsn 
820825830 
TyrGluIleIleAspSerGlyValTyrGlyHisGlyArgSerLeuArg 
835 840845 
LeuProPhePheGlyLysIleAspGluAsnGlyIleValSerArgArg 
850855860 
LeuValProPhePheValIleProAspAspCysAl aAspMetGluLys 
865870875880 
PheIleValAlaHisPheGluProLysAsnPheHisPheHisSerSer 
885890 895 
IleProLeuGluLysAlaAlaIleIleLeuLysAspIleGlyGlyGlu 
900905910 
TyrAlaGlyPhePheGluArgLysIleThrValAsnArgAspIlePhe 
915920925 
PheGlyThrArgLeuSerLeuSerIleAlaLeuArgGluArgGlyVal 
930935940 
AspIleAsnAspCysAlaAlaIle ThrThrPheValThrAspHisIle 
945950955960 
LeuAspAspIleIleThrTyrValTyrGluHisIleProAspHisAla 
965 970975 
IleGluTyrGlnAsnLeuSerValSerCysCysValValLysSerAsp 
980985990 
TrpIleLeuLeuGlnLeuIleProAsnLysThrIleGl yTyrArgHis 
99510001005 
GlyPheThrCysValArgPheLysHisAlaArgAlaArgArgAlaSer 
101010151020 
AlaArgSerTyr LeuAlaLeuAsnValAspAlaHisGlyArgLeuCys 
1025103010351040 
ValCysValIleGlnGlnCysPheAlaAlaLysCysGlyAsnAsnLys 
1045 10501055 
LeuArgThrLeuPheThrValAspIleAspSerLysCysArgLeuGlu 
106010651070 
HisGln 
1074