Recombinant human thymopoietin proteins and uses therefor

The present invention provides novel nucleotide and amino acid sequences for human thymopoietin .alpha., .beta., and .gamma., methods of recombinantly expressing same, and diagnostic and therapeutic uses thereof.

FIELD OF THE INVENTION 
The present invention relates generally to human thymopoietin proteins and 
their use in diagnosis and therapy of various immune and nervous system 
conditions. 
BACKGROUND OF THE INVENTION 
Thymopoietin is a polypeptide produced by cells of the thymus and other 
cells, which has been implicated in various immune and nervous system 
pathways. There have been several attempts to isolate and sequence various 
species of thymopoietin. Thymopoietin was originally isolated as a 5 kDa, 
49 amino acid protein from bovine thymus [Goldstein et al, Nature, 
247:11-14 (1974). See also, Schlesinger and Goldstein, Cell, 5:361-365 
(1975).] Later work described by T. Audhya et al, Biochemistry, 
20(21):6195-6200 (1981) purported to provide the complete sequences for 
bovine thymopoietins. Three 49 amino acid sequences were described 
therein. Zevin-Sonkin et al, Immunol. Lett., 31:301-310 (1992) report the 
isolation of a bovine cDNA using oligonucleotide probes based on the 
original 49 amino acid bovine TP protein sequence [Schlesinger and 
Goldstein, cited above], which encodes the originally determined sequence 
at the N-terminus of a larger open reading frame. 
The active site of thymopoietin, a pentapeptide of the sequence 
Arg-Lys-Asp-Val-Tyr [SEQ ID NO:7], was described by G. Goldstein et al, 
Science, 204:1309-1310 (1979) and in U.S. Pat. No. 4,190,646. There is a 
wealth of art describing analogs of the active site, termed thymopentin 
and their uses. 
Attempts to isolate and sequence thymopoietin continue. For example, 
European Patent Application 502,607 describes bovine thymopoietin or 
thymopoietin-like cDNA clones. 
Despite these publications and the knowledge of thymopoietin, to date, the 
cloning of the complete human thymopoietin gene and its recombinant 
expression has not been described. There remains a need in the art for a 
convenient method of producing human thymopoietin, fragments thereof, and 
polynucleotide sequences encoding the protein. 
SUMMARY OF THE INVENTION 
In one aspect, the invention provides three novel polynucleotide sequences 
encoding human thymopoietin proteins referred to as .alpha., .beta. and 
.gamma., isolated from other cellular materials with which they are 
naturally associated, and having a biological activity associated with 
immune function. These polynucleotide sequences are illustrated in FIG. 1 
[SEQ ID NO:1], FIG. 2 [SEQ ID NO:3] and FIG. 3 [SEQ ID NO:5]. Fragments of 
these sequences are also embodied by this invention. These sequences or 
fragments thereof may also be optionally associated with conventionally 
used labels for diagnostic or research use. 
In another aspect, the invention provides an expression vector which 
contains at least a polynucleotide sequence described above. In still 
another aspect, a host cell transformed with such an expression vector is 
provided. 
In still another aspect, the present invention provides a method for 
producing a recombinant human thymopoietin protein which involves 
transforming a host cell with an expression vector containing a 
recombinant polynucleotide encoding a human thymopoietin protein by 
incubating the host cell and expression vector, and following 
transformation, culturing the transformed host cell under conditions that 
allow expression of the human thymopoietin. 
In still another aspect, the present invention provides three proteins 
characterized by having activity in the immune system. These proteins are 
illustrated in FIGS. 1-3, and are designated herein as .alpha. SEQ ID NO: 
2, .beta. SEQ ID NO: 4, and .gamma. SEQ ID NO: 6, respectively. These 
proteins are characterized by being isolated from the cellular material 
with which they are naturally associated. Advantageously, one or more of 
these sequences is capable of being produced recombinantly. 
In yet another aspect, the present invention provides a pharmaceutical 
composition containing at least one of the thymopoietin proteins .alpha., 
.beta. or .gamma., and a pharmaceutically acceptable carrier. 
In another aspect, the invention provides a method of treating a subject 
with a disorder of the immune or nervous system by administering to the 
subject a pharmaceutical composition of the invention. 
In yet a further aspect, the invention provides a diagnostic reagent, such 
as a polyclonal or monoclonal antibody generated by use of one of these 
thymopoietin proteins or fragments thereof. 
In another aspect, the invention provides a diagnostic reagent, such as a 
DNA probe, i.e., an oligonucleotide fragment derived from the 
polynucleotide sequence encoding one of the proteins of the invention or 
from the complementary strand. The reagents may be optionally associated 
with a detectable label.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides novel recombinant human thymopoietin (rhTP) 
nucleic acid sequences and proteins, designated .alpha., .beta., and 
.gamma.. These sequences are provided in FIGS. 1-3 [SEQ ID NO: 1-6], 
respectively. Advantageously, the nucleic acid sequences are useful as 
diagnostic probes, in gene therapy, and in the production of thymopoietin 
proteins. The proteins are useful for a variety of therapeutic and 
diagnostic applications, as well as for generation of other therapeutic 
and diagnostic reagents. 
In the figures, the sequences are numbered differently than in the Sequence 
Listing. Specifically, in the figures, the sequences have been numbered so 
that amino acid +1 is the amino terminal proline of mature TP and 
nucleotide +1 is the first nucleotide of the proline codon. The initial 
Met, its codon, and the 5' end of the sequences all are designated in 
negative numbers. This is indicative of the fact that the initiating 
methionine is removed co-translationally by methionine aminopeptidase [R. 
A. Bradshaw, Trends Biochem. Sci., 14:276-279 (1989)]. In contrast, due to 
the limitations of the PatentIn program, the Sequence Listing does not 
contain any negative numbers. Thus, in the Sequence Listing, the 5' 
non-coding region begins with positive numbers and the first amino acid is 
Met. Throughout this application, fragments of the sequences will be 
referred to as in the figures, with the numbers of the Sequence Listing 
following in brackets. 
As used herein, the term ".beta. numbering system" reflects the fact that 
two common regions are shared by hTP.beta. and hTP.gamma. and are 
identified by reference to the amino acids of the hTP.beta. protein. 
Because hTP.beta. has a 109 amino acid insert (indicated in bold in FIG. 
2), discussed in detail below, between amino acids 220 and 221 of 
hTP.gamma., in the .beta.-numbering system, amino acid 330 of hTP.beta. is 
equivalent to amino acid 221 of hTP.gamma. (subtraction of the 109 
.beta.-specific amino acids results in correct numbering for .gamma.). See 
FIGS. 4B and 4C. 
The present invention provides the human thymopoietin .alpha., .beta., and 
.gamma. proteins. These proteins are characterized by the amino acid 
sequences of FIG. 1-3, respectively. Human TP.alpha. is 693 amino acids in 
length [SEQ ID NO:2] having a molecular weight of 75 kDa, hTP.beta. is a 
453 amino acid protein [SEQ ID NO:4] having a molecular weight of 51 kDa; 
and human TP.gamma. is a 344 amino acid protein [SEQ ID NO:6] having a 
molecular weight of 39 kDa. 
TPs .alpha., .beta., and .gamma. have identical N-terminal domains through 
Glu.sub.187 (indicated by an * in FIGS. 1-3). This region is termed 
.alpha..beta..gamma. [amino acids 2-188 of SEQ ID NO: 2, 4,6]. See FIGS. 
4A-4C. After Glu.sub.187, TP .alpha. [SEQ ID NO:2] diverges from TPs 
.beta. [SEQ ID NO:4] and .gamma. [SEQ ID NO:6]. This unique region from 
amino acid 188 through amino acid 693 of hTP.alpha. [189-694 SEQ ID NO:2] 
is termed simply .alpha.. A unique hTP.beta. region is found at amino acid 
221 through amino acid 329 [222-330 of SEQ ID NO:4]. TP.gamma. differs 
from TP.beta. only in missing the .beta.-specific domain containing amino 
acids 221-329 of TP.beta. (222-330 of SEQ ID NO:4]. The two regions common 
to hTP .beta. and hTP .gamma. are from amino acid 188-220 (.beta..gamma.1) 
[189-221 SEQ ID NO:4] and from amino acid 330-453 (.beta..gamma.2) 
[331-454 SEQ ID NO: 4], using the .beta. numbering system. In regions 
where the amino acid sequences of TPs .alpha. [SEQ ID NO:2], .beta. [SEQ 
ID NO:4], and .gamma. [SEQ ID NO:6] are identical, their nucleotide 
sequences are identical as well, consistent with their originating via 
alternative splicing of transcripts from a single gene. This was confirmed 
by sequencing of genomic clones. 
Included in this invention are fragments of the TP .alpha., .beta. and 
.gamma. proteins [SEQ ID NOS: 2, 4, 6]. Preferably, these fragments are at 
least about 3 amino acids in length and are characterized by being 
biologically active. These fragments are desirable for use in generating 
therapeutic or diagnostic antibodies or for other diagnostic purposes. 
Particularly desirable are the following fragments which have been found 
to be immunogenic sites. The following Table I makes use of the 
nomenclature above, e.g. .alpha..beta..gamma. hTP.sub.1-52 relates to 
amino acids 1-52 of .alpha., .beta. and .gamma. (amino acids 2-53 of SEQ 
ID NO: 2, 4 and 6). 
TABLE I 
______________________________________ 
SEQ 
Peptides SEQ ID NOS: Peptides ID NOS: 
______________________________________ 
.alpha..beta..gamma. hTP.sub.1-52 
2, 4, 6 .alpha. hTP.sub.425-443 
2 
(2-53) (426-444) 
.alpha..beta..gamma. hTP.sub.1-19 
2, 4, 6 .alpha. hTP.sub.518-538 
2 
(2-20) (519-539) 
.alpha..beta..gamma. hTP.sub.28-39 
2, 4, 6 .alpha. hTP.sub.604-622 
2 
(29-40) (605-623) 
.alpha..beta..gamma. hTP.sub.40-52 
2, 4, 6 .alpha. hTP.sub.188-197 
2 
(41-53) (189-198) 
.alpha..beta..gamma. hTP.sub.29-50 
2, 4, 6 .alpha. hTP.sub.188-202 
2 
(30-51) (189-203) 
.alpha..beta..gamma. hTP.sub.56-71 
2, 4, 6 .beta..gamma.1 hTP.sub.196-215 
4, 6 
(57-72) (197-216) 
.alpha..beta..gamma. hTP.sub.92-108 
2, 4, 6 .beta. hTP.sub.247-265 
4 
(93-109) (248-266) 
.alpha..beta..gamma. hTP.sub.168-187 
2, 4, 6 .beta. hTP.sub.312-329 
4 
(169-188) (313-330) 
.alpha. hTP.sub.233-253 
2 .beta. .gamma.2 hTP.sub.332-348 
4, 6 
(234-254) (333-349) 
.alpha. hTP.sub.342-362 
2 .beta..gamma.2 hTP.sub.397-412 
4, 6 
(343-363) (398-413) 
______________________________________ 
Also included in the invention are analogs of the .alpha., .beta., and 
.gamma. proteins provided herein. Typically, such analogs differ by only 
1, 2, 3 or 4 codon changes. Examples include polypeptides with minor amino 
acid variations from the illustrated amino acid sequences of .alpha., 
.beta. or .gamma. (FIGS. 1-3; SEQ ID NOS: 2, 4, 6); in particular, 
conservative amino acid replacements. Conservative replacements are those 
that take place within a family of amino acids that are related in their 
side chains and chemical properties. 
Additionally, the .alpha., .beta., and .gamma. proteins [SEQ ID NOS: 2, 4, 
6] of the invention may be altered, for example to improve production or 
to confer some other desired property upon the protein. For example, the 
transmembrane region of the protein, identified herein, may be removed, 
fully or in part, to obtain a soluble form of the protein. Alternatively, 
a TP protein of the invention may be truncated or modified to prevent 
localization to the nucleus or into the nuclear membrane. For example, the 
TP.alpha. may be modified to remove the putative nucleus localization 
motif at amino acids 189-195 [aa 190-196 of SEQ ID NO:2]. The carboxy 
terminal transmembrane localization motifs of TP.beta. and TP.gamma. can 
also be removed, e.g., at aa 411-431 [aa412-432 of SEQ ID NO: 4] 
(indicated by double underlining in FIGS. 2 and 3). 
Without being bound by the theory of the mechanism by which these rhTP 
proteins function, the inventors believe that each protein has unique 
characteristics. Each of the proteins plays a role in cellular physiology, 
especially in the immune system. As illustrated in the Examples below, TP 
mRNA expression was detected in all tissues examined, suggesting that some 
TP function(s) may be important in many or all cell types. However, TP 
mRNA expression was highest in adult thymus and in fetal liver, a major 
fetal site for production of T cell precursors. This suggests that TPs may 
play important roles in T cell development and function. 
Human TPs .alpha., .beta., and .gamma. [SEQ ID NOS: 2, 4, 6] do not appear 
to contain a cleavable hydrophobic amino-terminal signal peptide for 
directing the nascent peptide into the ER/Golgi pathway for protein 
secretion. The apparent absence of classical N-terminal hydrophobic 
cleavable signal sequences for secretion in TP .alpha., .beta., and 
.gamma. suggests that the proteins [SEQ ID NOS: 2, 4, 6] may be largely 
localized intracellularly and may have important intracellular functions. 
However, preliminary analysis of conditioned media from human and mouse 
T-cell lines using a TP immunoassay is consistent with the presence of one 
or more forms of extracellular TP. Extracellular TP may be generated by an 
alternative secretion pathway such as that used by interleukin-1 or the 
fibroblast growth factors, which also have no classical signal sequences 
[A. Rubartelli et al, Biochem. Soc. Trans., 19:255-259 (1991)]. 
TPs .beta. and .gamma. [SEQ ID NOS: 4 and 6] contain a hydrophobic domain 
near their carboxy termini, which may be a transmembrane signal-anchor 
domain. This putative transmembrane region is found at amino acid 
sequences 410-430, using the .beta. numbering system [411-431 of SEQ ID 
NO:4]. In contrast, TP .alpha. [SEQ ID NO: 2] does not appear to contain a 
membrane-spanning domain and is expected to be a soluble protein. 
Preliminary analysis of subcellular localization by immunofluorescence 
microscopy confirms the localizations suggested above, i.e., TPB and 
TP.gamma. being localized to the nuclear membrane and TP.alpha. being 
localized within the nucleus. 
Examination of TP .alpha., .beta., and .gamma. sequences [SEQ ID NOS: 2, 4, 
6] for additional motifs revealed potential phosphorylation sites for 
several protein kinases. Of particular interest is a consensus sequence 
for tyrosine phosphorylation in TP.alpha. [SEQ ID NO: 2] at Tyr.sub.626 
(indicated by underlining in FIG. 1). Typically, phosphorylation on 
tyrosine serves to regulate activities of many proteins, particularly 
proteins involved in controlling cell growth and differentiation. 
The nucleic acid sequences encoding these proteins are themselves useful 
for a variety of diagnostic and therapeutic uses, including gene therapy. 
Thus, the present invention also provides the nucleic acid sequences 
encoding hTP.alpha., .beta. and .gamma. [SEQ ID NOS: 2, 4, 6] and 
fragments thereof. The nucleic acid sequences of the invention are 
characterized by the DNA sequences of FIG. 1-3 [SEQ ID NOS: 1, 3, 5], 
respectively. Note that the first approximately 53 nucleotides of the 
TP.gamma. sequence of FIG. 3 may either be an alternatively spliced 
original TP.gamma. sequence, or alternatively may represent a non-TP 
cloning artifact. 
In addition to the fragments encoding the peptide sequences of Table I, 
other fragments of these sequences may prove useful for a variety of uses. 
Desirably, these fragments are at least about 15 nucleotides in length and 
encode a desired amino acid sequence, e.g. an epitope, a therapeutically 
useful peptide, or the like. These nucleotide sequences of the invention 
may be isolated as in Example 1, described below. Alternatively, these 
sequences may be constructed using conventional genetic engineering or 
chemical synthesis techniques. 
According to the invention, the nucleic acid sequences [SEQ ID NOS: 1, 3, 
5] coding for, as well as the encoded .alpha., .beta., and .gamma. 
proteins [SEQ ID NOS: 2, 4, 6] described above and provided in FIGS. 1-3, 
may be modified. Utilizing the sequence data in these figures, it is 
within the skill of the art to obtain other polynucleotide sequences 
encoding the proteins of the invention. Such modifications at the nucleic 
acid level include, for example, modifications to the nucleotide sequences 
which are silent or which change the amino acids, e.g. to improve 
expression or secretion. Alternatively, the amino acid sequence may be 
modified to enhance protein stability or other characteristics, e.g. 
binding activity or bioavailability. In still another alternative, the 
polynucleotide and/or protein sequences may be modified by adding readily 
assayable tags to facilitate quantitation, where desirable. Nucleotides 
may be substituted, inserted, or deleted by known techniques, including, 
for example, in vitro mutagenesis and primer repair. Also included are 
allelic variations, caused by the natural degeneracy of the genetic code. 
For example, in one of the hTP.alpha. cDNA clones isolated, nucleotide 
1792 is a G, which changes amino acid 598 from Gln to Glu (compare to SEQ 
ID NO:1 in which nucleotide 1792 is a C). Note, also, nucleotide 579 is C 
in the .beta. clone .lambda.T.6 and in a genomic clone, but T in the 
sequenced subclone of .lambda. clone .lambda.T.206, in both cases encoding 
leucine. 
In addition to isolated nucleic acid sequences [SEQ ID NOS: 1, 3, 5] 
encoding the thymopoietin proteins .alpha., .beta., and .gamma. [SEQ ID 
NOS: 2, 4, 6] described herein, this invention also encompasses other 
nucleic acid sequences, such as those complementary to the illustrated DNA 
sequences. Useful DNA sequences also include those sequences which 
hybridize under high or moderately high stringency conditions [see, T. 
Maniatis et al, Molecular cloning (A Laboratory Manual), Cold Spring 
Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences 
illustrated in FIG. 1-3. An example of a highly stringent hybridization 
condition is hybridization at 4XSSC at 65.degree. C. followed by a washing 
in 0.1XSSC at 65.degree. C. for an hour. Alternatively, an exemplary 
highly stringent hybridization condition is in 50% formamide, 4XSSC at 
42.degree. C. Other, moderately high stringency conditions may also prove 
useful, e.g. hybridization in 4XSSC at 55.degree. C., followed by washing 
in 0.1XSSC at 37.degree. C. for an hour. Alternatively, an exemplary 
moderately high stringency hybridization condition is in 50% formamide, 
4XSSC at 30.degree. C. 
Once constructed, or isolated, as described in further detail in Example 1 
below, these DNA sequences or suitable fragments are preferably employed 
to obtain proteins of this invention. 
The DNA sequences of the invention are inserted into a suitable expression 
system to obtain the proteins of the invention. Desirably, the 
polynucleotide sequence is operably linked to a heterologous expression 
control sequence permitting expression of the human thymopoietin protein. 
Numerous types of appropriate expression systems are known in the art for 
mammalian (including human) expression, as well as insect, yeast, fungal, 
and bacterial expression, by standard molecular biology techniques. 
Bacterial expression systems, using such host cells as E. coli, are 
desirable for expression of thymopoietin. 
Mammalian cell expression vectors are also desirable for expression. The 
mammalian cell expression vectors described herein may be synthesized by 
techniques well known to those skilled in this art. The components of the 
vectors, e.g. replicons, selection genes, enhancers, promoters, and the 
like, may be obtained from natural sources or synthesized by known 
procedures. 
The transformation of these vectors into appropriate host cells can result 
in expression of the selected thymopoietin proteins. Other appropriate 
expression vectors, of which numerous types are known in the art for 
mammalian expression, can also be used for this purpose. 
Suitable cells or cell lines for this method are mammalian cells, such as 
Human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell 
line or murine 3T3 cells derived from Swiss, Balb-c or NIH mice. The 
selection of suitable mammalian host cells and methods for transformation, 
culture, amplification, screening, and product production and purification 
are known in the art. [See, e.g., Gething and Sambrook, Nature, 
293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 
5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446]. Another 
suitable mammalian cell line is the CV-1 cell line. 
Similarly useful as host cells suitable for the present invention are 
bacterial cells. For example, the various strains of E. coli (e.g., HB101, 
MC1061, and strains used in the following examples) are well-known as host 
cells in the field of biotechnology. Various strains of B. subtilis, 
Pseudomonas, other bacilli and the like may also be employed in this 
method. 
Many strains of yeast cells known to those skilled in the art are also 
available as host cells for expression of the polypeptides of the present 
invention. Additionally, where desired, insect cells may be utilized as 
host cells in the method of the present invention. [See, e.g. Miller et 
al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references 
cited therein]. Fungal cells may also be employed as expression systems. 
The host cells transformed with the one or more vectors carrying the 
thymopoietin DNA, e.g. by conventional means, may then be cultured under 
suitable conditions to obtain expression of the desired protein. The 
method of this present invention therefore comprises culturing a suitable 
cell or cell line, which has been transformed with a DNA sequence coding 
for thymopoietin, the coding sequence under the control of a 
transcriptional regulatory sequence. The expressed protein is then 
recovered, isolated, and purified from the culture medium (or from the 
cell, if expressed intracellularly) by appropriate means known to one of 
skill in the art. 
For example, the proteins may be isolated following cell lysis in soluble 
form, or extracted in guanidine chloride. For example, a currently 
preferred method for purification of hTP.alpha. [SEQ ID NO: 2] is by lysis 
of the E. coli by freezing and thawing followed by sonication, and 
extraction of the recombinant protein with solutions containing 20 mM Tris 
HCl, pH 7.6, 1M urea or 1M guanidine HCl. In addition, molecular sieving, 
e.g. using a 300 kDa sieve [BioRad TSK-250] column, may be used. 
If desired, the TP proteins of the invention may be produced as a fusion 
protein. For example, it may be desirable to produce such TP fusion 
proteins, to enhance expression of the protein in a selected host cell, or 
to improve purification. Suitable fusion partners for the rhTP proteins of 
the invention are well known to those of skill in the art and include, 
among others, .beta.-galactosidase and poly-histidine. 
Other uses for the polynucleotide sequences of this invention include 
diagnostic and therapeutic uses. For example, the novel recombinant hTP 
nucleic acid sequences or genes of the invention, or suitable fragments 
thereof, are useful in gene therapy for correcting abnormalities, for 
example, those associated with an immune or nervous system disorder. 
Another example involves incorporating a desired hTP nucleic acid sequence 
of the invention into a suitable vector or other delivery system. Suitable 
delivery systems are well known to those of skill in the art. Vectors 
containing such sequences may be administered, thus, treating deficiencies 
of TP via in vivo expression of the proteins of the invention. Such 
delivery systems enable the desired hTP gene to be incorporated into the 
target cell and to be translated by the cell. In such a manner, a 
recombinant hTP protein of the invention can be provided to a cell, 
particularly a cell in an individual having a condition characterized by a 
deficiency in TP. 
These polynucleotide sequences of this invention may also be associated 
with detectable labels or components of label systems conventionally used 
in diagnostic or therapeutic methods. As diagnostic agents the 
polynucleotide sequences may be employed to detect or quantitate normal or 
mutant hTP mRNA or detect mutations in TP DNA in a patient sample. 
The TP.alpha., .beta. and .gamma. proteins [SEQ ID NOS: 2, 4, 6] of the 
invention and compositions containing these proteins demonstrate a variety 
of regulatory effects on the mammalian immune system. For example, 
peptides of this invention offer treatment therapies for chronic 
infection, autoimmune disorders, and certain affective psychiatric or 
neurological disorders, as well as other conditions characterized by a 
disorder of the immune system. Because of the immunomodulatory 
characteristics of the subject proteins, they are therapeutically useful 
in the treatment of humans, and possibly animals, since they are capable 
of effecting changes in the immune system of the mammal. 
These proteins have therapeutic uses in humans. For example, the rhTP 
proteins in a pharmaceutical composition of the present invention may be 
administered in vivo to raise levels of circulating TP in an individual 
requiring same, e.g., a patient suffering from disorders, e.g., stress 
related to insufficient levels of circulating hTP. Alternatively, the rhTP 
proteins of the invention may be administered in such a way as to produce 
a localized response. It is anticipated that these rhTP proteins will have 
longer half-lives than thymopentin. 
Also, the proteins according to the present invention may be used to 
diminish the effects of aging on the immune system. As the thymus shrinks 
with age, the level of thymopoietin decreases. Thus, administration of 
proteins of this invention which have biological activity similar to 
thymopoietin can help reduce the effects of aging related to inefficient 
or non-functioning immune systems. 
The invention further provides pharmaceutical compositions and a method for 
treatment of conditions resulting from disorder of the immune system 
and/or nervous system of a subject, which comprises administering to said 
subject a therapeutically-effective amount of at least one of the proteins 
or pharmaceutical compositions of this invention. Such pharmaceutical 
compositions of the invention contain one or more of the above-described 
proteins or acid- or base-addition salts thereof. Optionally, such 
compositions may further contain conventional therapeutic or other agents 
useful in treating the immune or other disorder. The subject proteins or 
pharmaceutical compositions containing the proteins or their acid or basic 
salts are generally considered to be useful when cellular immunity is an 
issue and particularly when there are deficiencies in immunity. The 
pharmaceutical compositions of the invention are also useful in treating 
imbalances and dysfunctions in the central nervous system. 
As used herein, the term "therapeutically-effective amount" means an amount 
which is effective to treat the conditions referred to above. A protein of 
the present invention is generally effective when parenterally 
administered in amounts above about 0.01 .mu.g protein per kg of body 
weight (.mu.g/kg), and preferably from about 1 .mu.g/kg to about 10 mg/kg. 
To prepare the pharmaceutical compositions of the present invention, a 
protein of this invention is combined as the active ingredient in intimate 
admixture with a pharmaceutical carrier according to conventional 
pharmaceutical compounding techniques. This carrier may take a wide 
variety of forms depending on the form of preparation desired for 
administration, e.g., sublingual, rectal, nasal, or parenteral. The 
presently preferred route of administration is parenteral. 
For parenteral products the carrier will usually comprise sterile water, 
although other ingredients may be included, e.g., to aid solubility or for 
preservation purposes. Injectable suspensions may also be prepared, in 
which case appropriate liquid carriers, suspending agents, and the like 
may be employed. 
Both the nucleic acid and amino acid sequences of the invention are useful 
for generating reagents for use in diagnostic assays. The nucleic acid 
sequences, or suitable fragments thereof, are also useful for detecting 
thymopoietin mRNA levels, and gene mutations. Further, antibodies, 
including monoclonal, polyclonal, and recombinant antibodies, may be 
generated to these peptide sequences which may similarly be useful for 
measuring thymopoietin levels. Such monoclonal antibodies may be generated 
using the standard Kohler and Milstein technique as well as well known 
modifications thereof. Alternatively, other known techniques for the 
generation of monoclonal or recombinant antibodies may be employed using 
fragments of the proteins or polynucleotide sequences of this invention to 
generate antibodies suitable for both therapeutic and diagnostic 
application. 
Thus, the invention provides a method for diagnosing an immune or nervous 
system disorder, and/or detecting a condition associated with increased or 
decreased levels of thymopoietin using conventional diagnostic assay 
methods. Such a diagnostic method may be performed using a monoclonal or 
polyclonal antibody directed against an epitope of protein .alpha., 
.beta., or .gamma., or a DNA probe of the invention, in an appropriate 
assay system. 
The following examples illustrate the preferred methods for isolating and 
expressing the novel sequences of the invention. In view of the disclosure 
of these sequences, other methods for obtaining them are available to the 
art and are therefore encompassed in this invention. These examples are 
illustrative only and do not limit the scope of the invention. 
EXAMPLE 1 
Isolation of Human Thymopoietin cDNA Clones 
Initial human thymopoietin cDNA clones were isolated from a commercial cDNA 
library prepared from human thymus RNA in the vector lambda GT10 
(Clontech; Palo Alto, Calif.). The sequence of human thymopoietin .alpha. 
was determined from the overlapping cDNA clones .lambda.hTP-T.32 and 
.lambda.hTP-T.153, which together provide the complete open reading frame, 
and was verified in the genomic clone .lambda.SHG-1, obtained from a 
commercial genomic library in vector .lambda.FIXII [Stratagene]. Isolation 
of the clones from which the TP proteins .alpha., .beta. and .gamma. of 
the invention were derived was performed as follows. 
The library was probed using two 95-mer oligonucleotides containing a 14 
nucleotide overlap based on the bovine thymopoietin sequence of 
Zevin-Sonkin et al, Immunol. Lett., 31:301-310 (1992). The sense 
oligonucleotide sequence was: GGGAATTCGC CGCCGAGATG CCGGAGTTCC TGGAAGACCC 
CTCGGTCCTG ACGAAAGAGA AGTTGAAGAG TGAGTTGGTC GCCAACAATG TGACG :SEQ ID NO:8. 
The antisense oligonucleotide sequence was: GGGAATTCAG CGCTTCAGGG 
CCGTCAGGTG CTGCAGGTAG AGCTGCACAT ACACGTCTTT GCGCTGCTCC CCGGCCGGGA 
GCGTCACATT GTTGG: SEQ ID NO:9. 
Clones .lambda.hTP-T.6 (hTP.beta.), .lambda.hTP-T.17 (hTP.beta.), and 
.lambda.hTP-T.32 (hTP.alpha.) were among the clones isolated in this 
initial screen. Clone .lambda.hTP-T.153 (hTP.alpha.) was among the clones 
isolated in a subsequent screen in which the probe was a 0.3 kb fragment 
isolated from the 3' end of .lambda.hTP-T.32 by digestion with the 
restriction enzymes Bam HI and Eco RI. Clones .lambda.hTP-T.206 
(hTP.gamma.) and .lambda.hTP-T.209 (hTP.beta.) were among the clones 
isolated in a screen in which the probe was two overlapping 
oligonucleotides derived from the 3' end of .lambda.hTP-T.17, the sense 
oligonucleotide being SEQ ID NO:10 :TCTATCAAGC TATGGAAACC AACCAAGTAA 
ATCCCTTCTC TAATT and the antisense oligonucleotide being SEQ ID NO: 11: 
CATTCAGTTG GATTTTCTAG GGTCAACATG AAGAGAATTA GAGAAGGGAT. 
The sequence of human thymopoietin .gamma. was determined from clone 
.lambda.hTP-T.206. The sequence of human thymopoietin .beta. was 
determined from the overlapping clones .lambda.hTP-T.6, .lambda.hTP-T.17, 
and .lambda.hTP-T.209. The clone numbers are based solely on the order of 
isolation from the library. 
EXAMPLE 2 
Analysis of TP Clones 
Sequences were determined using Sequenase Version 2.0 (United States 
Biochemical) or Taq polymerase (Perkin-Elmer), on the original clone DNA, 
or on fragments subcloned into plasmid vectors. All sequences reported 
here were determined on both strands of at least one clone, and, except 
for the 3' untranslated sequences of TPs .beta. and .gamma., have been 
confirmed in one or more additional clones. 
The sequences of human TP .alpha., .beta. and .gamma. are similar but not 
identical to the bovine sequence of Zevin-Sonkin et al, cited above, 
between amino acids 1-81, but show no further similarity beyond this 
point. Sequencing of the human TP gene [SEQ ID NO:1,3,5] in a genomic 
clone has revealed that the DNA sequence encoding amino acid 81 lies in 
the middle of an exon with no nearby potential splice donor sites, 
indicating that a TP containing C-terminal sequence similar to the bovine 
sequence is not produced from the human TP gene [SEQ ID NO:1,3,5]. 
Protein sequences were searched for motifs in release 9 of the Prosite 
database [A. Bairoch, Nucl. Acids Res., 21:3097-3103 (1993)] using 
MacPattern [R. Fuchs, Comput. Appl. Biosci., 7:105-106 (1991)]. This 
analysis revealed several potential phosphorylation sites for protein 
kinases, including KTYDAASY, amino acids 619-626 of TP.alpha. [620-627 of 
SEQ ID NO:2], which matches a consensus sequence for phosphorylation by 
some tyrosine kinases ([K/R]X.sub.2/3 [D/E]X.sub.2/3 Y) [T. Patschinsky et 
al, Proc. Natl. Acad. Sci., U.S.A., 79:973-977 (1982)]. 
Hydropathy analysis was performed by the method of D. M. Engelman et al, 
Ann. Rev. Biophys. Biophys. Chem., 15:321-353 (1986) as implemented in 
MacVector (Eastman Kodak Chemical Co., software version 4.1) and revealed 
that TPs .beta. and .gamma. [SEQ ID NOS: 4 and 6] contain a very 
hydrophobic region close to their carboxy termini that may function as a 
transmembrane domain. No compelling similarities to previously known 
protein or nucleic acid sequences other than TP were revealed. 
EXAMPLE 3 
Expression of Recombinant Human TP in Bacteria 
The open reading frames (ORFs) for recombinant human thymopoietin cDNAs 
.alpha., .beta., and .gamma. [SEQ ID NOS: 1, 3, 5] have been expressed in 
E. coli using inducible T7 RNA polymerase-dependent pET expression vectors 
[Novagen; Studier et al, Meth. Enzymol., 185:60-89 (1990)] as follows. 
To construct an hTP.alpha. expression vector, the ORF was amplified by PCR 
from .lambda.hTP-T.32 and an overlapping Bam HI/Hind III fragment from 
.lambda.hTP-T.153. Primers that introduced an Nhe I site at the 5' end and 
an Xho I site at the 3' end were used, allowing insertion into the vector 
pET-17b (Novagen) between the Nhe I and Xho I sites. This construct, 
called pEThTPe, pETTII, or pET17b-hTP.alpha., encodes hTP.alpha. as a 
fusion protein with three additional amino acids, Met Ala Ser at the amino 
terminus, followed by the hTPe sequence [SEQ ID NO: 2] beginning with Met 
Pro Glu. 
To construct an hTP.beta. expression vector, the open reading frame was 
amplified by PCR from .lambda.T.17, using primers that introduced an Nde I 
site at the 5' end and a BamHI site at the 3' end, allowing ligation into 
the vector pET-3a. The resulting expression plasmid, called pEThTP.beta., 
pETTIa or pET3ahTP.beta., encoded hTP.beta. [SEQ ID NO: 4] and contained 
no additional amino acids. 
pETHTP.gamma., pETTIb, or pET3ahTP.gamma. was constructed as described for 
pEThTP.beta., except the open reading frame was amplified from 
.lambda.hTP-T.206. 
For expression, the plasmids were transformed into E. coli strain BL21(DE3) 
[Novagen] which contains the T7 RNA polymerase gene integrated into the 
chromosome and under the control of the lacUV5 promoter. Induction of 
transcription from the lacUV5 promoter by addition of isopropyl 
.beta.-D-thioglucoside [IPTG; Gibco-BRL] produces the T7 RNA polymerase, 
which in turn transcribes the hTP genes which are under the control of a 
T7 RNA polymerase-dependent promoter. Cells were grown in M9 medium 
supplemented with 1% casamino acid [Difco] and 100 .mu.g/mL ampicillin or 
carbenicillin [Sigma]. When the cell density reached an optical density of 
0.3 to 0.5 at 600 nm (at approximately 4 hours), the T7 RNA polymerase was 
induced by addition of IPTG to 1 mM, and the cells were grown for an 
additional 4 hours or overnight. 
To confirm that the bacteria had been transformed with the appropriate 
plasmids and that the correct proteins were being produced, lysates of E. 
coli strains expressing the recombinant TPs were compared to lysates of 
the human T cell line CEM [American Type Culture Collection, ATCC #CCL 
119]. Mammalian cell extracts were prepared by lysing cells in 1% NP-40, 
20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 
plus the following protease inhibitors (Boehringer Mannheim):10 .mu.g/ml 
aprotinin, 0.3 mM pepstatin, 0.1 mM Pefabloc, 1 .mu.g/ml E-64. After 
passage through a 27 gauge needle 10 times to reduce viscosity and 
centrifugation to remove insoluble material, sample buffer [U. Laemmli, 
Nature, 227:658-680 (1970)] was added. E. coli extracts were prepared by 
direct lysis in sample buffer. Proteins were separated by SDS-PAGE in 10% 
gels (Novex) buffered with tricine, under reducing conditions. Proteins 
were transferred to nitrocellulose (Novex), and TPs were detected after 
incubation with an affinity-purified rabbit antiserum raised against a 
synthetic peptide consisting of amino acids 1 to 19 of the common amino 
terminal region of TPs .alpha., .beta. and .gamma. [2-20 of SEQ ID NO: 2, 
4, 6] and peroxidase-linked goat anti-rabbit Ig (Pierce) using an enhanced 
chemiluminescence system (Amersham). 
The molecular masses of the TPs .alpha., .beta. and .gamma. [SEQ ID NOS: 2, 
4, 6] were determined by comparison to marker proteins in separate 
experiments. Recombinant hTPs .alpha., .beta., and .gamma. [SEQ ID NOS: 2, 
4, 6] produce 75 kDa, 51 kDa and 39 kDa proteins that co-migrated with the 
major thymopoietin proteins expressed in the human T cell line CEM. See 
Example 4. 
EXAMPLE 4 
Characterization of TP Proteins 
A. Western Blot Analysis 
Recombinant TP .alpha., .gamma., and .gamma. [SEQ ID NOS: 2, 4, 6] 
expressed in E. coli were compared with the TP proteins expressed in the 
human T cell line CEM by immunoblotting as described above. 
CEM cells express three major intracellular proteins detected with an 
antiserum against TP amino acids 1-19, with apparent molecular masses of 
75, 51, and 39 kDa. The 75 kDa, 51 kDa, and 39 kDa CEM proteins are the 
sizes predicted from the cDNA sequences for TPs .alpha., .beta., and 
.gamma., respectively, and co-migrate with recombinant TPs .alpha., 
.beta., and .gamma.. 
B. Northern Blot Analysis 
Poly(A).sup.+ RNA from the human T cell line CEM (ATCC) was prepared by 
extraction with acid guanidinium thiocyanate-phenol-chloroform [P. 
Chomczynski et al, Anal. Biochem., 162:156-159 (1987)] using RNAzol 
(Cinna/Biotecx), followed by selection on oligo-dT columns as described 
[Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edit., Cold 
Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1989). 
C. TP mRNAs in T Cell Lines 
Probes for detection of TP mRNAs were partially overlapping 
oligonucleotides that were radiolabelled by extension of 3' ends to 
generate the complete double stranded sequence. Oligonucleotide sequences 
used were the sense and antisense (complementary) sequences as follows, 
.alpha./.beta./.gamma. sense: nucleotides 1 to 87 [208 to 294 of SEQ ID NO: 
1,3,5], antisense: 156 to 64 [363 to 271 of SEQ ID NO: 1,3,5]; 
.alpha.-specific sense: 1488 to 1587 [1695 to 1795 of SEQ ID NO:1], 
antisense: 1587 to 1570 [1794 to 1777 of SEQ ID NO: 1]; 
.beta.-specific sense: 849 to 898 [1089 to 1139 of SEQ ID NO:4], antisense: 
929 to 879 [1169 to 1119 of SEQ ID NO: 3]; 
.beta./.gamma.-specific sense: 1286 to 1330 [1527 to 1571 of SEQ ID NO:3], 
antisense: 1365 to 1316 [1605 to 1556 of SEQ ID NO: 3] 
Three distinct major human TP mRNAs, estimated to be 4.4 kb, 4.1 kb, and 
4.0 kb, were detected in CEM cells. All three mRNAs were detected when 
blots were probed with an oligonucleotide containing sequences encoding 
amino acids 1 to 52 of the human TPs [2-53 of SEQ ID NO: 2, 4, 6], 
sequences that are present in TPs .alpha., .beta., and .gamma.. As none of 
the cDNAs isolated contain complete 3' untranslated regions, the lengths 
of TP .alpha., .beta., and .gamma. mRNAs could not be determined simply 
from the lengths of the cDNAs. Only the .about.4.4 kb mRNA was detected 
with the .beta.-specific probe, only the .about.4.0 kb mRNA was detected 
with the .alpha.-specific probe, and the .about.4.1 kb mRNA was detected 
with the .beta./.gamma.-specific probe but not with s-specific or 
.beta.-specific probes, suggesting that the 4.4 kb mRNA encodes TP.beta., 
the 4.0 kb mRNA encodes TP.alpha., and the 4.1 kb mRNA encodes TP.gamma.. 
D. Expression of TP mRNAs in adult and fetal tissues 
Poly(A).sup.+ RNA from human tissues and blots of human tissue mRNAs were 
purchased from Clontech. Glyoxylated poly(A).sup.+ RNAs were separated on 
1% agarose gels and blotted to nylon membranes (Gibco BRL). Hybridization 
and washing conditions were as described in Sambrook et al, cited above. 
Sizes of mRNAs were determined by comparison to RNA size markers (Gibco 
BRL). 
TP mRNAs were detected in all tissues examined, with highest expression in 
adult thymus and in fetal liver. In some tissues, TP mRNAs of slightly 
different sizes than the thymus mRNAs were resolved when electrophoresis 
times were extended. Whether such differences result from different 5' or 
3' untranslated regions or additional distinct patterns of alternative 
splicing of coding exons is not yet known. Expression of TPs .alpha., 
.beta., and .gamma. SEQ ID NOS: 2, 4, 6] in many tissues, with 
particularly high expression in thymus, has also been observed in rodents, 
and initial analysis of rat TP cDNAs suggests a high level of sequence 
conservation between rat and human TP .alpha., consistent with important 
functions of TPs in thymus and other tissues. 
EXAMPLE 5 
Expression of Recombinant Human TP in Mammalian Cells 
hTPs .alpha., .beta., and .gamma. [SEQ ID NOS: 2, 4, 6] were expressed in 
mammalian cells by PCR amplification of the open reading frames and 
insertion into the mammalian expression vector pCMV6, a derivative of 
pCMV1 ]S. Andersson et al, J. Biol. Chem., 264:8222-8229 (1989)] between 
the Kpn I and Sal I sites for TP.alpha. and the Kpn I and Not I sites for 
TP.beta. and TP.gamma.. The resulting vectors are transfected into human 
embryonal kidney 293 cells [American Type Culture Collection, Accession 
#CRL 1573] by conventional techniques using calcium phosphate 
precipitation. The transfected cells are cultured in DMEM medium at 
37.degree. C. until confluent. 
The proteins are then isolated from the cell culture by lysis and 
conventional purification techniques and authenticated by Western blotting 
and SDS/PAGE. 
EXAMPLE 6 
Production of Site-Specific Antibodies to the HTP Sequence--Synthesis of 
(HFP.sub.1-19)-Lysine Core 
The antibodies described below were found to be capable of recognizing the 
specific peptide sequence within a larger synthetic peptide fragment or 
natural HTP molecule. 
An octameric branched lysine lattice was synthesized as described [Posnett 
et al, J. Biol. Chem., 263:1719-1725 (1988)] and the protected 
hTP.sub.1-19 fragment was synthesized by growth from both the .alpha.- and 
.epsilon.-amino groups. An Applied Biosystems model 430A peptide 
synthesizer was used employing standard protocols and software version 
1.4. All amino acids were double-coupled and the end-NH.sub.2 program was 
used to remove the terminal Boc-groups. The protected peptide-resin was 
treated with liquid hydrogen fluoride, in the presence of p-cresol, 
p-thiocresol, and dimethylsulfide as scavengers, at 0.degree. C. for 1 
hour with constant stirring. Excess HF was removed by vacuum and the 
residue treated with ether to remove scavenger products. The peptide was 
extracted (3.times.50 mL) with 50% acetic acid and the solvents evaporated 
in vacuo, and the product freeze-dried. 
The crude peptide was initially purified on an Amberlite IRA-68 
ion-exchange column; further purification was accomplished by 
reversed-phase HPLC on a preparative C.sub.18 column. The solvents used 
were: water containing 0.1% trifluoroacetic acid (TFA) (buffer A) and 
CH.sub.3 CN--H.sub.2 O (4:1) containing 0.1% TFA (buffer B). A linear 
gradient of 15-30% buffer B over 100 minutes was used. The appropriate 
fractions containing the peptide were pooled, the solvents evaporated in 
vacuo, and the product freeze-dried. The purified peptide gave 
satisfactory amino acid analysis. This peptide was used as an immunogen to 
raise antibodies as described in Example 7. 
EXAMPLE 7 
Generation and Routine Testing of Antisera Against Specific Protein 
Sequences 
A. Generation of Antiserum 
In order to produce reagents for use in immunoassays for both research and 
clinical diagnostic purposes, animals, usually rabbits, are repeatedly 
exposed to a compound in order to initiate an immune response that results 
in the formation of specific antibodies against that substance. By 
selecting specific regions of the hTP protein, e.g., those peptides 
disclosed in Table I above, and synthesizing these regions as smaller 
peptides, antibodies can be generated that specifically recognize the 
selected peptide and, the larger hTP as well. 
To greatly increase the antigenicity of the selected hTP peptide and assure 
greater exposure of the sequence of interest a polylysine core compound is 
designed which employs the multiple reactive sites on lysine to create a 
network of lysine molecules with repeats of the small hTP peptide as the 
final layer. Thus the odds of antibodies being generated against the 
specified hTP peptide sequence are greatly enhanced. 
Antisera are produced by injecting emulsions comprised of the polylysine 
core compound and an adjuvant into laboratory animals, preferably rabbits 
or sheep (mice are preferred for monoclonal preparation), to help 
stimulate the immune response. The injections are given in multiple sites 
and at regular time intervals in order to create repeated exposures from 
several routes. After sufficient exposure to stimulate an immune response, 
e.g., about 40 days, sera is collected from the rabbits and tested for the 
presence of antibodies against the injected peptide sequence. 
B. Testing of Antiserum Titers 
Enzyme-Linked Immunoassay (ELISA): In order to determine the concentration 
of specific antibodies present in the sera against the peptide of 
interest, serial dilutions of the test sera are added to wells of a 
microtiter plate that has been coated with the peptide used to generate 
the antiserum. After allowing time for the antibodies to bind to the 
coated peptide, the unbound sera is washed from the plate. A solution 
containing enzyme-linked antibodies that recognize immunoglobulins of the 
species in which the antisera was generated (e.g., anti-rabbit IgG 
antibodies) is added to the wells. These "anti-rabbit" antibodies bind to 
the rabbit antibodies that are bound to the peptide coated plate; thus, 
enzyme molecules (horseradish peroxidase) are effectively placed at each 
site where an antibody initially bound to the peptide coated plate. The 
unbound "anti-rabbit" antibodies are then washed from the plate. A 
substrate, which when converted by the enzyme to a different molecular 
form results in a color reaction, is added to the wells. 
The intensity of the color change is quantitated and used to determine the 
relative concentration of antibodies that bound to the peptide coated 
peptide. For purposes of comparison, the amount of antibody present 
(titer) is expressed as the concentration of antiserum required to produce 
a final color reaction with optical density of 1.0. This intensity 
generally represents a maximal response. Antisera showing sufficient titer 
are further characterized to determine both their full specificity and 
their utility in the various immuno-applications. 
C. Results 
Rabbits immunized with multiple antigenic peptides corresponding to amino 
acid sequences derived from the cDNAs of the invention yielded antiserums 
with the following titers (titer yielding 1.00D unit by ELISA): 
______________________________________ 
Peptides Titers SEQ ID NO 
______________________________________ 
.alpha..beta..gamma. 1-19 
8 .times. 10.sup.6 
(2-20) 2, 4, 6 
.alpha..beta..gamma. 28-39 
4 .times. 10.sup.5 
(29-40) 2, 4, 6 
.alpha..beta..gamma. 29-50 
1.2 .times. 10.sup.7 
(30-51) 2, 4, 6 
.alpha..beta..gamma. 40-52 
1.6 .times. 10.sup.7 
(41-53) 2, 4, 6 
.alpha..beta..gamma. 56-71 
1.5 .times. 10.sup.6 
(57-72) 2, 4, 6 
.alpha..beta..gamma. 92-108 
8 .times. 10.sup.6 
(93-109) 2, 4, 6 
.alpha. 168-187 
2 .times. 10.sup.5 
(169-188) 2, 4, 6 
.alpha. 233-253 
2 .times. 10.sup.6 
(234-254) 2 
.alpha. 342-362 
8 .times. 10.sup.6 
(343-363) 2 
.alpha. 425-443 
2.5 .times. 10.sup.5 
(426-444) 2 
.alpha. 518-538 
3 .times. 10.sup.6 
(519-539) 2 
.alpha. 604-622 
1.5 .times. 10.sup.6 
(605-623) 2 
.alpha. 188-197 
2.5 .times. 10.sup.5 
(189-198) 2 
.beta..gamma.1 196-215 
1 .times. 10.sup.6 
(197-216) 4, 6 
.beta. 247-265 
6 .times. 10.sup.6 
(248-266) 4 
.beta. 312-329 
3 .times. 10.sup.6 
(313-330) 4 
.beta..gamma.2 332-348 
3 .times. 10.sup.6 
(333-349) 4 
(224-240) 6 
.beta..gamma.2 397-412 
3 .times. 10.sup.6 
(398-413) 4 
(289-304) 6 
______________________________________ 
EXAMPLE 8 
Preparation of Monoclonal Antibodies Specific for Thymopoietin 
A. Immunization 
Synthetic peptide sequences (derived from the predicted protein sequences 
of each of three thymopoietin cDNAs) of approximately 20 amino acid 
residues were built on a branched core of seven lysine residues according 
to the method of Tam [see, e.g., Posnett et al, cited above]. These 
structures are referred to as multiple antigenic peptides (MAP). In 
particular, mice were immunized with the HTP.alpha..beta..gamma. sequence 
specified by residues 29-50 (GEQRKDVYVQLYLQHLTARNRP).sub.8 K.sub.7 G 
[30-51 of SEQ ID NO: 2, 4, 6]. 
Balb/c mice, 8-12 weeks of age, were injected with 50 .mu.g of MAP 
suspended in 200 .mu.l of adjuvant which was divided between the 
subcutaneous and peritoneal routes. The adjuvant for the first injection 
was either Ribi.TM. (Ribi ImmunoChem, Hamilton, Mont.) or complete 
Freund's adjuvant. For subsequent injections, Ribi.TM. or incomplete 
Freund's adjuvant was used. A minimum of four injections (but more often 
6-10) were given at no less than two week intervals. Sera were collected 
from animals 5 days following a booster injection in order to monitor 
antibody response. The reactivity of test sera with the specific MAP 
immunogen was measured by ELISA. Sera with high titers to the specific MAP 
were tested by western blot for binding to the native TP present in 
lysates of the T cell line CEM [ATCC; CCL 119]. Only mice which had serum 
showing high titers to the specific MAP and detectable binding to native 
TP were considered for fusion. 
B. Fusion 
Splenocytes from immunoresponsive mice, in particular, a mouse immunized 
with HTP.sub.29-50 MAP, were mixed with P3X63Ag8U1 (HGPRT myeloma) cells 
[obtained from Dr. Matthew D. Scharff, Einstein University, Bronx, N.Y.] 
at a ratio of 1:1. Cell fusion was accomplished by treating the pelleted 
cells with 40% polyethylene glycol 4000 essentially as described in G. 
Kohler and C. Milstein, Nature, 256:495 (1975). Hybridomas were grown in 
HAT selection medium as 1000 independent cultures and supernatants from 
the cultures were screened for TP-specific MAb production about 2 weeks 
after fusion. 
C. Hybridoma Selection 
Selection for hybridomas producing TP-specific monoclonal antibodies was 
achieved by testing culture supernatants in ELISA systems in which the 
antigen on the plate was either bovine serum albumin (BSA) or the 
immunizing peptide. Supernatants negative for BSA and positive for the 
immunizing peptide were tested on additional synthetic peptides or 
enriched preparations of native TP and the hybridomas producing 
supernatants positive for only HTP.sub.29-50 containing synthetic peptides 
and the TP-enriched native materials were chosen for subcloning. Hybridoma 
clones arising from a single cell were isolated by two successive rounds 
of limit dilution plating. For the HTP.alpha..beta..gamma..sub.29-50 
lysine core immunogen, three independent hybridomas (885-1.7B8, 885-1.6E10 
& 885-1.1C6) were identified and cloned. 
D. MAb Characterization 
Anti-TP monoclonal antibodies, purified from murine ascites fluid, were 
shown to be specific for native Tp by the immunostaining profile observed 
on western blots of cell lysates prepared from the early T cell line CEM. 
Three proteins of apparent molecular sizes of 75 kDa, 51 kDa and 39 kDa 
(the sizes predicted by the TP cDNA sequences and verified by expression 
of the TP cDNA's in E. coli) were detected by the anti-HTP.sub.29-50 
monoclonal antibodies. Preincubation of the antibodies with the synthetic 
HTP.sub.29-50 peptide but not with an irrelevant synthetic peptide 
resulted in the loss of immunostaining of the protein bands. This suggests 
that the protein bands recognized by the monoclonal antibodies are TP 
proteins. 
E. Other TP-Specific MAbs 
Other monoclonal antibodies specific for one or more of the TP proteins, 
were obtained by immunization with MAP immunogens. These include those 
reported in Table II below. 
TABLE II 
______________________________________ 
MAP MAb TP Proteins 
______________________________________ 
HTP.alpha..beta..gamma. 1-19 
850-1.10A8 
.alpha..beta..gamma. 
850-1.10F8 
.alpha..beta..gamma. 
HTP.beta. 312-329 
937-1.6G11 
.beta. 
937-1.2B11 
.beta. 
HTP.alpha. 233-253 
923-2.9F5 .alpha. 
______________________________________ 
Numerous modifications and variations of the present invention are included 
in the above-identified specification and are expected to be obvious to 
one of skill in the art. Such modifications and alterations to the 
compositions and processes of the present invention are believed to be 
encompassed in the scope of the claims appended hereto. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2490 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 205..2286 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GTTCGTAGTTCGGCTCTGGGGTCTTTTGTGTCCGGGTCTGGCTTGGCTTTGTGTCCGCGA60 
GTTTTTGTTCCGCTCCGCAGCGCTCTTCCCGGGCAGGAGCCGTGAGGCTCGGAGGCGGCA120 
GCGCGGTCCCCGGCCAGGAGCAAGCGCGCCGGCGTGAGCGGCGGCGGCAAAGGCTGTGGG180 
GAGGGGGCTTCGCAGATCCCCGAGATGCCGGAGTTCCTGGAAGACCCCTCG231 
MetProGluPheLe uGluAspProSer 
15 
GTCCTGACAAAAGACAAGTTGAAGAGTGAGTTGGTCGCCAACAATGTG279 
ValLeuThrLysAspLysLeuLysSerGluLeuValAlaAsn AsnVal 
10152025 
ACGCTGCCGGCCGGGGAGCAGCGCAAAGACGTGTACGTCCAGCTCTAC327 
ThrLeuProAlaGlyGluGlnArgLysAspValTyr ValGlnLeuTyr 
303540 
CTGCAGCACCTCACGGCTCGCAACCGGCCGCCGCTCCCCGCCGGCACC375 
LeuGlnHisLeuThrAlaArgAsnArgProPro LeuProAlaGlyThr 
455055 
AACAGCAAGGGGCCCCCGGACTTCTCCAGTGACGAAGAGCGCGAGCCC423 
AsnSerLysGlyProProAspPheSerSerAsp GluGluArgGluPro 
606570 
ACCCCGGTCCTCGGCTCTGGGGCCGCCGCCGCGGGCCGGAGCCGAGCA471 
ThrProValLeuGlySerGlyAlaAlaAlaAlaGly ArgSerArgAla 
758085 
GCCGTCGGCAGGAAAGCCACAAAAAAAACTGATAAACCCAGACAAGAA519 
AlaValGlyArgLysAlaThrLysLysThrAspLysProArg GlnGlu 
9095100105 
GATAAAGATGATCTAGATGTAACAGAGCTCACTAATGAAGATCTTTTG567 
AspLysAspAspLeuAspValThrGluLeuThrAsn GluAspLeuLeu 
110115120 
GATCAGCTTGTGAAATACGGAGTGAATCCTGGTCCTATTGTGGGAACA615 
AspGlnLeuValLysTyrGlyValAsnProGly ProIleValGlyThr 
125130135 
ACCAGGAAGCTATATGAGAAAAAGCTTTTGAAACTGAGGGAACAAGGA663 
ThrArgLysLeuTyrGluLysLysLeuLeuLys LeuArgGluGlnGly 
140145150 
ACAGAATCAAGATCTTCTACTCCTCTGCCAACAATTTCTTCTTCAGCA711 
ThrGluSerArgSerSerThrProLeuProThrIle SerSerSerAla 
155160165 
GAAAATACAAGGCAGAATGGAAGTAATGATTCTGACAGATACAGTGAC759 
GluAsnThrArgGlnAsnGlySerAsnAspSerAspArgTyr SerAsp 
170175180185 
AATGAAGAAGGAAAGAAGAAAGAACACAAGAAAGTGAAGTCCACTAGG807 
AsnGluGluGlyLysLysLysGluHisLysLysVal LysSerThrArg 
190195200 
GATATTGTTCCTTTTTCTGAACTTGGAACTACTCCCTCTGGTGGTGGA855 
AspIleValProPheSerGluLeuGlyThrThr ProSerGlyGlyGly 
205210215 
TTTTTTCAGGGTATTTCTTTTCCTGAAATCTCCACCCGTCCTCCTTTG903 
PhePheGlnGlyIleSerPheProGluIleSer ThrArgProProLeu 
220225230 
GGCAGTACCGAACTACAGGCAGCTAAGAAAGTACATACTTCTAAGGGA951 
GlySerThrGluLeuGlnAlaAlaLysLysValHis ThrSerLysGly 
235240245 
GACCTACCTAGGGAGCCTCTTGTTGCCACAAACTTGCCTGGCAGGGGA999 
AspLeuProArgGluProLeuValAlaThrAsnLeuProGly ArgGly 
250255260265 
CAGTTGCAGAAGTTAGCCTCTGAAAGGAATTTGTTTATTTCATGCAAG1047 
GlnLeuGlnLysLeuAlaSerGluArgAsnLeuPhe IleSerCysLys 
270275280 
TCTAGCCATGATAGGTGTTTAGAGAAAAGTTCTTCGTCATCTTCTCAG1095 
SerSerHisAspArgCysLeuGluLysSerSer SerSerSerSerGln 
285290295 
CCTGAACACAGTGCCATGTTGGTCTCTACTGCAGCTTCTCCTTCACTG1143 
ProGluHisSerAlaMetLeuValSerThrAla AlaSerProSerLeu 
300305310 
ATTAAAGAAACCACCACTGGTTACTATAAAGACATAGTAGAAAATATT1191 
IleLysGluThrThrThrGlyTyrTyrLysAspIle ValGluAsnIle 
315320325 
TGCGGTAGAGAGAAAAGTGGAATTCAACCATTATGTCCTGAGAGGTCC1239 
CysGlyArgGluLysSerGlyIleGlnProLeuCysProGlu ArgSer 
330335340345 
CATATTTCAGATCAATCGCCTCTCTCCAGTAAAAGGAAAGCACTAGAA1287 
HisIleSerAspGlnSerProLeuSerSerLysArg LysAlaLeuGlu 
350355360 
GAGTCTGAGAGCTCACAACTAATTTCTCCGCCACTTGCCCAGGCAATC1335 
GluSerGluSerSerGlnLeuIleSerProPro LeuAlaGlnAlaIle 
365370375 
AGAGATTATGTCAATTCTCTGTTGGTCCAGGGTGGGGTAGGTAGTTTG1383 
ArgAspTyrValAsnSerLeuLeuValGlnGly GlyValGlySerLeu 
380385390 
CCTGGAACTTCTAACTCTATGCCCCCACTGGATGTAGAAAACATACAG1431 
ProGlyThrSerAsnSerMetProProLeuAspVal GluAsnIleGln 
395400405 
AAGAGAATTGATCAGTCTAAGTTTCAAGAAACTGAATTCCTGTCTCCT1479 
LysArgIleAspGlnSerLysPheGlnGluThrGluPheLeu SerPro 
410415420425 
CCAAGAAAAGTCCCTAGACTGAGTGAGAAGTCAGTGGAGGAAAGGGAT1527 
ProArgLysValProArgLeuSerGluLysSerVal GluGluArgAsp 
430435440 
TCAGGTTCCTTTGTGGCATTTCAGAACATACCTGGATCCGAACTGATG1575 
SerGlySerPheValAlaPheGlnAsnIlePro GlySerGluLeuMet 
445450455 
TCTTCTTTTGCCAAAACTGTTGTCTCTCATTCACTCACTACCTTAGGT1623 
SerSerPheAlaLysThrValValSerHisSer LeuThrThrLeuGly 
460465470 
CTAGAAGTGGCTAAGCAATCACAGCATGATAAAATAGATGCCTCAGAA1671 
LeuGluValAlaLysGlnSerGlnHisAspLysIle AspAlaSerGlu 
475480485 
CTATCTTTTCCCTTCCATGAATCTATTTTAAAAGTAATTGAAGAAGAA1719 
LeuSerPheProPheHisGluSerIleLeuLysValIleGlu GluGlu 
490495500505 
TGGCAGCAAGTTGACAGGCAGCTGCCTTCACTGGCATGCAAATATCCA1767 
TrpGlnGlnValAspArgGlnLeuProSerLeuAla CysLysTyrPro 
510515520 
GTTTCTTCCAGGGAGGCAACACAGATATTATCAGTTCCAAAAGTAGAT1815 
ValSerSerArgGluAlaThrGlnIleLeuSer ValProLysValAsp 
525530535 
GATGAAATCCTAGGGTTTATTTCTGAAGCCACTCCACTAGGAGGTATT1863 
AspGluIleLeuGlyPheIleSerGluAlaThr ProLeuGlyGlyIle 
540545550 
CAAGCAGCCTCCACTGAGTCTTGCAATCAGCAGTTGGACTTAGCACTC1911 
GlnAlaAlaSerThrGluSerCysAsnGlnGlnLeu AspLeuAlaLeu 
555560565 
TGTAGAGCATATGAAGCTGCAGCATCAGCATTGCAGATTGCAACTCAC1959 
CysArgAlaTyrGluAlaAlaAlaSerAlaLeuGlnIleAla ThrHis 
570575580585 
ACTGCCTTTGTAGCTAAGGCTATGCAGGCAGACATTAGTCAAGCTGCA2007 
ThrAlaPheValAlaLysAlaMetGlnAlaAspIle SerGlnAlaAla 
590595600 
CAGATTCTTAGCTCAGATCCTAGTCGTACCCACCAAGCGCTTGGGATT2055 
GlnIleLeuSerSerAspProSerArgThrHis GlnAlaLeuGlyIle 
605610615 
CTGAGCAAAACATATGATGCAGCCTCATATATTTGTGAAGCTGCATTT2103 
LeuSerLysThrTyrAspAlaAlaSerTyrIle CysGluAlaAlaPhe 
620625630 
GATGAAGTGAAGATGGCTGCCCATACCATGGGAAATGCCACTGTAGGT2151 
AspGluValLysMetAlaAlaHisThrMetGlyAsn AlaThrValGly 
635640645 
CGTCGATACCTCTGGCTGAAGGATTGCAAAATTAATTTAGCTTCTAAG2199 
ArgArgTyrLeuTrpLeuLysAspCysLysIleAsnLeuAla SerLys 
650655660665 
AATAAGCTGGCTTCCACTCCCTTTAAAGGTGGAACATTATTTGGAGGA2247 
AsnLysLeuAlaSerThrProPheLysGlyGlyThr LeuPheGlyGly 
670675680 
GAAGTATGCAAAGTAATTAAAAAGCGTGGAAATAAACACTAGTAAAATT2296 
GluValCysLysValIleLysLysArgGlyAsn LysHis 
685690 
AAGGACAAAAAGACATCTATCTTATCTTTCAGGTACTTTATGCCAACATTTTCTTTTCTG2356 
TTAAGGTTGTTTTAGTTTCCAGATAGGGCTAATTACAAAATGTTAAGCTTCTACCCATCA2416 
AATTACAGTATAAAAGTAATTGCCTGTGTAGAACTACTTGTCTTTTCTAAAGATTTGCGT2476 
AGATAGGAAGCCTG2490 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 694 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetProGluPheLeuGluAspProSerValLeuThrLysAspLysLeu 
151015 
L ysSerGluLeuValAlaAsnAsnValThrLeuProAlaGlyGluGln 
202530 
ArgLysAspValTyrValGlnLeuTyrLeuGlnHisLeuThrAlaArg 
3 54045 
AsnArgProProLeuProAlaGlyThrAsnSerLysGlyProProAsp 
505560 
PheSerSerAspGluGluArgGluProTh rProValLeuGlySerGly 
65707580 
AlaAlaAlaAlaGlyArgSerArgAlaAlaValGlyArgLysAlaThr 
8590 95 
LysLysThrAspLysProArgGlnGluAspLysAspAspLeuAspVal 
100105110 
ThrGluLeuThrAsnGluAspLeuLeuAspGlnLeuVal LysTyrGly 
115120125 
ValAsnProGlyProIleValGlyThrThrArgLysLeuTyrGluLys 
130135140 
LysLeuLeuL ysLeuArgGluGlnGlyThrGluSerArgSerSerThr 
145150155160 
ProLeuProThrIleSerSerSerAlaGluAsnThrArgGlnAsnGly 
165170175 
SerAsnAspSerAspArgTyrSerAspAsnGluGluGlyLysLysLys 
180185190 
GluHisLysLysValLysSe rThrArgAspIleValProPheSerGlu 
195200205 
LeuGlyThrThrProSerGlyGlyGlyPhePheGlnGlyIleSerPhe 
210215 220 
ProGluIleSerThrArgProProLeuGlySerThrGluLeuGlnAla 
225230235240 
AlaLysLysValHisThrSerLysGlyAspLeuProArgGlu ProLeu 
245250255 
ValAlaThrAsnLeuProGlyArgGlyGlnLeuGlnLysLeuAlaSer 
260265270 
G luArgAsnLeuPheIleSerCysLysSerSerHisAspArgCysLeu 
275280285 
GluLysSerSerSerSerSerSerGlnProGluHisSerAlaMetLeu 
290 295300 
ValSerThrAlaAlaSerProSerLeuIleLysGluThrThrThrGly 
305310315320 
TyrTyrLysAspIleValGluAs nIleCysGlyArgGluLysSerGly 
325330335 
IleGlnProLeuCysProGluArgSerHisIleSerAspGlnSerPro 
340345 350 
LeuSerSerLysArgLysAlaLeuGluGluSerGluSerSerGlnLeu 
355360365 
IleSerProProLeuAlaGlnAlaIleArgAspTyrValAsn SerLeu 
370375380 
LeuValGlnGlyGlyValGlySerLeuProGlyThrSerAsnSerMet 
385390395400 
ProP roLeuAspValGluAsnIleGlnLysArgIleAspGlnSerLys 
405410415 
PheGlnGluThrGluPheLeuSerProProArgLysValProArgLeu 
420425430 
SerGluLysSerValGluGluArgAspSerGlySerPheValAlaPhe 
435440445 
GlnAsnIleProGlySerGluLe uMetSerSerPheAlaLysThrVal 
450455460 
ValSerHisSerLeuThrThrLeuGlyLeuGluValAlaLysGlnSer 
465470475 480 
GlnHisAspLysIleAspAlaSerGluLeuSerPheProPheHisGlu 
485490495 
SerIleLeuLysValIleGluGluGluTrpGlnGlnVal AspArgGln 
500505510 
LeuProSerLeuAlaCysLysTyrProValSerSerArgGluAlaThr 
515520525 
GlnI leLeuSerValProLysValAspAspGluIleLeuGlyPheIle 
530535540 
SerGluAlaThrProLeuGlyGlyIleGlnAlaAlaSerThrGluSer 
54555 0555560 
CysAsnGlnGlnLeuAspLeuAlaLeuCysArgAlaTyrGluAlaAla 
565570575 
AlaSerAlaLeuGlnIleAl aThrHisThrAlaPheValAlaLysAla 
580585590 
MetGlnAlaAspIleSerGlnAlaAlaGlnIleLeuSerSerAspPro 
595600 605 
SerArgThrHisGlnAlaLeuGlyIleLeuSerLysThrTyrAspAla 
610615620 
AlaSerTyrIleCysGluAlaAlaPheAspGluValLysMetAlaAla 
625630635640 
HisThrMetGlyAsnAlaThrValGlyArgArgTyrLeuTrpLeuLys 
645650655 
A spCysLysIleAsnLeuAlaSerLysAsnLysLeuAlaSerThrPro 
660665670 
PheLysGlyGlyThrLeuPheGlyGlyGluValCysLysValIleLys 
67 5680685 
LysArgGlyAsnLysHis 
690 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1743 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 238..1599 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGTTGGTGCGAGCTTCCAGCTTGGCCGCAGTTGGTTCGTAGTTCGGCTCTGGGGTCTTTT60 
GTGTCCGGGTCTGGCTTGGCTTTGTGTCCGCGAGTTTTTGTTCCGCTCCGCAGC GCTCTT120 
CCCGGGCAGGAGCCGTGAGGCTCGGAGGCGGCAGCGCGGTCCCCGGCCAGGAGCAAGCGC180 
GCCGGCGTGAGCGGCGGCGGCAAAGGCTGTGGGGAGGGGGCTTCGCAGATCCCCGAG237 
ATGCCGGAGTTCCTGGAAGACCCC TCGGTCCTGACAAAAGACAAGTTG285 
MetProGluPheLeuGluAspProSerValLeuThrLysAspLysLeu 
151015 
AAGAGTGAGTTGGTCGCCAAC AATGTGACGCTGCCGGCCGGGGAGCAG333 
LysSerGluLeuValAlaAsnAsnValThrLeuProAlaGlyGluGln 
202530 
CGCAAAGACGTGTACGTCCAG CTCTACCTGCAGCACCTCACGGCTCGC381 
ArgLysAspValTyrValGlnLeuTyrLeuGlnHisLeuThrAlaArg 
354045 
AACCGGCCGCCGCTCCCCGCCGGC ACCAACAGCAAGGGGCCCCCGGAC429 
AsnArgProProLeuProAlaGlyThrAsnSerLysGlyProProAsp 
505560 
TTCTCCAGTGACGAAGAGCGCGAGCCCACC CCGGTCCTCGGCTCTGGG477 
PheSerSerAspGluGluArgGluProThrProValLeuGlySerGly 
65707580 
GCCGCCGCCGCGGGCCGGAGCCGA GCAGCCGTCGGCAGGAAAGCCACA525 
AlaAlaAlaAlaGlyArgSerArgAlaAlaValGlyArgLysAlaThr 
859095 
AAAAAAACTGATAAACCCAGA CAAGAAGATAAAGATGATCTAGATGTA573 
LysLysThrAspLysProArgGlnGluAspLysAspAspLeuAspVal 
100105110 
ACAGAGCTCACTAATGAAGAT CTTTTGGATCAGCTTGTGAAATACGGA621 
ThrGluLeuThrAsnGluAspLeuLeuAspGlnLeuValLysTyrGly 
115120125 
GTGAATCCTGGTCCTATTGTGGGA ACAACCAGGAAGCTATATGAGAAA669 
ValAsnProGlyProIleValGlyThrThrArgLysLeuTyrGluLys 
130135140 
AAGCTTTTGAAACTGAGGGAACAAGGAACA GAATCAAGATCTTCTACT717 
LysLeuLeuLysLeuArgGluGlnGlyThrGluSerArgSerSerThr 
145150155160 
CCTCTGCCAACAATTTCTTCTTCA GCAGAAAATACAAGGCAGAATGGA765 
ProLeuProThrIleSerSerSerAlaGluAsnThrArgGlnAsnGly 
165170175 
AGTAATGATTCTGACAGATAC AGTGACAATGAAGAAGACTCTAAAATA813 
SerAsnAspSerAspArgTyrSerAspAsnGluGluAspSerLysIle 
180185190 
GAGCTCAAGCTTGAGAAGAGA GAACCACTAAAGGGCAGAGCAAAGACT861 
GluLeuLysLeuGluLysArgGluProLeuLysGlyArgAlaLysThr 
195200205 
CCAGTAACACTCAAGCAAAGAAGA GTTGAGCACAATCAGAGCTATTCT909 
ProValThrLeuLysGlnArgArgValGluHisAsnGlnSerTyrSer 
210215220 
CAAGCTGGAATAACTGAGACTGAATGGACA AGTGGATCTTCAAAAGGC957 
GlnAlaGlyIleThrGluThrGluTrpThrSerGlySerSerLysGly 
225230235240 
GGACCTCTGCAGGCATTAACTAGG GAATCTACAAGAGGGTCAAGAAGA1005 
GlyProLeuGlnAlaLeuThrArgGluSerThrArgGlySerArgArg 
245250255 
ACTCCAAGGAAAAGGGTGGAA ACTTCAGAACATTTTCGTATAGATGGT1053 
ThrProArgLysArgValGluThrSerGluHisPheArgIleAspGly 
260265270 
CCAGTAATTTCAGAGAGTACT CCCATAGCTGAAACTATAATGGCTTCA1101 
ProValIleSerGluSerThrProIleAlaGluThrIleMetAlaSer 
275280285 
AGCAACGAATCCTTAGTTGTCAAT AGGGTGACTGGAAATTTCAAGCAT1149 
SerAsnGluSerLeuValValAsnArgValThrGlyAsnPheLysHis 
290295300 
GCATCTCCTATTCTGCCAATCACTGAATTC TCAGACATACCCAGAAGA1197 
AlaSerProIleLeuProIleThrGluPheSerAspIleProArgArg 
305310315320 
GCACCAAAGAAACCATTGACAAGA GCTGAAGTGGGAGAAAAAACAGAG1245 
AlaProLysLysProLeuThrArgAlaGluValGlyGluLysThrGlu 
325330335 
GAAAGAAGAGTAGAAAGGGAT ATTCTTAAGGAAATGTTCCCCTATGAA1293 
GluArgArgValGluArgAspIleLeuLysGluMetPheProTyrGlu 
340345350 
GCATCTACACCAACAGGAATT AGTGCTAGTTGCCGCAGACCAATCAAA1341 
AlaSerThrProThrGlyIleSerAlaSerCysArgArgProIleLys 
355360365 
GGGGCTGCAGGCCGGCCATTAGAA CTCAGTGATTTCAGGATGGAGGAG1389 
GlyAlaAlaGlyArgProLeuGluLeuSerAspPheArgMetGluGlu 
370375380 
TCTTTTTCATCTAAATATGTTCCTAAGTAT GTTCCCTTGGCAGATGTC1437 
SerPheSerSerLysTyrValProLysTyrValProLeuAlaAspVal 
385390395400 
AAGTCAGAAAAGACAAAAAAGGGA CGCTCCATTCCCGTATGGATAAAA1485 
LysSerGluLysThrLysLysGlyArgSerIleProValTrpIleLys 
405410415 
ATTTTGCTGTTTGTTGTTGTG GCAGTTTTTTTGTTTTTGGTCTATCAA1533 
IleLeuLeuPheValValValAlaValPheLeuPheLeuValTyrGln 
420425430 
GCTATGGAAACCAACCAAGTA AATCCCTTCTCTAATTTTCTTCATGTT1581 
AlaMetGluThrAsnGlnValAsnProPheSerAsnPheLeuHisVal 
435440445 
GACCCTAGAAAATCCAACTGAATGG TATCTCTTTGGCACGTTCAACTT1629 
AspProArgLysSerAsn 
450 
GGTCTCCTATTTTCAATAACTGTTGAAAAACATTTGTGTACACTTGTTGACTCCAAGAAC1689 
TAAAAATAATGTGATTTCGCCTCAATAAATGTAGTATTTC ATTGAAAAGCAAAC1743 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 454 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetProGluPheLeuGluAspProSerValLeuThrLys AspLysLeu 
151015 
LysSerGluLeuValAlaAsnAsnValThrLeuProAlaGlyGluGln 
202530 
ArgLysAspValTyrValGlnLeuTyrLeuGlnHisLeuThrAlaArg 
354045 
AsnArgProProLeuProAlaGlyThrAsnSerLysGlyProProAsp 
50 5560 
PheSerSerAspGluGluArgGluProThrProValLeuGlySerGly 
65707580 
AlaAlaAlaAlaGlyArgSe rArgAlaAlaValGlyArgLysAlaThr 
859095 
LysLysThrAspLysProArgGlnGluAspLysAspAspLeuAspVal 
100 105110 
ThrGluLeuThrAsnGluAspLeuLeuAspGlnLeuValLysTyrGly 
115120125 
ValAsnProGlyProIleValGlyThrThrArgLysLeu TyrGluLys 
130135140 
LysLeuLeuLysLeuArgGluGlnGlyThrGluSerArgSerSerThr 
145150155160 
P roLeuProThrIleSerSerSerAlaGluAsnThrArgGlnAsnGly 
165170175 
SerAsnAspSerAspArgTyrSerAspAsnGluGluAspSerLysIle 
180185190 
GluLeuLysLeuGluLysArgGluProLeuLysGlyArgAlaLysThr 
195200205 
ProValThrLeuLysGlnAr gArgValGluHisAsnGlnSerTyrSer 
210215220 
GlnAlaGlyIleThrGluThrGluTrpThrSerGlySerSerLysGly 
225230235 240 
GlyProLeuGlnAlaLeuThrArgGluSerThrArgGlySerArgArg 
245250255 
ThrProArgLysArgValGluThrSerGluHisPhe ArgIleAspGly 
260265270 
ProValIleSerGluSerThrProIleAlaGluThrIleMetAlaSer 
275280285 
S erAsnGluSerLeuValValAsnArgValThrGlyAsnPheLysHis 
290295300 
AlaSerProIleLeuProIleThrGluPheSerAspIleProArgArg 
305 310315320 
AlaProLysLysProLeuThrArgAlaGluValGlyGluLysThrGlu 
325330335 
GluArgArgValGluAr gAspIleLeuLysGluMetPheProTyrGlu 
340345350 
AlaSerThrProThrGlyIleSerAlaSerCysArgArgProIleLys 
355360 365 
GlyAlaAlaGlyArgProLeuGluLeuSerAspPheArgMetGluGlu 
370375380 
SerPheSerSerLysTyrValProLysTyrValProLeuAlaAsp Val 
385390395400 
LysSerGluLysThrLysLysGlyArgSerIleProValTrpIleLys 
405410415 
IleLeuLeuPheValValValAlaValPheLeuPheLeuValTyrGln 
420425430 
AlaMetGluThrAsnGlnValAsnProPheSerAsnPheLeuHisVal 
435440445 
AspProArgLysSerAsn 
450 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2392 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 241..1275 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCCTGCTACCAAGGCCCAGCTATGGCCCCAGGGTTGAAAAGTTATGAGGGTCAGGGGTCT60 
TTTGTGTCCGGGTCTGGCTTGGCTTTGTGTCCGCGAGTTTTTGTTCCGCT CCGCAGCGCT120 
CTTCCCGGGCAGGAGCCGTGAGGCTCGGAGGCGGCAGCGCGGTCCCCGGCCAGGAGCAAG180 
CGCGCCGGCGTGAGCGGCGGCGGCAAAGGCTGTGGGGAGGGGGCTTCGCAGATCCCCGAG240 
ATGCCGGAGTTCCTGGAAGACCCCT CGGTCCTGACAAAAGACAAGTTGAAGAGTGAGTTG300 
GTCGCCAACAATGTGACGCTGCCGGCCGGGGAGCAGCGCAAAGACGTGTACGTCCAGCTC360 
TACCTGCAGCACCTCACGGCTCGCAACCGGCCGCCGCTCCCCGCCGGCACCAACAGCAAG420 
GGGCCCCCGGACTTCTCCAGTGACGAAGAGCGCGAGCCCACCCCGGTCCTCGGCTCTGGG480 
GCCGCCGCCGCGGGCCGGAGCCGAGCAGCCGTCGGCAGGAAAGCCACAAAAAAAACTGAT540 
AAACCCAGACAAGAAGATAAAGATGATCTAGATGTAACAGAGC TCACTAATGAAGATCTT600 
TTGGATCAGCTTGTGAAATACGGAGTGAATCCTGGTCCTATTGTGGGAACAACCAGGAAG660 
CTATATGAGAAAAAGCTTTTGAAACTGAGGGAACAAGGAACAGAATCAAGATCTTCTACT720 
CCTCTGCCAACAATTTCT TCTTCAGCAGAAAATACAAGGCAGAATGGAAGTAATGATTCT780 
GACAGATACAGTGACAATGAAGAAGACTCTAAAATAGAGCTYAAGCTTGAGAAGAGAGAA840 
CCACTAAAGGGCAGAGCAAAGACTCCAGTAACACTCAAGCAAAGAAGAGTTGAGCACAAT 900 
CAGGTGGGAGAAAAAACAGAGGAAAGAAGAGTAGAAAGGGATATTCTTAAGGAAATGTTC960 
CCCTATGAAGCATCTACACCAACAGGAATTAGTGCTAGTTGCCGCAGACCAATCAAAGGG1020 
GCTGCAGGCCGGCCATTAGAACTCAGTGATTTCAGG ATGGAGGAGTCTTTTTCATCTAAA1080 
TATGTTCCTAAGTATGTTCCCTTGGCAGATGTCAAGTCAGAAAAGACAAAAAAGGGACGC1140 
TCCATTCCCGTATGGATAAAAATTTTGCTGTTTGTTGTTGTGGCAGTTTTTTTGTTTTTG1200 
GTCTATCAAG CTATGGAAACCAACCAAGTAAATCCCTTCTCTAATTTTCTTCATGTTGAC1260 
CCTAGAAAATCCAACTGAATGGTATCTCTTTGGCACGTTCAACTTGGTCTCCTATTTTCA1320 
ATAACTGTTGAAAAACATTTGTGTACACTTGTTGACTCCAAGAACTAAAAATAA TGTGAT1380 
TTCGCCTCAATAAATGTAGTATTTCATTGAAAAGCAAACAAAATATATATAAATGGACTT1440 
CATTAAAATGTTTTTGAACTTTGGACTAGTAGGAGATCACTTTGTGCCATATGAATAATC1500 
TTTTTTAGCTCTGGAACTTTTTGTAGGCT TTATTTTTTTAATGTGGGCATCTTATTTCAT1560 
TTTTGAAAAAATGTATATGTTTTTTGTGTATTTGGGAAACGAAGGGTGAAACATGGTAGT1620 
ATAATGTGAAGCTACACATTTAAATACTTAGAATTCTTACAGAAAAGATTTTAAGAATTA1680 
TTC TCTGCTGAATAAAAACTGCAAATATGTGAAACATAATGAAATTCAGTAAGAGGAAAA1740 
GTAACTTGGTTGTACTTTTTGTAACTGCAACAAAGTTTGATGGTGTTTATGAGGAAAAGT1800 
ACAGCAATAATCTCTTCTGTAACCTTTATTAATAGTAATGTTGTTGT AGCCCTATCATAC1860 
TCACTTTTTAAGACACAGTATCATGAAAGTCCTATTTCAGTAAGACCCATTTACATACAG1920 
TAGATTTTTAGCAGAGATCTTTTAGTGTAACATACATATTTTAGAGAATTGTTGGCTAGC1980 
TGTACATGTTTTGAAAAGCTG TTTAGCTAGCTATAAGGCTATAATTGGAAATTTGTATTT2040 
TTTATTTACAGCAAAACATTTATTCAGTCATCCAGTTTGCTACCAAAATATGTTTTAGAT2100 
AAGTGTGTGTATGTTTGTTTAGAAGTTAGAAATTGTAAACACTGGTCTTATGTTTCATTT216 0 
GGATTCATTATTGCATTGTCTTGTTACCAGAAACAAATTTTGCCGAGCTTTTTTTGCCCT2220 
ATATTTCCCAGCATAATTTGATTAGAAAGTACAAAAAGGGCCGGGCGCGGTGGCTTACGC2280 
CTGTAATCCCAGCACTTTGGGAGGCCAGGGCGGGTGGATC ACGAGGTCAGGAGATCGGGA2340 
CCATCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAAAAAAAAAAAAA2392 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 345 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii ) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetProGluPheLeuGluAspProSerValLeuThrLysAspLysLeu 
151015 
LysSerGluLeuValAlaAsnAsnValThrLeuPro AlaGlyGluGln 
202530 
ArgLysAspValTyrValGlnLeuTyrLeuGlnHisLeuThrAlaArg 
354045 
Asn ArgProProLeuProAlaGlyThrAsnSerLysGlyProProAsp 
505560 
PheSerSerAspGluGluArgGluProThrProValLeuGlySerGly 
657 07580 
AlaAlaAlaAlaGlyArgSerArgAlaAlaValGlyArgLysAlaThr 
859095 
LysLysThrAspLysProArg GlnGluAspLysAspAspLeuAspVal 
100105110 
ThrGluLeuThrAsnGluAspLeuLeuAspGlnLeuValLysTyrGly 
115120 125 
ValAsnProGlyProIleValGlyThrThrArgLysLeuTyrGluLys 
130135140 
LysLeuLeuLysLeuArgGluGlnGlyThrGluSerArgSerSerThr 
145150155160 
ProLeuProThrIleSerSerSerAlaGluAsnThrArgGlnAsnGly 
165170175 
Se rAsnAspSerAspArgTyrSerAspAsnGluGluAspSerLysIle 
180185190 
GluLeuLysLeuGluLysArgGluProLeuLysGlyArgAlaLysThr 
195 200205 
ProValThrLeuLysGlnArgArgValGluHisAsnGlnValGlyGlu 
210215220 
LysThrGluGluArgArgValGluArgAsp IleLeuLysGluMetPhe 
225230235240 
ProTyrGluAlaSerThrProThrGlyIleSerAlaSerCysArgArg 
245250 255 
ProIleLysGlyAlaAlaGlyArgProLeuGluLeuSerAspPheArg 
260265270 
MetGluGluSerPheSerSerLysTyrValProLysTyr ValProLeu 
275280285 
AlaAspValLysSerGluLysThrLysLysGlyArgSerIleProVal 
290295300 
TrpIleLysIl eLeuLeuPheValValValAlaValPheLeuPheLeu 
305310315320 
ValTyrGlnAlaMetGluThrAsnGlnValAsnProPheSerAsnPhe 
325330335 
LeuHisValAspProArgLysSerAsn 
340345 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ArgLysAspValTyr 
15 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 95 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GGGAATTCGCCGCCGAGATGCCGGAGTTCCTGGAAGACCCCTCGGTCCTGACGAAAGAGA60 
AGTTGAAGAGTGAGTTGGTCGCCAACAATGTGACG95 
(2) INFORMATION FOR SEQ ID NO:9: 
(i ) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 95 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GGGAATTCAGCGCTTCAGGGCCGTCAGGTGCTGCAGGTAGAGCTGCACATACACGTCTTT60 
GCGCTGCTCC CCGGCCGGGAGCGTCACATTGTTGG95 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TCTATCAAGCTATGGAAACCAACCAAGTAAATCCCTTCTCTAATT45 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: cDNA 
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CATTCAGTTGGATTTTCTAGGGTCAACATGAAGAGAATTAGAGAAGGGAT50