JPH0348696A - Human lymphocyte surface antigen and gene coding the same - Google Patents

Abstract

NEW MATERIAL:A human lymphocyte surface antigen having an aminoacid sequence of the formula and the same active substance. USE:A reagent for researching autoimmune disease patients and a material for preparing a treating agent of the autoimmune diseases, cancer, infectious diseases, etc. PREPARATION:For example, a monocytic leukemia cell is cultured in a CO2 incubator, treated with a lithium chloride/guanidine thiocyanate solution, homogenized, layered on a cesium chloride solution and subsequently centrifuged to separate RNA, which is treated with an oligo dT cellulose column to prepare poly A<+> RNA. The poly A<+>RNA is used to prepare cDNA with a reverse transcriptase and further prepare cDNA library by a conventional method. The cDNA library is screened with a probe to select a DNA coding a human lymphocyte surface antigen, which is treating by a conventional method to prepare a manifestation vector. The vector is introduced into a host, transformed and cultured to provide the human lymphocyte surface antigen of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a purified protein constituting a human lymphocyte surface antigen and a gene encoding the same. More specifically, the present invention provides a purified human lymphocyte antigen corresponding to the mouse lymphocyte antigen Ly6 and a gene encoding the same.

[Prior art] Ly6 antigen is a surface antigen found on mouse lymphocytes, and is mainly present on the surface of 1928 cells, NK cells, monocytes, etc., in the form of a (PI anchoring protein) anchored to the cell membrane with phosphatidylinositol. exists in Ly6
is strongly involved in the activation of lymphocytes, and the monoclonal antibody against L)/6 activates the function of mouse lymphocytes in some cases alone, and in other cases by performing treatments such as crosslinking.

On the other hand, in flow cytometer analysis using antibodies against L/6 antigen, for example, MRL/α mice (MR
In autoimmune disease model mice such as L/Mp-4, r/Q, ), the number of L16-positive cells and L per cell
It is known that the number of y6 antigens is significantly increased.

The Ly6 antigen in mice forms a family consisting of several species, and the existence of subtypes with slightly different sequences depending on the genetic lineage of the mouse has been reported. However, the similarity (homology) between them is quite large.

From the above, it can be seen that Ly6 is strongly involved in at least one of the mechanisms of lymphocyte activation, and that the number of Ly6-positive cells increases as a phenomenon associated with autoimmune diseases. Therefore, a more detailed study of Ly6 itself and the mechanism of lymphocyte activation involved in it will lead to the development of anticancer and anti-infective drugs that utilize lymphocyte activation, while suppressing Ly6 function. It is hoped that this will lead to the development of immunosuppressive drugs.

However, a human lymphocyte antigen corresponding to the mouse lymphocyte Ly6 antigen has not yet been identified. Attempts at cloning by hybridization of the corresponding human gene using the mouse gene as a probe, which is a common technique in such cases, have not been successful.

The present inventors first found that monoclonal antibody F4, which was selected from antibodies against mouse LGL (large granular lymphocytes) that the present inventors had independently created, recognized the mouse lymphocyte antigen Ly6 through immunochemical analysis. We were confident that it was an antibody. This was later confirmed by the Br1an 5eed method (
Brian 5eed, Nature, 329°8
40-842 (1987); Br1an 5eed
et al., Proc, Natl.

Acad, Sci, LISA, 84.3365-
3369 (1987)), the cDNA cloned by the present inventors using the F4 antibody as a foothold was
This was confirmed by proving that it is the known Ly6 (Ly6C1) gene.

Therefore, as a first step, the present inventors used the above-mentioned F4 antibody to analyze human peripheral blood lymphocytes and various human leukemia cell lines using a flow cytometer. showed no specific binding.

This revealed that the F4 antibody was not a clue for cloning the human lymphocyte antigen corresponding to mouse Ly6.

At the same time, the present inventors analyzed the mRNA of human peripheral blood lymphocytes and various human leukemia cell lines by the Northern method using the mouse Ly6C1 cDNA cloned by the present inventors as a probe, but no specific positive signal was observed. I couldn't. At the same time, cDNA libraries of human peripheral blood lymphocytes and several human leukemia cell lines were screened by hybridization using the radioisotope-labeled mouse Ly6C2 cDNA as a probe, but no specific positive clones were obtained. Ta. From this, in the hybridization method using mouse L16 cDNA as a probe,
Cloning the human lymphocyte antigen corresponding to mouse Ly6 has proven difficult or impossible.

On the other hand, there is a suggestion that MEM43, one of the monoclonal antibodies against human lymphocytes, may recognize a lymphocyte antigen corresponding to mouse Ly6, based on the behavior of the cells and antigens it recognizes (Stefanova, 1
．． et al., Molecular 1mmunology,
26°153-161 (1989)).

Furthermore, the cell extract fraction of human lymphocytes was added to MEM43.
It has been reported that a homogeneous purified protein can be obtained by purification using an antibody, and that the sequence of the N-terminal 17 amino acids of the protein is LQCYNCPNPTADCKTAV (the above-mentioned document).

Recently, there has been a proposal to name the antigen recognized by MEM43 antibody CD59 (4th International
ional Workshop and Conference
ence on Iluman Leukocyte
Differentiatlon, and Stepha
nShaw, Nature, 338.539-54
0 (1989)). These research results merely suggest that the antigen recognized by the MEM43 antibody may be a human lymphocyte antigen corresponding to mouse Ly6;
Specification of the human antigen recognized by the EM43 antibody is extremely insufficient.

[Problems to be Solved by the Invention] In view of this situation, the present invention aims to obtain a purified human lymphocyte antigen protein corresponding to mouse LV6 and a gene encoding the same.

[Means for Solving the Problems] The present inventors obtained the entire gene sequence by cloning the gene based on the N-terminal amino acid sequence of the antigenic protein recognized by the MEM43 antibody shown in FIG. 3(a). At the same time, by clarifying the entire amino acid sequence, we clarified the complete nature of the protein, and this revealed that this protein is mouse Ly6
It was confirmed that the antigen corresponds to a human lymphocyte antigen, and thus the present invention was completed. That is, the present invention provides a human lymphocyte surface antigen having the amino acid sequence shown in FIG. 1, its equivalent, and a gene encoding the antigen.

An example of the gene sequence of the present invention is shown in FIG.

The present invention will be explained in detail below.

DNA oligomers as probes and primers are shown in FIGS. 3(b), (C), (d) based on FIG. 3(a).
) Three types of Ly61, Ly2, and Ly3 shown in (1) were synthesized. Ly61 is mRNA encoding the amino acid sequence
This is the sequence on the A chain side, and Ly2 and Ly3 were synthesized as sequences complementary to mRNA. In the figure, ■ indicates deoxyinosine, and the complementary strand of ■ is I. Two types of bases arranged vertically were synthesized as a mixture.

Next, various human lymphoid cells were cultured, and an RNA fraction containing mRNA was extracted using a conventional method.

The cells used are as follows.

Human peripheral blood lymphocyte fraction was stimulated with LPS (lipopolysaccharide), human tonsil lymphocytes were stimulated with ConA
(concanavalin A), human tonsil lymphocytes stimulated with TPA/PHA, human monocytic leukemia cell line J111, human promyelocytic leukemia cell line HL6
0, human acute myeloid leukemia cell line KGI, human promyelocytic leukemia cell line ML 3 (Minowada, J.
rLeukemiaJ (Gunz, F. &Hend
pI), 119-139.
Grune & 5tratton.

New York (1982)), human T-cell leukemia cell line RPM I 8402 (Sahai 5r1v)
astere, J., Nat., Canecr In.
5t, 55.11-14 (1975)), the human lymphocytic leukemia cell line Molt4, and the tonsillar lymphocyte cell line BecII. Among the above cell lines, Jllll,
HL60, KGI, Molt4 and BecII are A
It can be done manually by TCC.

After electrophoresing the obtained RNA in a formalin-containing RNA gel, the RNA was transferred to a nylon membrane, and after fixation, Figure 3 (C)
Northern analysis was performed using radioisotope-labeled DNA oligos 7-Ly2 and Ly3 of (d) as probes. Human peripheral blood lymphocyte fraction and Jl
The positive signal was about 900 bases long and 1
It was observed at 300 base length and 1800 base length. Therefore, these two types of cells are Ly2 and Ly3D
It was found that mRNA with a sequence complementary to the NA oligomer was synthesized, but since the positive signal was weak, the amount was compared to normal mRNA, such as β-actin mRNA.
It was estimated that the amount was extremely small compared to NA.

First, by the Br1an 5eed method (mentioned above),
Derived from the above two types of cells (peripheral blood lymphocytes and Jllll)
A cDNA library was constructed using the mRNA of . The size of the library (library size) was approximately 50,000 to 100,000. These libraries (cD
The plasmid with NA attached to the vector entered the host E. coli) was plated on an agar medium and screened by colony hybridization using oligomers Ly2 and Ly3 as probes in the usual manner, but no specific positive clones were found. was not obtained. It was strongly assumed that the reason was that the target clone was not included in the cDNA library because the target mRNA was too small compared to the size of the library.

Therefore, they decided to apply a unique modification to the PCR (polymerase chain reaction) technique. Common references for PCR include Erlich, H.A., et al.

Nature, 33L 481-462 (1988
).

PCR usually requires a clear, known short (15 to 20 bases) sequence separated by a certain distance (the length of the gene), but for the gene of the target protein of the present invention, such a The sequence is unknown, only the N-terminal amino acid sequence (17 amino acids) is known.

Therefore, the present inventors made efforts to use oligomer Ly61 shown in FIG. 3(b), which is a mixture of multiple oligomers, as the first primer in the PCR reaction, and oligomer dT consisting of 15 bases as the second primer. there was.

Taq polymerase and four types of substrates (A.

T, G, C) Using deoxynucleoside triphosphate, double-stranded cDNA synthesized in a conventional manner from 2 μg of mRNA derived from the above two types of cells (LPS-stimulated peripheral blood lymphocytes and 7111) as a template. Thermal denaturation and
40 cycles of annealing and chain extension reactions were performed.

When this reaction mixture was subjected to agarose gel electrophoresis,
No clear band was observed in ethidium bromide staining, and it was unclear which band was specific, but after transfer to a nylon membrane, Southern hybridization was performed using the labeled Ly2 oligomer as a probe.
A clear positive band was observed around about 400 bp. Therefore, this reaction mixture was once reacted with DNA polymerase Flenow fragment to blunt the ends, and then
Adapter (DN
Two A oligomers) were combined and partitioned by ultracentrifugation in a KOAc concentration gradient. The divided fractions were analyzed by agarose gel electrophoresis and Southern hybridization, and the fractions positive with the labeled Ly2 oligomer probe (
fraction) was connected to the CDM8 vector, and E. coli MC106
1/P3 was transformed.

Approximately 120,000 transformants (derived from peripheral blood lymphocytes) and approximately 170,000 (derived from J111) thus obtained were transferred to the Ly
2 oligomer as a probe by a conventional colony hybridization method, two positive clones derived from peripheral blood lymphocytes and four positive clones derived from 1111 were obtained. Among them, one clone, Pl, was selected, and its DNA was obtained by a conventional method and the insert was examined, and it was found to be approximately 350 bp. This insert was excised and purified, this DNA fragment was labeled with a radioisotope using the nick translation method, and the differences with other positive clones were examined by hybridization. Since these clones combined with other positive clones, these clones It was estimated that all of them had almost the same sequence.

Therefore, the DNA base sequence of the clone P1 was analyzed. The obtained base sequence is shown in FIG. In FIG. 2, a portion of the vector and a portion of the adapter of the CDM8 are not shown, but only the insert portion is shown.

When the DNA base sequence shown in Figure 2 was translated into an amino acid sequence in both directions using three frames each, an open reading frame was created in which only the first frame in the direction shown in Figure 2 was long. The amino acid sequence is shown in FIG. 4 together with the DNA base sequence. The first 16 amino acids are the 3rd
17 N-terminal sequences of human lymphocyte-derived proteins purified and determined based on MEM43 shown in Figure (a)
Of the amino acids, positions 2 to 17 were exactly the same. Therefore, it can be concluded that the amino acid sequences shown in FIGS. 1 and 4 are the amino acid sequences of proteins recognized by MEM43. Due to the synthesis of the primer (oligomer Ly61) used in the PCR reaction, the DNA sequence in Figure 4 begins with the second Q (glutamine).
It is clear from the above that L (leucine) is placed in front of it, so L is added to the beginning of FIG. 4 (before Q).

It is concluded from several facts described below that the protein having the amino acid sequence shown in FIGS. 1 and 4 is a human lymphocyte antigen corresponding to the mouse Ly6 antigen.

(2) The N-terminal sequence completely matches the N-terminal sequence of the protein purified based on MEM43. From immunochemical cell and antigen analysis using MEM43 antibody, ME
It has been suggested that the antigen recognized by the M43 antibody is likely to be a human lymphocyte antigen corresponding to mouse Ly6.

■ There is approximately 25% similarity (mature peptide portion) with the amino acid sequence of mouse Ly6C2a shown in Figure 5, especially in the N-terminal and central portions.The size is also approximately the same. The fact that there is only 25% homology explains well why the human gene could not be identified using mouse cDNA as a probe.The amino acid sequence of the present invention was aligned with that of mouse Ly6C2a, and maximal matching was performed. The result is shown in Figure 5.

The gene sequence of mouse Ly6C2a is available from the gene sequence data bank LA S L -Ge of Los Alamos Laboratory in the United States.
n Sequence registered in Bank (ID name: MLISLY
6C2A) and translated it into an amino acid sequence.

The amino acid sequence shown at the bottom of Figure 5 represents the mature protein and begins with L (leucine). Mouse Ly
The original report on the gene sequence of 6C2a is available in Pa1free, R.
Journal of Ia+mun by , G, E, et al.
ology, 140.305-310 (1988)
It is.

As shown in FIG. 5, the amino acid sequence of the present invention shows 33% similarity with the amino acid sequence of mouse Ly6C2a when L (leucine) is added to the N-terminus. Furthermore, the number and arrangement of C (cysteine) are almost exactly the same, which strongly suggests that both proteins are analogs with the same function.

Furthermore, in the amino acid composition of the human protein of the present invention,
The sum of D (aspartic acid) and E (glutamic acid) is 11, and the sum of K (lysine), R (arginine), and H (histamine) is eight. On the other hand, in mouse Ly6C2a, the sum of D and E is 10, and the sum of K, R, and H is 9, and the ratios of acidic and basic amino acids are very similar.

In addition, both have very similar numbers of amino acids and molecular weights.

■ The protein amino acid sequence of the present invention is
Fig. 6 shows the hydrophobicity distribution by the method, and Fig. 7 shows that of the mouse L''/6C2a.

As can be seen from Figures 6 and 7, they are very similar except for a part of the central part, and in particular, the C-terminal part has a strong hydrophobic part in common, which is phosphatidyl. It is strongly shown that it is an inositol anchored protein (PI anchoring protein). Mouse L
This has been demonstrated for y6C2a, and it is therefore strongly suggested that the protein of the present invention is also a lymphocyte surface antigen. This hydrophobicity/! 11Aqueous analysis:
The GENETYX program of software development ■ was used.

From the above, it is concluded that the protein of the present invention and its gene are human lymphocyte surface antigens corresponding to the mouse lymphocyte surface antigen Ly6.

The present invention involves the complete characterization of a surface antigen (designated CD59) corresponding to murine Ly6 on human lymphocytes, and therefore can be defined as an important invention that will form the basis for future development studies. . In mouse Ly6, a family containing subtypes with slight sequence differences has been identified, and therefore, it is natural to expect that such a family will be identified in humans as well, and the gene sequence of the present invention is a promising candidate for this purpose. It is a means.

Therefore, it goes without saying that the present invention also includes the gene sequences and amino acid sequences of these human CD59 families, ie, those with the same efficacy.

For example, the protein of the present invention has about 20 hydrophobic amino acids lined up at its C-terminus, which is an element for it to be a cell membrane surface protein. If it is artificially prepared using techniques such as genetic recombination technology, it may be possible to suppress lymphocyte activation mediated by the human CD59 antigen.

Such application development is a technology that is made possible by the protein described by the present invention and, above all, by its gene, and therefore naturally falls within the scope of the present invention.

Therefore, although the human lymphocyte surface antigen of the present invention has the amino acid sequence shown in FIG. Included in the present invention.

Furthermore, the lymphocyte surface antigen shown in the present invention is mouse L
In the case of y6, it is known that it is involved in lymphocyte activation and is strongly related to autoimmune diseases, so the present invention aims to scientifically elucidate the function of the CD59 antigen in humans and to develop it industrially. It provides the most important material for use.

In other words, if antibodies (including monoclonal antibodies) are created based on the protein of the present invention, it can be understood that the antibodies will play an important role in the study of lymphocyte function, and at the same time, the antibodies can be used to investigate autoimmune diseases. It is useful in diagnosing immune overresponses such as

In addition, if the antibody activates human lymphocytes, the antibody can be applied to anti-cancer or anti-infectious disease treatment, and if the antibody suppresses the function of human lymphocytes In addition, the antibody can also be applied to the treatment of immune hyperresponse diseases (autoimmune diseases).

[Examples] The present invention will be explained in more detail below with reference to Examples.

In the examples below, some of the methods used are conventional and their experimental formulations are detailed in the apparatus shown below.

Au5ubcl, F. M. et al., 'Curren
tProtocols inMolecular B
iology"John Wiley & 5ons,
1987 and Maniati S, T. et al.
'Mo1e(Lllar Cloning: A La
boratory ManuaBiCol1d Sprin
g Harbor Laboratory, 1982.

Hereinafter, in the examples, the above-mentioned device will be referred to as the bottom holder.

Example 1 Preparation of mRNA The cell line Jllll (human monocytic leukemia cells) was cultured in Dulbecco's MEM medium containing 10% tallow serum (Lasui). Cell line HL60 (human promyelocytic leukemia cell line), KGI (human acute myeloid leukemia cell line), ML3 (human promyelocytic leukemia cell line), RPMI
8402 (human T cell leukemia cells), Mo1t4 (
human lymphoblastic leukemia cells), and BecII (
The tonsil lymphocytes were cultured in RP M11640 medium containing 10% tallow serum and 2-mercaptoethanol at a final concentration of 5 x 10-5 M (Rasui).

The human peripheral blood lymphocyte fraction is obtained by collecting blood from a normal person, collecting the fee coat fraction cells by centrifugation, which are obtained by a conventional method including centrifugation in a heparin-treated centrifuge tube, and collecting the cells by centrifugation.
Suspended in PM11640 medium, 10 μg/ml LPS
was added and cultured for 15 hours. Human tonsil lymphocytes were collected using a conventional method, suspended in RPMI 1640 medium, and treated with ConA (10 μg/ml) or TP.
A: Add PHA (5mg/ml: 1%),
It was cultured for 20 hours.

All cell cultures were performed in a CO2 incubator. A lithium chloride/guanidine thiocyanate solution (50 g of guanidine thiocyanate dissolved in 58 ml of 25% lithium chloride solution, passed through a 0.45μ filter, and then 2 ml of 2-mercaptoethanol added) was added to the cells; The resulting viscous solution was subjected to a polytron twice for 40 seconds to disrupt the chromosomal DNA.

This cell homogenate was layered on 1/3 volume of 5.7M cesium chloride (containing 100mM EDTA) solution,
Centrifugation was performed at 35.00 rpm for 20 hours at 20°C. The total RNA precipitated at the bottom of the centrifuge tube was diluted with a small amount of lithium chloride/
It was dissolved in a guanidine thiocyanate solution, 1/10 volume of 3M sodium acetate solution and 2.5 volumes of ethanol were added, and the mixture was allowed to stand overnight at -20°C to cause precipitation.

Human peripheral blood lymphocytes, human tonsils, Jllll, ML3, K
For GI and HL60, the total RNA was then subjected to oligo dT cellulose column chromatography according to a conventional method to select and collect polyA RNA.

The number of cells used in the experiment is as follows.

Jlll: 3X108 cells, human peripheral blood lymphocytes: 1.
2x109 cells, human tonsil: 5x108 cells, ML3
: 1.4×109 cells, KGI: 2×108 cells, H
L60: 3X10B cells, RPMI8402: 4
X107 cells, Molt4: 4 x 107 cells, Bec
II-5X107 cells.

Example 2 Detection of human Ly6 mRNA (A) Synthesis of DNA probe Figure 3 (a) Based on the N-terminal amino acid sequence of the antigen protein recognized by the MEM43 antibody shown above, Figure 3 (
Three types of oligonucleotides (oligomers) Ly61, Ly2, and Ly3 shown in b) to (d) were synthesized. Ly
2.Ly3 shows the sequence of its complementary strand.

Therefore, the sequence of the DNA oligomer actually synthesized and used is as follows.

L V 2: ACIGCIGTTTTACAA
TCIGCIGTIGGATTCGG
G As a probe for Northern hybridization, L
y2 and Ly3 were used. 5′ hydroxyl group with T4 kinase and γ
(“2P) Phosphorylation labeling using ATP.

(B) Northern hybridization Total RNA (10 to 20 μg) or poly ARNA (2 to 5 μg) obtained in Example 1 was electrophoresed on a 1% agarose gel containing formalin, and then transferred to a nylon membrane (Amarjam “Bihonto N”). After transferring, it was fixed with ultraviolet light (2 minutes and 30 seconds) with the surface in contact with the gel facing down. After prehybridization was performed at 42°C for 1 hour, hybridization was also performed at 42°C overnight. Washing was also carried out at 42°C. The composition of the hybridization solution was 5 x
S S P E, 5 XDenhardt's,
0. 1% SDS.

100 μg/ml salmon sperm DNA (heat denatured), washing solution is 2×SSC, 0.1% SDS. These methods were carried out in accordance with the above-mentioned method.

As a result of autoradiography, positive bands were observed at approximately 900 base length, 1300 base length, and 1800 base length in polyA RNA of J111 cells using both Ly2 and Ly3 probes.

Although the signal was very weak compared to J111 cells, it was also observed in polyA RNA of human peripheral blood lymphocytes.

It could not be detected in other cells.

Example 3 Preparation of eDNA library PolyA prepared from Jllll and human peripheral blood lymphocytes
Using 5 μg of RNA, seed
A cDNA library was prepared by the method of N. 5eed (mentioned above). Double-stranded cDNA synthesis or cD
NA synthesis kit [Amarjam (A [Ilersham]
), and the procedure was carried out according to its protocol.

A phosphorylated adapter (12mer: CTTTAG) was added to the double-stranded cDNA using T4 DNA ligase.
AGCACA and 8[l1erCTCTAAAG) were ligated. To remove excess adapters and short cDNAs, perform 5-20% calcium acetate density gradient centrifugation (50%
,00Orpm.

(3 hours at 22°C). A syringe needle was inserted into the bottom of the centrifuge tube, and 0.4 ml fractions were fractionated. C the fractions up to the 6th
DM8 vector (Br1an 5eed mentioned above)
Bst) (1 site, T 4 DNA IJ was ligated with Garsenyori. The size of the cDNA library obtained by transforming Escherichia coli MC1061/P3 strain (mentioned above) using a conventional method is as follows: It was about 100,000, and about 50,000 for peripheral blood lymphocytes.

These libraries are Ly2 and L created in Example 2.
Screening was performed by colony hybridization using the y3 DNA probe. Recombinant E. coli was plated so that approximately 2,000 colonies appeared per plate. Two replica plates were made for each plate. 5 colonies on replica plate
After transferring to No. 41 paper (manufactured by Whatmann), the plasmid DNA was fixed on the filter by performing alkali denaturation, neutralization, and washing operations. Prehybridization was performed at 42°C for 1 to several hours, followed by hybridization at 42°C overnight. Washing was also carried out at 42°C. These operations were performed in accordance with the above-mentioned scribe.

Autoradiography results, Ly2 probe, Ly
It was found that all three probes had strong non-specific binding.

About 200,000 clones were screened for the J111 library, and about 50,000 clones were screened for the human peripheral blood lymphocyte library, but no specific positive clones were obtained.

Example 4 Amplification of human Ly6c DNA by PCR method A cDNA synthesis kit (
(described above) to synthesize double-stranded cDNA. synthesized c
1/3ffi of DNA (double-stranded cDNA of 100 ng or more)
A) was used in one PCR (polymerase extension chain reaction, previously mentioned) experiment. Oligomer Ly61 in Figure 3(b) was used as the first primer in the PCR reaction, and oligomer dT (
15mer) was used. The reaction solution composition is as follows.

Heat denaturation at 94°C for 1 minute using a DNA thermal cycler-PJlooO (PerkinElmer Cetus)
The reaction was carried out for 40 cycles under the conditions of annealing at 40°C for 2 minutes and chain extension reaction at 72°C for 3 minutes.

When 115 volumes of this reaction mixture were subjected to 1% agarose gel electrophoresis, no clear band was observed in ethidium bromide staining. After the gel was denatured twice with alkaline for 30 minutes and neutralized for 15 minutes, a nylon membrane [Amasham, 1lyb
ond N)) and fixed with ultraviolet light (2
minutes and 30 seconds). Southern hybridization was performed using the Ly2 oligomer synthesized in Example 2 as a probe. This operation was carried out in accordance with the above-mentioned transcription. Prehybridization was performed at 42°C for 1 to several hours, followed by hybridization at 42°C overnight.

Washing was also carried out at 42°C. As a result of autoradiography, a clear band was observed around a length of about 400 bases.

The remaining 415 volumes of the reaction mixture was treated with phenol:chloroform (1:1) twice and with chloroform once, and then subjected to ethanol precipitation to recover DNA. This DN
When a further 40 cycles of reaction were carried out using 115 amounts of A and the same reaction composition and reaction conditions as above, an increase in the number of positive bands was observed in Southern hybridization.

Example 5 Cloning of cDNA that intersects with Ly2 probe
7 The reaction mixture obtained in Example 4 (40 cycles twice) was treated with phenol:chloroform (1:1) twice and chloroform once, and then subjected to ethanol precipitation to recover DNA. DNA polymerase (Flenow fragment) treatment was performed at 22° C. for 30 minutes to blunt the ends.

The same phosphorylated adapter used in Example 3 was used with T
4 DNA ligase (16°C, overnight). Potassium acetate density gradient ultracentrifugation (described above) was performed to remove excess adapters and primers used in the PCR reaction. 0.4 ml portions were distributed into 12 fractions as described above. 1/10 amount of each was subjected to 1% agarose gel electrophoresis, and Southern hybridization (described above) was performed using labeled Ly2 oligomer as a probe. As a result of autoradiography, a positive band (approximately 4
00 base length) was observed.

DNA was extracted from the 8th fraction by ethanol precipitation.
This DNA was transferred to B of the CDM8 vector (described above).
The stX1 site was ligated with T4 DNA ligase (1
6°C, -overnight). When Escherichia coli MCl061/P3 (described above) was transformed using the usual method, in the case of Jlll, approximately 1
20,000 transformants were obtained, approximately 17 in the case of peripheral blood lymphocytes.

For each of about 2,500 libraries, the above-mentioned L
Screening using the colony hybridization method (mentioned above) using the y2 oligomer as a probe revealed that 4 oligomers derived from J111 (J-2, J-6, J-7, J-9
), two positive clones (P-1, P-2) derived from peripheral blood lymphocytes were obtained. Among them, one clone P-1 was selected and its DNA was obtained according to the procedure described above. this DNA
was cut with Xho I and the length of the insert cDNA was determined to be approximately 350 bases long. Cut out this insert and clean the scene [Bio No. 101 (Bio
101)). The DNA fragment was labeled with α(32P)CTP using the nick translation method. This operation was performed in accordance with the above-mentioned bottom placement. This labeled DNA fragment was used as a probe for hybridization (prehybridization, hybridization at 65°C).
When examined for differences with other positive clones, it was found that the clones combined with other positive clones. Therefore, it was estimated that all these clones had almost the same sequence.

Example 6 Determination of base sequence of cDNA clone CDM8 vector sea fence primer (5'
TAATACGACTCACTATA3'5' CT
Sequence analysis was performed using the dideoxy method using deoxy-7-diazaguanine triphosphate.

The obtained base sequence is shown in FIG. Following the sequence of the Ly61 primer used in the PCR reaction, there is a sequence corresponding to the Ly2 probe, and these are among the base sequences (Figure 3 (a)) deduced from the N-terminal amino acid sequence of the antigen protein recognized by MEM43. It was consistent with one of the following.

Furthermore, the translated amino acid sequence corresponding to the base sequence was as shown in FIG. 1, and 16 amino acids from glutamic acid to valine were completely identical.

[Effects of the Invention] By using the cDNA obtained by the present invention as a probe, it is possible to determine whether a family consisting of similar genes exists as in mice, how it is related to lymphocyte activation, and whether autoimmunity It is expected that important progress will be made in research on the human immune system, such as whether it is related to immune overload in diseases. At the same time, the human lymphocyte antigen of the present invention can be suppressed to produce a therapeutic agent for autoimmune diseases, or activated to produce an anticancer drug or an anti-infective drug. It is hoped that this will lead to development. In other words, it can be said that the present invention greatly opens up possibilities for research and industrial application of human lymphocyte antigens corresponding to mouse Ly6.

[Brief explanation of drawings]

FIG. 1 shows the amino acid sequence of the human lymphocyte surface antigen of the present invention, and FIG. 2 shows an example of the gene sequence encoding the same. Figure 3 (a) shows the N-terminal amino acid sequence of the antigenic protein recognized by the MEM43 antibody and the DNA sequence that can specify this amino acid sequence, and Figures 3 (b), (c), (d)
) are the synthetic oligomers Ly61 and Ly2 used in the present invention.
, shows the sequence of Ly3. Those arranged vertically indicate a mixture of nucleobases, and ■ indicates deoxyinosine. FIG. 4 shows the amino acid sequence of the human lymphocyte surface antigen of the present invention, together with its gene sequence. FIG. 5 shows the amino acid sequence of the present invention and mouse Ly6C2a
This is the maximum matching of the amino acid sequences of In the figure, the * mark indicates a matching amino acid sequence, and the ten mark indicates cysteine. FIG. 6 shows the hydrophobicity distribution of the amino acid sequence of the present invention by the Doo l i method, and FIG. 7 shows the hydrophobicity distribution of mouse 6C2a by the same method.

Claims (2)

[Claims] (1) A human lymphocyte surface antigen having the following amino acid sequence and its equivalent. Leu_1GlnCysTyrAsnCysProAs
nProT_1h_0rAlaAspCysLysTh
rAlaValAsnCysS_2e_0rSerAs
pPheAspAlaCysLeuIleThrL_3
y_0sAlaGlyLeuGlnValTyrAsn
LysCysT_4r_0pLysPheGluHis
CysAsnPheAsnAspV_5a_0lThr
ThrArgLeuArgGluAsnGluLeuT
_6h_0rTyrTyrCysCysLysLysA
spLeuCysA_7s_0nPheAsnGluG
lnLeuGluAsnGlyGlyT_8h_0rS
erLeuSerGluLysThrValLeuLe
uL_9e_0uValThrProPheLeuAl
aAlaAlaTrpS_1e_0r_0LeuHis
P_1r_0o_3 (2) A gene encoding the human lymphocyte surface antigen or its equivalent according to claim (1). (3) The gene according to claim (2), which has the following base sequence. CAGTGCTAC^1A^0ACTGTCCTA^2
A^0CCCAAACTGC^3T^0GACTGCAA
A^4A^0CAGCCGTCA^5A^0TTGTT
CATC^6T^0GATTTTGAT^7G^0CG
TGTCTCA^8T^0TACCAAAGC^9T^
0GGGTTAAC^1A^0G^0TGTATAAC
^1A^1A^0GTGTTGGA^1A^2G^0T
TTGAGCA^1T^3T^0GCAATTTC^1
A^4A^0CGACGTCA^1C^5A^0ACC
CGCTT^1G^6A^0GGGAAAT^1G^
7A^0GCTAACGT^1A^8C^0TACTG
CTG^1C^9A^0AGAAGGAC^2C^0T
^0GTGTAACT^2T^1T^0AACGAAC
A^2G^2C^0TTGAAAAT^2G^3G^0
TGGGACAT^2C^4C^0TTATCAGA^
2G^5^A0AAACAGTT^2C^6T^0TC
TGCTGG^2T^7G^0ACTCCATT^2T
^8C^0TGGCAGCA^2G^9C^0CTGG
AGCC^3T^0T^0CATCCCTA^3A^1
G^0TCAACACC^3A^2G^0GAGAGC
TT^3C^3T^0CCCAAA^3^4^0