JPH03112486A - Hla-b35 gene and dna probe, and transformant cell - Google Patents

Abstract

PURPOSE:To provide the title gene having exon expressed by a specific base sequence, useful for the research, assay and diagnosis of HLA-B35 alloantigen. CONSTITUTION:The objective HLA-B35 gene having exon expressed by a base sequence of the formula (where the description of bases from 521th to 1089th are omitted) made up of 1089 bases (A is adenine; C is cytosine; G is guanine; T is thymine). When labelled with a marker, cDNA coinciding with part of the base sequence of the present HLA-B35 gene can be applied to DNA probe to be used for DNA typing. Said marker is e.g. peroxidase, iron porphyrin derivative. Moreover, since the HLA-B35 gene is capable of coding HLA-B35 antigen, transformant cells capable of manifesting HLA-B35 antigen can be obtained by transducing this gene into eucaryote cells.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to the HLA-835 gene, DNA probe, and transformed cells, and particularly relates to HLA-835 gene, DNA probe, and transformed cells.
n 1 eukocyte antigen) -835
DNA encoding antigen allotypes and patient class in DNA typing DNA probes used in extracting and identifying the gene itself and D of the present invention
The present invention relates to transformed cells that express an allotype of HLA-835 antigen on the cell surface by introducing NA into human and non-human eukaryotic cells. Cells expressing the HLA-B35 alloantigen can be administered to animals as an immunogen and are therefore useful for developing monoclonal antibodies specific to the HLA-B35 antigen. Furthermore, cells expressing the HLA-B35 antigen by introducing the gene of the present invention into human eukaryotic cells are useful as panel cells used for determining the specificity of antisera.

[Conventional technology]

During human organ transplantation, HLA antigen plays an important role in transplant immunity as an antigen that determines whether or not the graft will colonize. Among these, class I antigens are involved in antigen recognition by cytotoxic T cells in cell-mediated immunity, as well as in herpes vulgaris (CW6), herpes vulgaris (AIO), herpes 0 (A29), and Behcet's (BS). ?) and other diseases have been revealed. (Ozawa+
A, +0hkido.

io, ↑5ujt, X, J, Am, ^caa, nerm
atot+Sat, 205.1981') HLA antigens are also called major histocompatibility antigens because they are closely related to rejection phenomena and transplant immunity, and the gene group encoding them is the major &U.
It is called the textile compatibility complex (MMC).

HLA is human MMC, and HLA class ■ antigens are membrane proteins present on cell membranes, including p, B, and C.
It is controlled by three genetic loci. class! The antigen has a molecular weight of 44 kd, and the sugar chain (α chain) has a molecular weight of 12 kd.
It has a structure non-covalently associated with the protein (β2-microglobulin), and the α chain is located on the N-terminal side located outside the cell, and the α1. α2. There are three domains of α3, followed by an intracellularly embedded region (transmem
brain region), regions located within cells (cytoplasmic domains) are lined up. β2-microglobulin consists of one domain and is linked to the α chain.

The HLA gene region exists over a distance of 2.5 centimorgans (cM) on the short arm of human chromosome 6, and includes a class II cM region (1,OcM) and a class ■ region (DR-B) from the centromere side. between = 0.7 cM), class I region (
0.8c M).

(Jordan+B, R, et al, Immuno
1, Rev. +84.73.1985) Methods for testing ILA class I antigen include HLA-DNA typ. by serological methods and recombinant DNA technology.
There is ing.

As a serological method, the complement-dependent cytotoxicity test (T
erasaki, P, 1. and McClella
nd, J., and D.

Nature (London), 204. pp9
98-1000.1964) is the standard method.

This method uses alloantiserum and uses rabbit serum as complement to detect lymphocyte cytotoxicity (lymphocytotoxicity).
), but the antiserum is an alloserum from a pregnant woman with multiple births, and the antiserum shows cross-reactivity (cross-reactlon).
class! It is difficult to obtain antibodies that react monospecifically with allotypes, and since antisera are obtained from humans, it is impossible to provide a stable supply of serum with reproducibility. Antisera against alloantigens that are expressed in low amounts are difficult to obtain. Since there is no such thing, it is desired to develop a monoclonal antibody to replace allo serum.

Approximately 150 monoclonal antibodies were submitted at the 1984 International Histocompatibility Workshop.
(Fauchet, R., Bonder, J.G.,
Kennedy, L. J., et al: HL A-
A, B, Monoclonal ant
ibodies, Hist.

coa+patib11ity Testing 19
84 (A1bert+1!-D.

Baur+ M,P,,MayrJ,R,ed,)+
Springer-Verlag.

Berlin, Heidelberg, Nen Yo
rk+Tokyo: 211+1984) Anti-HLA monoclonal antibodies can be produced by fusing mouse myeloma cells with lung cells obtained by immunizing a mouse with -i human lymphocytes. (Oi, V, T,
+Herzenbergi, A.:Ima+unogl
obulin-producing hybrid
cell 1ines.

5 selected methods in IIIlm
unology, 351.1981) However,
To date, monoclonal antibodies suitable for HLA typing - for example, monoclonal antibodies against HLA-B antigen allotypes - 8w4,8
w5゜B7. B8. B13. Currently, only monoclonal antibodies that monospecifically bind to B27 have been obtained, and the development of monoclonal antibodies that have monospecificity to many other allotypes is expected. Furthermore, there are many alloantigens for which even reactive monoclonal antibodies have not yet been obtained. For such alloantigens, monoclonal antibodies that have cross-reactivity with other alloantigens are also effective. Recently, further improvements have been made in methods for producing anti-HLA monoclonal antibodies.

In the antibody production method using peripheral lymphocytes, which are dominated by human T lymphocytes, as an immunogen, many human-derived antigens are present, and therefore, many antigen sites other than the target HLA allotype antigen are produced. This is an improvement in the ability to obtain monoclonal antibodies that show reactivity to the immunized animal, and the transformation is achieved by introducing the HLA antigen gene clone into eukaryotic cells derived from the immunized animal and administering the transformed cells to the immunized animal. This is a method for obtaining lung cells that efficiently produce monoclonal antibodies against human HLA antigens expressed on cells.
(Monoclonal antibodies to
HLA-DP-transfecteds+
use L cell: Proc, Natl, A
cad, sc! ,+USA+83゜pp 3417-
3421.1986) For those for which antisera or monoclonal antibodies specific to HLA class ■ antigen allotypes have not been obtained, DNA typing is an effective testing method. Psoriasis vulgaris is an HLA class ■antigen C.
w5. It is known as a disease that shows a very high correlation with Cw7. (Ozasea et al.: 5olle
recent advances in HL A
and 5kindeases+J, As, Ac
ad, Dermatol, 4+ 205+1981
) Therefore, Ozawa et al.
Sodoyer+R,et al,+complete
nucleotide sequence of a
gene encoding a function
lhuman class I histo
Compatibi order antigen (HLA
-Cw3), EMBOJ, 3:879.1
Using the Pvu II fragment of 984) as a probe, DNA typing was performed on 21 people with Cw6°Cw7 and 16 healthy volunteers.
awa.

^, et al: DN A typing
in psoriasisVu1garis+ Ill
:Proceeding of the 3rd
As1aOceania Histocompatib
llity ga orkshop andConfere
nce+ in press) As a result, the signal sequence, the αl domain and the Pvu domain, which corresponds to a part of the α2 domain.
A patient-specific band was detected when the n 1.7 kb fragment was used as a probe.

[Problem to be solved by the invention]

Conventional techniques have not been able to obtain many isolated HLA genes whose nucleotide sequences have been specified, which has limited the application of HLA antigen allotype analysis and DNA typing. In addition, there were few good immunogens for producing anti-HLA monoclonal antibodies.

The present invention was made in view of these circumstances, and aims to provide a novel HLA-835 gene, a DNA probe, and transformed cells.

[Means and effects for solving the problem] The inventor is HLA
As a result of continuing research on the subject, they succeeded in isolating the HLA-B35 gene and completed the present invention.

The HLA-835 gene of the present invention has an exon represented by the following base sequence.

ATGCGGGTCACGGCGCCCGAACCG
TCCTCCTGCTGCTCTGGGGGGCAGT
GGCCCTGACCGAGACCTGGGCCGGC
TCCC＾CTCCATGAGGTATTTCTACA
CCGCCATGTCCCGGCCCGGCCGCGG
GGAGCCCCGCTTCATCGCAGTGGGC
TACGTGGACGACACCCAGTTCGTGA
GGTTCGACAGCGACGCCGCGAGTCC
GAGGACGGAGCCCCGGGCGCCATGG
ATAGAGCAGGAGGGGCCGGAGTATT
GGGACCGGAACACACAGATCTTCAA
GACCAACACACAGACTTACCGAGAG
AGCCTGCGGAACCTGCGCGGCTACT
ACAACCAGAGCGAGGCCGGGTCTCA
CATCATCCAGAGGATGT-ATGGCTG
CGACCTGGGGCCCGACGGGCGCCTC
CTCCGCGGGGCATGACCAGTCCGCCCT
ACGACGGCAAGGATTACATCGCCCT
GAACGAGGACCTGAGCTCCTGGACC
GCGGCGGACACCGCGGCTCAGATCA
CCCAGCGCAAGTGGGAGGCGGCCCG
TGTGGCGGAGCAGCTGAGGGCCTAC
CTGGAGGGCCTGTGCGTGGAGTGGC
TCCGCAGATAACCTGGAGAACGGGAA
GGAGACGCTGCAGGCCGCGGACCCC
CCAAAGACACAGCTGACCCACCACC
CCGTCTCTGACCATGAGGCCACCCT
GAGGTGCTGGGCCCTGGGCTTCTAC
CCTGCGGAGATCACACTGACCTGGC
AGCGGGATGGCGAGGACCAAAACTCA
GGACACTGAGCTTGTGGAGACCAGA
CCAGCAGGAGATAGAACCTTCCAGA
ACTGGGCAGCTGTGGTGGTGCCTTC
TGGAGAAGAGCAGAGATACACATGC
CATGTACAGCATGAGGGGCTGCCGA
AGCCCCTCACCCTGAGATGGGAGCC
ATCTTCCCAGTCCCACCATCCCCCATC
GTGGGCATTGTTGCTGGCCTGGCTG
TCCTAGCAGTTGTGGTCATCGGAGC
TGTGGTCGCTACTGTGATGTGTAGG
AGGAAGAGCTCAGGTGGAAAAGGAG
GGAGCTACTCTCAGGCTGCGTCCAG
CGACAGTGCCCAGGGCTCTGATGTG
TCTCTCACAGCTTGA In the above base sequence and the base sequence described in Claim 1 of this specification, A is adenine,
C represents cytosine, G represents guanine, and T represents thymine.

A cDNA that matches a part of the base sequence of the HLA-835 gene of the present invention can be labeled with a marker substance and applied to a DNA probe used for DNA typing. Peroxidase is used as a marker substance. Iron porphyrin derivatives, luminescent substances such as luciferase, fluorescent substances, 2.3
- Phosphorescent substances such as butadione and radioactive substances such as 3tp, 'ss3 are commonly used. These marker substances and methods for labeling single-stranded DNA with the marker substances are known. (^nnualReview Biop
hys, Bioeng, pp175-195. (19
81).

Rigby et al., J. Mo1. Biol...
113,237. (1977)) Since this HLA-B35 gene encodes the HLA-835 antigen, transformed cells expressing the HLA-B35 antigen can be obtained by introducing it into eukaryotic cells.

〔Example〕

The present invention will be explained based on examples.

Isolation of chromosomal DNA of HLA-835 HLA-A24/A26. B35/Bw52゜C-/C
-, 5 x 10' Japanese peripheral blood lymphocytes (PBL) with DR2/DR- hablotypes were suspended in 10- phosphate buffered saline (PBS) and heated at 1500 rpm+.
It was centrifuged at The supernatant was discarded, the suspension was resuspended in 10 ad PBS, centrifuged at 1500 rpm, and the supernatant was completely removed.

Next, PBL was added to 2d(7)LST buffer (20mMTr
is solution P H7,4,10+sM NaCl,3mM
MgCIg) was suspended, centrifuged at 1500 rpm, and the supernatant was removed.

5%5ocose, 4%NF-4Q cooled to 4°C
LST11 solution 1- containing LST11 was added to suspend PBL and left for 5 minutes. Then, centrifuge at 1500 rpm, remove the supernatant, and cool 51111 ACE buffer (50111
M sodium acetate, 10mM EDTA) was added and stirred. After adding 0.5 d of 10% SDS solution, 5 d of phenol solution was added and shaken for 4 minutes. After that 200Or
Centrifuged at pm for 10 minutes and placed the upper layer into a new test tube.

Add the same amount of chloroform solution as the upper layer solution to this test tube 4
After shaking for a minute, it was centrifuged at 2000 rpm for 10 minutes, and the upper layer was placed in a new test tube.

Two times the amount of 99% ethanol was slowly added to the test tube from which the upper layer had been removed and mixed to separate human chromosomal DNA into threads. 30ug of isolated human chromosomal DNA was treated with the restriction enzyme EcoRI.
Incubated with 9Q units (U) at 37°C for 3 hours.

Human chromosomal DNA cut with EcoRI was added to the end of a 0.8% agarose gel and electrophoresed, and DNA fragments ranging from 6.0 kb to 8.5 kb were separated using the DE paper separation method, and HLA class I genomic DNA was separated. I got it.

Creation of genomic DNA library 0.4 isolated human DNA! The arm of λ phage (λZAP treated with EcoRI (Stratag
ene)) was added, T4 ligase was added, and the mixture was incubated at 4°C for 18 hours. This sample is packaged with a packaging kit (Gigapa).
ck-gold (manufactured by Stratagene) in vitro. In order to confirm whether the packaging was successful, the titer of the packaged phage sample was examined using the following procedure.

(Measurement of sample titer) Take a small amount of B B 4 E. coli and add 40W1 L
B medium (NaC 15g, yeast extract 5
10 g of tryptone dissolved in 11 H, O) was placed in a 50-centrifuge tube and cultured with shaking at 37°C overnight. Add 1 l of cultured bacterial medium to the centrifuge tube, add 3 l of LB medium and 80 l of IMMgSO
added. 0.2m of the bacterial culture medium thus obtained and a dilution series of 2μ of the packaging sample! (Standard solution, 10-fold diluted solution, 100-fold diluted solution, 1000-fold diluted solution) into 6
It was added to a test tube of Ili and incubated at 37°C for 15 minutes. 2.5-0.7% soft agar kept at 45°C is added to each test tube and mixed, and then layered on a 1.5% agar plate with a diameter of 90 m. After culturing at 37°C for 8 hours, the number of plaques was counted.

HLA class ■ Cloning of genomic DNA Spread the library stock solution in 3M medium (NaC115.8g, Mg5Oa
IHzo 2g, 50aeffi IMTri
sCl p H7,5,5+d 2% gelati
n is 1! ) (dissolved to 10%). - The next screening was performed using the same procedure as for measuring the titer of the samples described above, and phage plaques were formed in 90 plates.

A nitrocellulose membrane (manufactured by Ge1a+anScience) was placed on the soft agar layer and left for 5 minutes.Next, the nitrocellulose membrane was soaked in aenature solution (0.1M NaO
Place the phage side up on a chromatography paper soaked in H, 1,5M Na c 1) and leave it for 5 minutes.Next, place the nitrocellulose membrane on a chromatography paper containing twice the amount of SSC solution. After standing for 5 minutes, the nitrocellulose membrane was dried at room temperature.

Finally, the plotting was completed by leaving it in a vacuum dryer at 80°C for 120 minutes.

HLA-B7 cDNA used in hybridization
A probe was prepared as follows.

HLA-B7cDNA1340bp (
Orr H, T, etal; Blochemist
ry, 1B, 5711.1979) is Pstlsit
Plasmid pI) poo contained in PBR32B in e
l (Dr Jeisman, Vale Llnive
(obtained from rsity) was cut with Pstl, electrophoresed on agarose gel, and 1340bp H
LA-B7 cDNA isolated 0 isolated HLA-B7
200 ng of cDNA was identified with 3-P-dCTP using the unidirectional translation method. Labeled cDNA
In order to separate "P-dCTP" and unbound "P-dCTP," it was applied to a Sephadex 050 column and the first peak with radioactivity was collected.

The HLA class I gene was cloned using the following procedure.

The nitrocellulose membrane on which the phage library was blotted was placed in a hybridization bag. Prehybridization solution (50x Denhar
dt's 5solution O, 8m+10mFor
mawide+ 0.2m 10% SDS, 0
．． 5m of 4■/d salmon sperm DNA, 6.6ae
20x SSC, 1.9 dH1O) was added, the bag was sealed and incubated at 42°C for 2 hours.*2 XIQI'
IcpIII HLA-B7 cDNA probe and 4mg
/-Put 0.5 m of salmon sperm DNA into a glass test tube 1
After heating in a 00°C water bath for 10 minutes, it was rapidly cooled in ice. This glass test tube contains 0.8-50 times Denhar.
dt'5olution.

10d formamide, 0.2m! 10%S
DS, 6.6m! 20x SSC solution, 2d 50
A mixture of %Dextran 5ulfate was added to prepare a hybridization solution. The prehybridization solution was discarded from the hybridization bag, the hybridization solution was added, the bag was sealed, and the bag was incubated at 42°C for 18 hours. Take out the nitrocellulose membrane from the bag and double the ssc, o, i%S.
The nitrocellulose membrane was washed with a washing solution consisting of DS at 37°C for 30 minutes twice and then at 52°C for 30 minutes twice, and then dried. -hat dried nitrocellulose membrane
After fixing on man 3 M M filter paper and placing it in an X-ray film cassette and exposing it to X-ray film for 3-18 hours,
The film was exposed. After confirming the positive plaques that appeared as black dots, pipette the positive plaques from the 90mm dish and place them in a 1.5d centrifuge tube containing 1atSM buffer and incubate for 1 hour at 37'C.Next, add a few drops of chloroform. 15000rpm
After centrifugation for several seconds, the supernatant was used as phages for secondary screening.

The supernatant was diluted with 3M medium to produce 100-500 plaques on 90 plates, and then blotted onto a nitrocellulose membrane in the same manner as the primary screening.
Hybridization was performed using the L, A-B7 cDNA probe and positive plaques were taken.

The positive plaques obtained in the secondary screening were taken out with a pipette, placed in a 1,5-centrifuge tube containing ladsM medium, incubated at room temperature for 1 hour, and then a few drops of chloroform were added and centrifuged at 1500 rpm for several seconds. The supernatant after centrifugation was used as a phage for the tertiary screening, and screening was performed in the same manner as the secondary screening. It was confirmed by hybridization that all the plaques were positive plaques, and the cloning was completed.

Purification of DNA The genomic gene incorporated into the λ phage obtained in the tertiary screening was converted into a plasmid using a helper phage by the following procedure. Take the λ phage clone from the tertiary screening plate and add 500 pl of 3M medium and 20
The mixture was mixed with ul of chloroform to form a λZAP solution. 10. Add 200 pZ λZAP solution to a centrifuge tube. Ill's I
R408 helper phage with a concentration of
Add Blue E and coli, mix and heat at 37°C15
Incubate for a minute. Next, add 57 double concentration YT medium (5g NaCl, 5g yeast extract, 9g Bact) to this centrifuge tube.

- Trypton! (dissolved in water) and shaken at 37°C for 4 hours.

Next, after heating at 70°C for 20 minutes, the centrifuge tube was heated at 100°C.
Centrifugation was performed at 0 g for 5 minutes. Transfer the supernatant to a new tube,
This test tube contains 200117 8X10 pieces/-XL
1-Add Blue E and coli to 37'C15
Incubate for a minute. 100μ of the solution in this test tube
1 was poured onto an ampicillin plate and allowed to stand overnight at 37°C. The next day, colonies were taken, planted in LB soil containing ampicillin, and shaken overnight at 37°C. Next, 5 d of this culture solution was added to 400 m of LB medium containing ampicillin and incubated at 37°C under O
The cells were cultured with shaking until Dhe, = 0.8. 2I
11 of 34■/H1 chloramphenicol was added, and the mixture was further cultured at 37°C for 12 hours.

Next, transfer this culture solution to a centrifuge tube and spin it at 5QOO rpm for 10
Centrifuge for a minute and discard the supernatant. 40m of 0, I to this centrifuge tube
MNaCl, 10mM Tris
-HCI (pH 7, 8) and 1 mM EDTA were added to suspend the precipitate, followed by centrifugation at 5000 rpm for 10 minutes and the supernatant was discarded. A 7m glucose-lysozyme solution was added to the centrifuge tube to resuspend the precipitate, and the tube was left at room temperature for 10 minutes. Furthermore, add 14d alkaline solution to the centrifuge tube,
The mixture was allowed to stand for 10 minutes under ice cooling, and then a 10.5 d potassium acetate solution was added thereto, stirred, and then allowed to stand in water for 10 minutes. This centrifuge tube was centrifuged at 150 rpm for 20 minutes at 4°C, and the supernatant was transferred to a new 50-centrifuge tube. Add 0.6 volume of isopropyl alcohol to this, mix, and then
Leave it for a minute. This centrifuge tube was centrifuged at 300 rpm for 10 minutes and the supernatant was discarded. After adding 100% ethanol to this centrifuge tube and drying the precipitate, 3.5 d of T E 7
74 street solution (10mM Tris C1, 1mM EDT
pH 8.0) was added to dissolve the precipitate. For 3.5 m of this DNA solution, 3.7 g CsC1,0,2m
A 10% aqueous solution of ethidium bromide was added to completely dissolve the cesium chloride. The resulting solution was transferred to a sealed tube, the tube was filled with 50% (w/w) cesium chloride solution, and the opening was sealed. Seal tube 1
Plasmid DNA was separated by centrifugation at 5°C, 55,000 rpm+ for 15 hours. This plasmid DNA I'i was collected with a syringe, and an equal volume of dibutanol saturated with water was added and mixed. It was centrifuged at 3000 rpm for 1 minute and the upper layer was discarded. This operation was repeated three times to remove ethidium bromide. Next, the plasmid DNA was dialyzed against TE loose fluid overnight to purify the DNA.

The HLA class ■ genomic DNA cloned from HLA class 1 by tk-
The gene was introduced into mouse L cells, and its expression on the cell surface was investigated using an anti-HLA class summer antibody.

Introduction of HLA class I gene into mouse L cells On the day before gene transfer, 5×10 − mouse L cells were placed in a culture flask and cultured in MEM medium containing 10% FC3.

A precipitate was prepared by the Ca-phosphate precipitation method using the following procedure.

(a) 20μg/100μm of HLA class ■Genomic DNA, 1gg/100μl of thymidine kinase gene, and 100μj! CaCIg・2Hz
O solution pH 7.0 and 700 μl of H and O were concentrated to make a total volume of 1 d.

(b) Mix Heps/NaCl solution and 100 times NaHPO solution at a ratio of 49:1, and mix the solution with 6
m in a test tube.

(c) Pour the solution prepared in (a) one drop at a time into the test tube (b), mix, and incubate for 20 minutes.
A NA precipitate was created.

Mouse L cells cultured on the previous day were washed once with Ca-free PBS, and Ca-DNA precipitate was added to the cells for 1 hour in a flask.
Incubated for 5 minutes.

Furthermore, 10ad of MEM medium containing 10% fetal calf serum (Fe2) was added and incubated in a CO incubator at 37°C for 6 hours.Next, a 34% PEG-5% DMSO solution prepared using FC3-free medium was added to the flask. After removing the medium inside, 34% PEG-5% D of 21t1
Add MSO solution and incubate for 3 minutes. FC3
After washing 3 times with medium without it, wash with medium containing 10% FC3 for 2
Washed twice. Next, 10ad! A medium containing 10% FC3 was added and cultured overnight. The next day, the medium was changed to EAT and cultured #. The HAT medium was replaced every 2 to 3 days.

After about 14 to 21 days, it was confirmed that the cells proliferated and formed colonies after the tk gene was introduced. After each colony was picked using a cloning ring, each clone was cultured.

Confirmation of HLA class I antigen expressed on transformed cells 1×10 6 of each cloned colony were washed twice with PBS and suspended in 100 μl of PBS, and 50a11 were placed in two test tubes each. 50 μl of anti-HLA class I monoclonal antibody W 6/32 in 2 tubes
(Parham P et al, J, Ima+uno
l.

(1979), 123. pp342-349), 5
01tl (D anti-HLA-DR monoclonal antibody L 2
43 CLampson LAet al, J, lm5
uno1. (1980), 125. pp293-299
) were added separately and incubated at 4°C for 30 minutes. Add 0.5yal (7) PBS to each test tube and 715
The mixture was centrifuged at 00 rpm for 5 minutes, and the supernatant was removed. This was repeated three times. Add 50-fold diluted FITC anti-mouse Ig antibody (manufactured by 5ilenus) to each test tube and incubate at 4°C.
Incubated for 0 minutes. 0.5d P B in each test tube
The process of adding S and centrifuging at 1500 rpm for 5 minutes to remove the supernatant was repeated three times. Finally, 1 dPBS was added to each test tube and the fluorescence intensity was measured using Spectrum ■ (manufactured by Ortho). For cells expressing HLA class ■ antigen on L cells, the fluorescence intensity with W6/32 antibody becomes stronger than that with L243 antibody, and expression can be confirmed.

By the above method, it was confirmed that the transformed cells into which two types of HLA class I genome gene clones 1.2 and 2.1 obtained from PBL were introduced expressed HLA class I antigen.

From the structure of the restriction enzyme cleavage gene using nine types of restriction enzymes, clone 1.2 was found to contain the HLA-8w52 gene (J, In
+muno1.142. pp306-311. (19
89)).

Clone 2.1 was similar to the HLA-8w52 gene, but the Pvull fragment was different from the HLA-8w52 gene. Therefore, clone 2.1 is HLA-83
It was strongly suggested that there are 5 genes.

Clone 2.1 was cloned together with a hygromycin resistance gene into the B lymphoblastoid cell line Hmy2cIR, which does not express HLA-A and B antigens (J, Exp, Med).

166, pp283-288. (1897)), and transformed cells were obtained using hygromycin B medium.

These transformed cells were further cultured, and flow cytometry using the W6/32 monoclonal antibody revealed that clone 281 transformed 1 (wy
2 CI R cell lines were obtained.

The alloantigenicity of this clone 2.1 transformed cell line Hmy2CIR was investigated by a complement-dependent cytotoxicity test.

Antiserum-A with specificity for HLA-B35, 8w53
OH223 and other various antisera listed in Table 1 were previously dispensed at 1 pl into each well of a microplate.Next, 3 x 10''/pl of target cells were dispensed at 1 pi into each well, and incubated at room temperature. Incubated for 30 minutes.

Next, 5 pl/well of rabbit complement was dispensed into each well and reacted for 60 minutes at room temperature.Next, 2 pl/well of 5% eosin aqueous solution was dispensed into each well and allowed to stand at room temperature for 5 minutes. did. 8 μl/well of 37% formaldehyde was added to each well to stop the reaction and fix the cells. After being left at room temperature for 30 minutes, the cells were observed under a microscope and the percentage of positive cells to total cells was determined. The results are shown in Table 1. The target cells were a cell line transformed by introducing clone 2.1 into Haiy2CIH, and untransformed H+wy2.
CIR cell line was used as a negative control.

Clone 2.1 Umbrella ul lfl tlWjJ AOH311B5.8w53 0
0*: Non-transformed Hsy2C[R Table 1 From the above results, it was confirmed that clone 2.1 was the HLA-835 gene.

The entire nucleotide sequence of the genomic DNA of clone 2.1 was determined by the dideoxy method. The results are shown in Figure 1.
-835 gene, for reference, HLA-B51,8
The base sequences of w52 and 8w5B are listed. HLA-B5
1,8w52 J, Immunol, 142. p
p306-311.1989, HLA-8w58 J,
Blol, Chew, 260. ppH92411
933, 1985, in which A represents adenine, C represents cytosine, G represents guanine, and T represents thymine.

Figure 2 shows the amino acid sequence of the peptide encoded by each exon region of the HLA-835 gene shown in Figure 1. Amino acids are indicated by single letter abbreviations, A is alanine, R is arginine, and N is asparagine. , D is aspartic acid.

C is cysteine, Q is glutamine, E is glutamic acid,
G is glycine, H is cystidine, ■ is isoleucine, L
represents leucine, represents lysine, M represents methionine, F represents phenylalanine, P represents proline, S represents serine, T represents threonine, W represents tryptophan, Y represents tyrosine, and ■ represents valine.

In the diagram, for reference, HLA-B35, 8w52°8w5B
, B8, 8w41, and 8w60 are listed in parallel.

HLA-B51,8w52 is J, II1munol+1
42゜pp306-311.1989. HLA-
B w 5 B is J, Biol.

Chew, 260. pp11924-11933.
1985. HL A-B9.8w41 is Proc
, Natl, Acad, Sci, (USA).

85, pp4005-4009.1988°HLA-8
w60 is Biochemistry, 22. pp396
13969, 1983.

In FIGS. 1 and 2, dashes indicate the same base or amino acid as the sequence of HLA-835.

Since the base sequence of the HLA-835 gene has been clarified by the above-mentioned example, it is strongly expected that the allotype of HLA-835 will be determined by DNA typing.

HLA-B35 and 8w58 have eight amino acid substitutions in the helical region of the α1 domain. On the other hand, the amino acid residues of other domains are all (identical. Therefore,
The following nucleotide sequence (1
) is considered to be a base sequence specific to the HLA-B 35 alloantigen.

(1') CGGAACACACAGATCTTCAA
GACCAACACACAGACTTACCGAGAG
The DNA used for the AGCCTGCGGAACCTGCGCGGC DNA probe has the above base sequence (1)
Preferably, a region containing the nucleotide sequence 147th to 269th, 147th to 248th, 183rd to 269th, 183rd to 248th of exon 2 is preferable.
A is listed as a candidate. DN whose base sequence was determined
A is synthesized using a DNA synthesizer or phosphotriester synthesis method. Alkaline phosphatase (3,1,3,1), polynucleotide kinase (2,7,1,78) and α-"PdATP are added to the synthesized DNA. In addition, by labeling 21p at the 5' end of single-stranded DNA, DNA
A probe is obtained.

〔Effect of the invention〕

The present invention is useful for research, testing, and diagnosis of HLA-B35 alloantigen.

[Brief explanation of drawings]

Figure 1 (1) and (■) are diagrams showing the base sequence of the HLA-835 gene, Figure 2 (1) and (It) are HLA-B 3
FIG. 5 is a diagram showing the amino acid sequences encoded by five genes. Patent applicant 2. -8 Olympus Optical Industry Co., Ltd.

Claims (1)

[Claims] 1. HL having an exon represented by the following base sequence
A-B35 gene [Gene sequence available] [Gene sequence available] In the above formula, A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. 2. HLA-B35 gene encoding the following amino acid sequence [Gene sequence available] [Gene sequence available] In the above formula, A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, Q is glutamine, E is glutamic acid, G is glycine, H is histicine, I is isoleucine, L is leucine, K is lysine, M is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is Tryptophan, Y represents tyrosine, and V represents valine. 3. A part of the HLA-B gene according to claim 1 and DNA
A DNA probe consisting of a marker substance bound to a portion of the DNA probe. 4. A transformed cell characterized by expressing the HLA-B35 antigen obtained by introducing the HLA-B35 gene according to claim 1 into a eukaryotic cell.