JPS60166624A - Adult t-cell leukemia virus antigen polypeptide - Google Patents

Abstract

PURPOSE:To produce the titled polypeptide useful for the detection of anti- ATLA antibody and the prevention of the ATLV infection, in large quantities at a low cost, by culturing a microorganism having a recombinant plasmid integrated with a DNA fraction coding the env gene of ATLV. CONSTITUTION:The DNA coding the surface antigen peptide gene (env gene) of adult T-cell leukemia virus (ATLV), e.g. the DNA from PATK100, is integrated in a vector DNA (e.g. PAM82) by the method of recombinant DNA to obtain a recombinant plasmid (e.g. PATYE307). The recombinant plasmid is integrated in a microbial cell (preferably yeast belonging to Saccharomyces cerevisiae by transformation, and the obtained transformed strain is cultured in a medium to produce and accumulate the ATLV antigen polypeptide coded with env gene in the cultured product. The objective antigen polypeptide is separated from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adult T-cell leukemia virus antigen polypeptide encoded by the env gene, a recombinant plasmid incorporating a DNA fragment encoding the peptide, a microorganism containing the plasmid, and the microorganism. This invention relates to a method for producing an adult artificial cell Shiromen disease virus antigen polypeptide encoded by the env gene.

Adult T cell
leukemia virus, hereinafter abbreviated as ΔTLV. ATLV is also known as human r-cell leukemia virus (I
Although it is also called ITI, V), ATLV is used here. ]
is adult T cell leukemia (adult T cell leukemia).
eukemia, hereinafter abbreviated as Δ1゛L. ) is a type C retrovirus isolated from a patient [Yoshikawa et al., P.
roc, Natl, 8cad.

Sci, ll5A, 79.2031-2036 (
]982)]oATT-The patient's prognosis is poor;
There are no effective treatments, and there are many reports that half of patients die within 10 months.

In recent years, an ATL-derived cultured strain, M.
The existence of antibodies that specifically react with T-1 cells was discovered [Hinuma et al., Proc. Natl., Acad., Sc.
i.

11s 8, 711.6476-6480 (1981)
). Since then, the presence of this antibody has been confirmed in all patients with ATL, and the antigen for this antibody is ATL-related antigen (8Tl).
, -associateddantigen, hereinafter AT
(abbreviated as LΔ). It was found that this antibody against ATLΔ (hereinafter abbreviated as anti-ATLA antibody) is present in 25% of normal healthy people in ATI-prone areas, and that the distribution of anti-ΔTLΔ antibody carriers corresponds to ATL-prone areas. ing. The main body of ATLΔ is this retrovirus antigen, and the presence of the ATLV genome in patient's peripheral blood lymphocytes has been proven, and even if the lymphocytes of normal people who are positive for anti-ΔTLΔ antibodies are cultured, A.T.
LV is detected.

ATL and ATLV are closely related; ATLV is AT
Although it is thought to be the causative virus of L, the route of infection is still unclear. Blood transfusion has been shown to be one route of infection.

In areas where ATL is prevalent, 25% of healthy people are positive for anti-ΔTLA antibodies, and these people are highly likely to be carriers of 8T[,■, so blood transfusions from these people should be avoided.

If anti-ATLΔ antibodies can be detected, such blood transfusions can be avoided and ATL can be detected early.

Currently, anti-ATLA antibodies are detected using acetone-fixed slides of ATL-derived cultured cell lines. However, simpler and faster detection of anti-ATL△ antibodies and ATL
Early detection is desired.

The inventors have proposed the detection of anti-ΔTLΔ antibodies and the detection of ATL.
We conducted research on a method for supplying AT LΔ in large quantities and at low cost, which is useful for preventing V infection. As a result, ATLV
Genome coat protein antigen (surface antigen) peptide gene (
env gene) into vector DNA using recombinant DNA techniques, and the resulting recombinant DNA
As a result of culturing yeast containing the env gene, it was found that a significant amount of the ΔTLV antigen polypeptide encoded by the env gene was produced and accumulated, and the present invention was completed.

One of the env gene products is a glycoprotein with a molecular weight of 62,000 (
gp62) has already been reported.
ri et al.: Gann, 74.790-793 (198
3)], and the entire DNA sequence of ΔTLV was determined by the present inventors [Patent Application No. 57-214287. Proc
.

Natl, 8cad, Sci, [, USA 80.
3618-3622 (1983)], the present invention is the first to produce an ATLV antigen polypeptide encoded by the env gene in a microorganism.

The ATLV surface antigen protein produced in the present invention contains the entire surface antigen protein, and thus contains all of the multiple antigenic determinants present on the surface antigen, and is useful for diagnosis.

It is considered suitable as an antigen for vaccines.

The present invention will be explained in detail below.

The present invention provides DNA encoding the env gene of ATLV.
Recombinant plasmid incorporating the fragment.

A microorganism containing the plasmid and an AT using the microorganism
A method for producing L V antigen peptide (env gene product) is provided.

The recombinant plasmid of the present invention is constructed as follows.

A recombinant plasmid can be constructed by incorporating DNA encoding the ATL V env gene into vector DNA using recombinant DNA techniques.

The DNA encoding the env gene of ATLV is
For example, pΔTK10 cloned by Shimizu et al.
0 [Shimizu et al., Proc, Natl, 8 cad.

Sci,, USA, 80.3613-3622 (1
983)) can be used. The ATLV genome consists of I-TR at both ends, gag, pol, env, p
It consists of at least four genes of X. Of these en
The protein encoded by the v gene is known to be a coat protein antigen (surface antigen) expressed by ATLV infection, so it can be used to diagnose ATLV infection, prevent ATLV infection, and treat leukemia caused by ATLV. Treatment.

Furthermore, the protein itself is expected to be used as a vaccine.

pATKl,00 is a clone containing the entire env gene, and can be used as a source of a DNA fragment encoding the env gene.

Any vector can be used as long as the inserted target DNA can be expressed in microorganisms. Preferably, a suitable promoter is used, such as the yeast 3-phosphoglycerate kinase (PGK> system,
tryptophan (TRPl) system, alcohol dehydrogenation yeast I (ΔD +-11) system.

Galactose kinase (GΔL1) system, glyceraldehyde 3-phosphate dehydrogenase (GΔP DH) system,
A plasmid having a promoter such as P'-60 and into which the target DNA can be inserted downstream can be used.

The plasmid is pYeHBs.

pM△36. pMΔ56. Although pMA82 etc. can be used, a specifically suitable plasmid is pΔ
I can give you the M82. pAM82 is Miy
anohara et al, Proc, Natl, 8cad, Sci.

IsA, 80. ] ~ 5 (1983). pAM82 contains a replication origin derived from pBR322, an ampicillin resistance (Δp11) gene, and arsl and 1eu2 genes derived from yeast.

This plasmid has a 2μ DNA replication origin and is capable of self-replication in E. coli and yeast and selection of plasmid retention.

Furthermore, pΔM82 contains the promoter region of the polypeptide (P-, -60> gene of the inhibitory yeast phosphatase of yeast, and the Xh
Foreign DNA can be expressed by inserting a gene having a translation initiation site into the o1 site.

DNA encoding the env gene of ΔTLV.

For example, DNA from pΔTK100 and vector DNA
For example, recombination with pΔM82 can be carried out using common recombinant DNA techniques in which both DNAs are digested using restriction enzymes and then ligated using T4 DNA ligase.

For ligation, fill-in reaction using DNA polymerase [K]enow fragment or D
This can be carried out by a method using NA rintsuno.

In the case of pATKl 00 and pΔM82 shown as specific examples, as shown in Fig. 1, the env of pΔTK100 is
After digesting the gene with the restriction enzyme PstT, the ends were made blunt with T4DNΔ polymerase, and the fragments obtained by successively digesting with H10d■ and Haem were transformed into pΔM82.
Recombinant plasmid pΔTYE307 can be constructed by inserting into the Xho1 site of .

The reaction conditions necessary for constructing the recombinant plasmid described in -F are as follows.

Digestion of DNA with restriction enzymes is usually 0.1 to 100 μg.
2 to 200 mmol (preferably 10 to 40 mmol) of DNA
mmol) of Tris-HCIl (p
t(6,0-9.5, preferably pH7,0-8.
0), 1-150 mmol NaCl! , 2-20 mmol (preferably 5-10 mmol) M g CE
2, restriction enzyme 0.1 to 300 tons (preferably 1μ
Using 1-3 units of enzyme per g of DNA, 18-4
At 2°C (preferably 32-38°C), for 15 minutes to 2
Perform the digestion reaction for 4 hours. The reaction is usually stopped at 55-75℃
(preferably 63 to 70°C) for 5 to 30 minutes, but a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

When linking DNA fragments, 2 to 200 mmol (preferably 10 to 70 mmol) of Tris-HCl (pt(
6,0-9.5, pH 7, Q-8.0), 2-20 mmol (preferably 5-10 mmol) of MgCR2,0
, 1-10 mmol (preferably 0.5-2 mmol)
ΔTP of T4 DNA ligase 0 in 1-50 mmol (preferably 5-10 mmol) dithiothreitol.
．． 1 to 37°C (preferably 3 to 2°C) using 1 to 10 units.
0℃) for 15 minutes to 72 hours (preferably 2 to 20 hours)
Perform the binding reaction.

Purification of DNA fragments, recombinant plasmids, etc. is performed by agarose gel electrophoresis.

By transforming a recombinant plasmid, ie, pΔTYE30T, into a microorganism and culturing the resulting transformant, ΔTl encoded by the env gene can be obtained.
, ■An antigen polypeptide can be obtained.

As the microorganism, yeast is preferably used, and specifically preferred is Saccharomyces cerevis.
iae AlI22(a,]eu2.his4.c
anL cir4) [:lto, If, et a
l.

J, Bacteriol, 153.163-168
(19B3) ] is used.

Transformation was performed using the method of H. lto et al. [:J, Bacl:e
riol, 153゜163-168 (1983)]
This can be done according to the following. The transformed strain is pATYE3
In the case of 07, it can be obtained as a leucine non-auxotrophic strain.

Expression of the ATLV antigen polypeptide by a transformed strain can be achieved, for example, by culturing this strain in Pulck-Boulder's minimal medium or its modified medium, then disrupting the cells and extracting with a phosphate buffer containing phenylmethylsulfonyl fluoride.
The resulting extract was subjected to gel electrophoresis in the presence of SDS,
The method of Towbin et al. [Towbin It, et al.
al,, Proc, Natl, Acad, Sc
i.

IIs 80, 4350-4354 (1979)]
This is confirmed by detecting the ATLV coat protein produced in yeast. It was confirmed that the polypeptide detected here was specifically immunoprecipitated with patient serum.
It can be seen that it possesses the properties as a ΔT L V antigen peptide.

Isolation of plasmids from yeast is described by 5tru et al., P.
roc, Natl, 8cad, Sci, USA, 76
1035 (1979).

The transformed strain of pATYE307 was purchased from Saccharom at the American Type Culture Collection.
yces cerevisiae C11l ATCC
International deposits have been made for each.

Examples of the present invention will be shown below.

Example 1 Plasmid pATK 100 [5eiki, M
, et al.

Proc, Natl, 8cad, Sci, ll5A 8
0, 3618-3622], 5
0mM NaCl! , IOmMTr i 5-HCI
(pH 7,5), 7mM MgCβ2 and 6mM 2
- It was dissolved in 1000 μβ of a buffer consisting of mercaptoethanol, and 200 units of restriction enzyme PstI (manufactured by Zenshuzo Co., Ltd.; unless otherwise specified, restriction enzymes are manufactured by the same company) was further added, and the mixture was allowed to react at 37° C. for 1 hour. Reactions were subjected to 0.9% agarose gel electrophoresis and stained with 1 μg/ml ethidium bromide to detect DNA fragments.
A DNA fragment of 2.425 base pairs (abbreviated as bp) containing the v gene was obtained. 41μg of this DNA fragment was added to 30mM
Tris-acetic acid (p I-17,9), 66mM potassium acetate, 10mM magnesium acetate, 0.5mM
Dithiothreitol, O, L m MdΔTP, 0.1
．． mM dTTP, 0. ], mM dGTP and 0.
In addition to 300μj2 of a solution containing 1mM clcTP, 4 units of T4DNA polymerase (manufactured by Zenshuzo Co., Ltd.) were added, and the mixture was reacted at 37°C for 10 minutes.

In this reaction, the cohesive end of the DNA fragment caused by PstI cleavage was repaired and became a blunt end.

40 μg of this DNA fragment was mixed with 50 mM NaCl, 10
mM Tris-HCj! (pH 7,5), 7mM
Dissolve gCβ2 in 200 μl of a buffer consisting of 6mM 2-mercaptoethanol, and add restriction enzyme Hin.
Add 50 units of dnI, react at 37°C for 1 hour, and en
A DNA fragment of L 743 bp containing the v gene was obtained. This 1.743 bp DNA fragment 2'/1-1g
50mM NaCl2, ], OmMTri 5-
H (J! (pH 7,5>, 7mM MgCjL and 6mM 2-mercaptoethanol) was dissolved in 250μβ of a buffer solution, 42 units of restriction enzyme Ha em were added, and the reaction was carried out at 37°C for 25 minutes. ．．
4% agarose gel electrophoresis and staining with 1 μg/m+ ethidium bromide to detect DN fragments.
A 1.590 bp DNA fragment containing the env gene was obtained.

On the other hand, vector plasmid pA MB2 (Miya
nohara.

A, et ayu, Proc, Natl, 8cad
, Sci, [ISA, 80.

1-5.1983 > 3μg in 100mM NaCl
1°10mM Tris-HCj2 (pH 7,5),
? The mixture was dissolved in 20 μl of a buffer consisting of mM Mgc, g2 and 6 mM 2-mercaptoethanol, 8 units of restriction enzyme Xhol were added, and the mixture was reacted at 37° C. for 1 hour. This reaction solution containing DNA fragments was diluted with 50mM Tri
5-H (1! (pH 7,8), 5mM MgCβ2.1
0mM 2-mercaptoethanol, 5μg/ml BS
Δ(bovine serum albumin), 0.1mM dΔTP, 0.
1mM dTTP, 0.1mM dGTP and 0.1
In addition to 25 μβ of a solution containing mM dCTP, add DNA
Add 3 units of polymerase I (Klenow fragment),
The reaction was carried out at 22°C for 30 minutes. In this reaction, Xh of the DNA fragment.

(2) A DNA fragment of about 1,000 bp was obtained in which the sticky ends caused by the cleavage were repaired and became blunt ends.

DNA fragment derived from pΔTK100 (1,590 bp) 0
．． 23 μg and a DNA fragment derived from pΔM82 (approximately 11.
0OObp) 0.84μg and 66mM Tris-
HCI (pH 7.5), 6.6mM Mg (1!2.1
．． In addition to 50μβ of a solution consisting of 0mM dithiothretol and 0.5mM ATP, T4DNA! J gauze (
3 units (manufactured by Zenshuzo Co., Ltd.) were added thereto, and a binding reaction was carried out at 4°C for 1 day.

The reaction solution was extracted with phenol and protein was removed. Using the solution obtained, E. coli HBIOI[:Boliver]
et al., Gene 275 (1977)]. The transformed strain was prepared using L-13roth (10 g of Bactodrypton in IR, 5 g of yeast extract, 1!5 g of Na).
The strain was selected as one that grows on a medium containing 25 μg/ml of ampicillin. A plasmid was extracted from one of the transformed strains by CoBirnboim.
Nucleic 8cids Re5earch
7.1513 (1979) and named pATYE307.

pATYE307 is the P of vector plasmid pAM82.
- It is a recombinant with the env gene integrated in the same transcription direction as the P-60 promoter, and has a structure that allows expression of the ATLV eT'Iv gene under the control of the P-60 promoter (see structure below). , Maxam-Gi
Albert's method [A, M, Maxam et al.: P
roc, Natl, 8cad, Sci, USA, 74
．． 560 (1977):] 1.

1--TGAAACGTAT to TAGCGCTGAT
To GTTTTGCTGTCGAGG11ogness
box TCGCCCCGCCG8TCCCA8AGA
To GA CACCAAC8CC←4 To this plus using pATYE307 Li+AlI
22 (a, Ieu2. his4. canl,
cir” (described in the above-mentioned literature). The transformed strain was transformed with 6.7 g/p Yeast nitro
gen base without amino ac
ids (manufactured by Difco), 20g/j? of glucose and 20 μg/ml of histidine.

The transformed strain containing pΔTYE307 was stored in the American Type Culture Collection at Saccharom.
yces cerevisiae CT 1 ] A
It has been deposited as TCC.

Example 2゜Transformed strain C11l and pAM8 obtained in Example 1
2 was introduced into the A322 strain, and the -82-1 strain was modified with a Purukholder's minimal medium (pH 5, (1) A medium containing the following components in I:1β: 20 g of Nigelcose, 2 g of asparagine, 1.5 g of KH2PO4, CaCL” 2H2
00,33g, Mg304・7H200,5g, (NH
4) 230. 2g.

K I Q, 1+++g, H3BO460ug, Mn5
Os30μg, ZTISO4・7H20300μg.

Cu5Oa ・5H2040μg, FeCj! 3.68
20 250μg, Na2M004 ・282025μ
g, Chiryomin 200 μg, Biri 1' + Shin 200 μg,
Nicotinic acid 200μg, Pantonic acid 200μg, Biothia 2μg, Inositol IQmg, Histidine 20
mg] and cultured overnight at 30°C with shaking (2Q Orpm). After collecting the bacteria and washing the cells with sterile water, reduce the KH2PO4 concentration of the production medium (improved pulquehol-goo minimal medium to 30 mg/l, and add 1.5 g/12 KC).
OD6. , to a concentration of 0.05, and shaken at 30°C for 20 to 24 hours (20 Orpm).
) was cultured. After collecting the bacteria and washing the cells with sterile water, the cells were washed with 50% water containing 1mM phenylmethylsulfonyl fluoride.
Suspend in 0.5 ml of mM phosphate buffer (pH 7,2),
An extract was prepared by grinding using glass beads. The extract was subjected to polyacrylamide gel electrophoresis in the presence of 0.1% SDS using the method of Jowbin et al. [:Tov+b
in, tt,, et al,, Proc, Nat
l, 8 cad.

Sci, USA, 76, 4350-4354 (1
979):] by “Western blotti
ng'' (protein binding) to detect the ATLV coat protein produced in yeast.The primary and secondary antibodies used in the above method were ATL patient serum and [+25■] labeled anti-human immunotherapy. Globulin (4Hers)
(manufactured by ham) was used.

As a result, the molecular weight was approximately 43. OOO protein was detected as a specific band. This band was not detected when normal human serum was used as the primary antibody, and yeast L-8 transformed with pAM82 used as a vector
Since this band was not detected in 2-1, it was found that this protein was an ATLV coat protein gene product.

[Brief explanation of drawings]

FIG. 1 is a flow sheet showing the construction of plasmid pATYE307. In the figure, H indicates the l(a e m site. The arrow → represents the direction of transcription by P-60, and -÷ represents the direction of env gene transcription. Procedural amendment document, March 1982, No. 2) To the Commissioner of the Japan Patent Office, Patent Application No. 16952 of 1982 2. Name of the invention: Adult subcellular leukemia virus antigen polypeptide 3. Relationship with the person making the amendment: Patent applicant postal code: 170 Detailed description of the invention in the specification column and Column 5 of the list of attached documents to the application, contents of amendment (1) Specification box page 11, line 13, [r after ΔTCCJ]
Add 20697J. Procedural amendments filed on March 20, 1980 Display of the case Patent Application No. 16952 of 1982 2 Title of the invention Adult subcellular leukemia virus antigen polypeptide 3 Person making the amendment Relationship to the case Patent applicant postal code 170 Column 5 of the detailed description of the invention in the specification of Ikebukuro 1-37-1, "Toshimaku Kamiikebukuro Principal Place: Toshima-ku, Tokyo" - Contents of the amendment (1) rProc on page 3, lines 12-13 of the specification, N
atl. ^CAD, Sci, J [Proceedings of
The (2) rGann, J on page 5, line 11 of the specification box is corrected to [Gann, J]. (3) On page 7, line 7 of the specification, 1 dehydrogenase yeast ■ is corrected to ``dehydrogenase I.'' (4) rpMA82J on page 7, line 13 of the specification
Corrected to AM82 J. (5) "Suppressed yeast" on page 7, line 24 of the specification is corrected to "suppressed enzyme." (6) “J, Bacter” on page 10, line 13 of the specification
iol, J to [Journal of Bacteriology (J. Bacteriol,) J. (7) Delete "each" on page 11, line 14 of the specification. (8) rGeneJ on page 14, line 12 of the specification is corrected to [Gene J]. (9) Enter [Nucleic Acids Research J] on page 14, line 18 of the specification box.
(each) Correct to J. OQ Statement box page 16, line 1, before “close” r 2
Add 0697J. (2) 77-

Claims (1)

[Scope of Claims] (1) An adult T-cell leukemia virus antigen polypeptide encoded by the adult-induced cell leukemia virus env gene. (2) A recombinant plasmid incorporating a DNA fragment encoding the env gene of adult hypocellular leukemia virus. (3) The recombinant plasmid according to claim 2, which has a P-60 promoter. (4) The recombinant plasmid according to claim 3, which is pATYE307. (5) A microorganism carrying a recombinant plasmid into which a DNA fragment encoding the env gene of the adult T-cell leukemia virus has been integrated is cultured in a medium, and the adult T-cell leukemia virus antigen encoded by the env gene is contained in the culture. A method for producing an adult T-cell leukemia virus antigen polypeptide encoded by the env gene, which comprises producing and accumulating the polypeptide and collecting the peptide from the culture. (6) The method according to claim 5, wherein the recombinant plasmid is a recombinant plasmid having a P-60 promoter. (7) The method according to claim 5, wherein the microorganism belongs to the genus Satucharomyces. (8) The method according to claim 7, wherein the microorganism belongs to Satucharomyces cerevisiae. (9) The method according to claim 5, wherein the recombinant plasmid is pATYE307. αQ A microorganism containing a recombinant plasmid into which a DNA fragment encoding the env gene of adult T-cell leukemia virus has been integrated. (11) The microorganism according to claim 10, wherein the recombinant plasmid is a recombinant plasmid having a 1-60 promoter. (12) The microorganism according to claim 11, wherein the microorganism belongs to Satucharomyces cerevisiae.