JPH08107786A - Adult t cell leukemic viral antigenic polypeptide - Google Patents

Abstract

PURPOSE: To obtain a microorganism containing a plasmid capable of mass-producing a protein for diagnosis, treatment, etc., of adult T cell leukemia(ATL) by integrating a DNA fragment, etc., prepared by cleaving an env gene of an adult T cell leukemia virus(ATLV) with HinfI and a β-galactosidase gene thereinto. CONSTITUTION: This microorganism comprises Escherichia coli C19 (ATCC39591) and Escherichia coli CI10 (ATCC39592) containing a recombinant plasmid, prepared by integrating a DNA fragment of 748bp obtained by cleaving an env gene of ATLV with HinfI or a DNA fragment of 551bp prepared by cleaving thereof with AluI and a gene capable of coding β-galactosidase thereinto and capable of mass-producing a fused protein useful for diagnosis of infection with the ATLV, prevention of the infection with the ATLV, treatment, etc., of the ATL caused by the ATLV at a low cost. The microorganism is obtained by transducing a recombinant plasmid pEH9 or pEA1 into a microorganism belonging to the Escherichia coli and transforming the microorganism.

Description

Detailed Description of the Invention

The present invention relates to adult T-cell leukemia virus (AT
LV is also referred to as human T-cell leukemia virus HTLV, but ATLV is used here). A microorganism containing a recombinant plasmid incorporating a DNA fragment encoding the env gene or a part thereof and adult T-cell leukemia using the microorganism. The present invention relates to a method for producing a fusion protein of an antigen polypeptide coded by a viral env gene or a part thereof and an enzyme such as β-galactosidase. Adult T cell leukemia (adult T cell leukemia, hereinafter referred to as A
It is a C-type retrosaurus isolated from a patient (abbreviated as TL) [Yoshida et al., Proc. Natl. Ac.
ad. Sci.), USA. 79 , 2031-2036 (1982)]. ATL
Patients have a poor prognosis, there is no effective treatment, and it is often reported that half of the patients die within 10 months.

In recent years, the presence of an antibody specifically reacting with MT-1 cells, which is an ATL-derived culture, was found in the serum of ATL patients [Hinuma et al., Proc. Natl. Acad. Sci. USA 7
8 6476-6480 (1981)]. Since then, the presence of this antibody was confirmed in all ATL patients, and the antigen for this antibody was ATL-
It is called an associated antigen (ATL-associated antigen, hereinafter abbreviated as ATLA). This antibody against ATLA (hereinafter abbreviated as anti-ATLA antibody) is present in 25% of normal healthy people in the ATL-prone zone, and anti-ATL
The distribution of A antibody carriers has been found to be consistent with ATL-prone areas. ATLA is mainly the viral antigen of ATLV, and in the peripheral blood lymphocytes of patients, ATL
The existence of the V genome has been proved, and anti-A
ATLV can also be detected by culturing lymphocytes of TLA antibody-positive persons.

There is a close relationship between ATL and ATLV.
TLV is considered to be the causative virus of ATL, but the route of infection is still unclear. Blood transfusion has been shown to be one of the routes of infection. Blood transfusions from these people should be avoided, as 25% of healthy people in areas of high ATL are positive for anti-ATLA antibodies and they are very likely carriers of ATLV.

If an anti-ATLA antibody can be detected, such blood transfusion can be avoided, and ATL can be detected early. Currently, detection of anti-ATLA antibody is performed using an acetone-fixed slide of an ATL-derived cultured cell line. But,
It is desired to detect anti-ATLA antibody and to detect ATL at an early stage more simply and quickly.

The present inventors have made a diagnosis of ATLV infection, AT
A study was conducted on a method capable of supplying ATLA in large quantities and at low cost, which is useful for preventing LV infection and treating leukemia caused by ATLV. As a result, the en of the ATLV genome
The DNA fragment of the region encoding the antigen peptide gene called v gene was incorporated into vector DNA by the recombinant DNA technique, and the resulting microorganism containing the recombinant DNA was cultured. The present invention has been completed by finding that the encoded ATLV antigen peptide or a fusion protein of the peptide and the enzyme such as β-galactosidase is produced and accumulated in a large amount.

It has already been reported that one of the env gene products is a glycoprotein (gp62) having a molecular weight of 620,000 [Hattori et al .: Gann, 74 , 790-793].
(1983)], and the entire DNA sequence of ATLV was determined by the present inventors [Japanese Patent Application No. 57-214287, Proc. Natl.
Acad. Sci. USA 80 , 3618-3622 (1983)] produced a ATLV antigen peptide encoded by the env gene or a part thereof and a fusion protein of the peptide and an enzyme such as β-galactosidase in a microorganism. This is the first time that the present invention has been carried out.

The present invention will be described in detail below. The present invention
D encoding the env gene of ATLV or a part thereof
Provided are a microorganism containing a recombinant plasmid incorporating an NA fragment, and a method for producing an antigen polypeptide encoded by the env gene of ATLV or a part thereof using the microorganism.

The recombinant plasmid of the present invention is constructed as follows. A recombinant plasmid can be constructed by incorporating DNA encoding the env gene of ATLV or a part thereof into vector DNA by a recombinant DNA technique. The DNA encoding the env gene of ATLV or a part thereof includes, for example, pATK08 cloned by Kiyoki et al. [Kiyoki et al., Proc.
Natl. Acad. Sci., USA, 80 , 3613-3622.
(1983)] can be used. The genome of ATLV is composed of LTRs at both ends and at least four genes of gag, pol, env and pX. Among them, the protein encoded by the env gene is presumed to be a glycoprotein involved in the infectivity of ATLV.
It is expected to be used for diagnosis of infection, prevention of ATLV infection, treatment of leukemia caused by ATLV, and use of the protein itself as a vaccine.

PATK08 is a clone containing all of the env gene and can be used as a source of a DNA fragment encoding the env gene. Any vector DNA can be used as long as the inserted target DNA can be expressed in a microorganism. Specific preferred plasmids include pORF1 and pORF
I can give you 2. These plasmids are Weinst
ock et al .: Proc. Natl. Acad. Sci. USA 80 , 443
No. 2-4436, 1983, which can be prepared by the method described in the literature.

DNA encoding the env gene of ATLV or a part thereof and vector DNA such as pORF1
Alternatively, recombination with pORF2 can be achieved by using restriction enzymes
After digestion of NA, this can be done using common recombinant DNA techniques in which T4 DNA ligase is used to ligate. The binding can also be carried out by a fill-in reaction using a DNA polymerase I.Klenow fragment or a method using a DNA linker.

PATK08 and pOR shown as specific examples
In the case of F2, as shown in FIG. 1, e of pATK08
After digesting the nv gene with the restriction enzyme HinfI, the ends are blunt-ended with DNA polymerase I (Klenow fragment), and this fragment is inserted into the SmaI site of pORF2 to construct the recombinant plasmid pEH9. In addition, pEA1 can be obtained by digesting the env gene of pATK08 with the restriction enzyme AluI and then inserting it into the site of pORF1. As shown in Fig. 1, pEH9 is located in the first half of the env gene.
A DNA fragment of 8 base pairs (hereinafter abbreviated as bp) is incorporated, and pEA1 is located at the latter half of the env gene.
A bp DNA fragment has been incorporated.

The reaction conditions necessary for preparing the above recombinant plasmid are as follows. The digestion of DNA with a restriction enzyme is usually 0.1 to 100 µg of DNA to 2 to 200 mmol (preferably 10 to 40 mmol) of tris-hydrochloric acid (pH
6.0-9.5, preferably pH 7.0-8.0), 1-150
Millimolar NaCl, 2 to 20 millimolar (preferably 5
Restriction enzyme 0.1-30 in MgCl _{2 (} ~ 10 mmol)
0 unit (preferably 1 to 3 units of enzyme for 1 μg of DNA) is used at 18 to 42 ° C. (preferably 32 to 38)
The digestion reaction is performed at 15 ° C. for 15 minutes to 24 hours. The reaction is usually stopped at 55 to 75 ° C (preferably 63 to 70 ° C)
Although heating is carried out for 5 to 30 minutes, a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

2 to 200 when binding DNA fragments
Tris-hydrochloric acid (pH 6.0 to 9.5, preferably pH 7.0 to 8.0) of 2 mmol (preferably 10 to 70 mmol), 2
~ 20 mmol (preferably 5-10 mmol) Mg
Cl _2, 0.1 to 10 mmol (preferably 0.5-2 mmol) ATP of 1 to 50 mmol (preferably 5-1
0 to 10 units of T4 DNA ligase in 0 mmol of dithiothreitol at 1 to 37 ° C (preferably 3
The binding reaction is performed at -20 ° C for 15 minutes to 72 hours (preferably 2 to 20 hours).

Purification of DNA fragments, recombinant plasmids, etc. is carried out by agarose gel electrophoresis. The ATLV antigen polypeptide encoded by the env gene or a part thereof can be obtained by transfecting a microorganism with a recombinant plasmid such as pEH9 or pEA1 and culturing the resulting transduced strain.

Escherichia coli is preferably used as the microorganism, and more specifically, a derivative M of Escherichia coli K-12 is preferably used.
H3,000 [GM Weinstock et al., Proc. Natl. Ac
ad.Sci. USA 80 , 4432-4436, 1983] or TK10
46 (ibid.) Is used. Transduction is SN Cohen
Et al. [Proc. Natl. Acad. Sci., USA 69 , 2110 (19
72)]. Transductants are selected by blue colony formation with the fusion protein with β-galactosidase.

The expression of the ATLV antigen peptide by the transduced strain is carried out, for example, by culturing this strain in L-Broth,
The cells were collected and 125 mM Tris-HCl (pH 6.
8), suspended in a buffer consisting of 2% SDS, 0.7M 2-mercaptoethanol and 0.0025% bromophenol blue, then heated, and the centrifuged supernatant is electrophoresed in a 7% polyacrylamide gel containing SDS. , The gel is stained with Coomassie blue for detection. It can be confirmed that the polypeptide detected here specifically immunoprecipitates with patient serum, and it is understood that the polypeptide retains the property as an ATLV antigen peptide. HC Birnboim et al .: Nucleic Acids Research 7 , 1513 (19)
79).

Transgenic strains of pEH9 and pEA1 are Es in the American Type Culture Collection.
cherichia coli CI9 ATCC 39591 and Escher
International deposits have been made as ichia coli CI10 ATCC 39592 respectively. Examples of the present invention will be shown below.

[0018]

【Example】

Example 1 Construction of plasmid pEH9 (plasmid containing the first half of the env gene): Plasmid pATK08 from E. coli ATCC32246 was added to Kiyoki et al., Proc. Natl. Aca.
It was collected by the method described in d. Sci. USA, 80 , 3613-3622, 1983. 20 μg of pATK08 was added to 50 mM N
aCl, 6 mM Tris-HCl (pH 7.5), 6 m
It was dissolved in 200 μl of a buffer solution consisting of M MgCl ₂ and ₂ mM _2- mercaptoethanol, and the restriction enzyme H was added.
60 units of infI (manufactured by Takara Shuzo Co., Ltd., with respect to restriction enzymes hereinafter unless otherwise specified, manufactured by the same company) were added and reacted at 37 ° C. for 3 hours. The reaction product was subjected to 1% agarose gel electrophoresis and stained with 5 µg / ml of ethidium bromide to detect a DNA fragment, and a DNA fragment of about 750 bp was recovered. About 1 μg of the recovered DNA fragment is added to 50 mM Tr
is-HCl (pH 7.5), 5 mM MgCl ₂ , 2 m
M dithiothreitol, 0.1 mM dATP, 0.1 mM
dTTP, 0.1 mM dGTP and 0.1 mM dC
In addition to 100 μl of a solution containing TP, 3 units of DNA polymerase I (Klenow fragment) [Bethesda Research Laboratory (hereinafter referred to as BRL)] was added,
The reaction was carried out at 15 ° C for 1 hour. In this reaction, the DNA fragment
Staggered end by HinfI cutting (staggard en
d) has been restored to a blunt end. The reaction was subjected to 1% agarose gel electrophoresis and 7
A 48 bp DNA fragment was recovered.

On the other hand, the vector plasmid pORF2 was
Using maI, linearization was carried out by the method described in the literature. 748
0.5 μg of bp DNA fragment and linearized pORF25
μg to 50 mM Tris-HCl (pH 7.5), 10 m
M MgCl ₂ , 20 mM dithiothreitol and 1 m
Add T4DNA to 0.5 ml of MATP.
5 units of ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and a binding reaction was carried out at 14 ° C for 3 days. The reaction mixture was extracted with phenol and deproteinized to obtain a solution, which was then subjected to a conventional method [Weinstock et al., Proc. Nat.
L. Acad. Sci. USA. 80 , 4432-4436, 1983], E. coli. MH 3,000 (ibid.) was transduced. Transduced E. coli MH 3,000 was mixed with L-Broth (10 g bactotryptone, 5 g yeast extract;
a medium containing 5 g of aCl) and 10 mg / ml of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (5-bromo-4-chloro-3-indolyl-β-D-gala
agar medium containing ctoside (hereinafter referred to as XG) (on L-Broth)
15 g of agar was included), and the mixture was cultured at 25 ° C. for 2 days.
A bacterium that produces a fusion protein containing β-galactosidase into which a desired DNA fragment is inserted is X.
G is decomposed to form a blue colony. A colony showing a blue color was selected and cultured at 25 ° C. for 3 hours using L-Broth. From cultured cells, HC Birnbiom et al .: Nucleic Acids
Plasmid 1 by the method described in Research 7 , 1513, 1979.
00 μg was collected. The resulting plasmid is called pEH9. The obtained pEH9 has a structure containing the first half of the env gene as expected, which is the method of Maxam et al.
Maxam & W. Gilbert, Methods in Enzymology 65 , 499-560 (1)
980)], and confirmed by analyzing the DNA inserted in the plasmid and the base sequence in the vicinity thereof.

Example 2 Construction of plasmid pEA1 (plasmid containing the latter half of the env gene): In the same manner as in Example 1, plasmid pATK
08 was collected. 20 μg of pATK08 is added to 50 mM
NaCl, 6 mM Tris-HCl (pH 7.5), 6
mM MgCl ₂ and 2 mM 2-mercaptoethano-
It was dissolved in 200 μl of a buffer solution consisting of 100 μl, and 60 units of the restriction enzyme AluI was further added, followed by reaction at 37 ° C. for 3 hours. The reaction mixture was subjected to 1% agarose gel electrophoresis and 5 μm.
A DNA fragment was detected by staining with g / ml of ethidium bromide, and a DNA fragment of about 551 bp was recovered.

On the other hand, pORF1 was linearized in the same manner as in Example 1. 0.5 μg of a 551 bp DNA fragment and 5 μg of linearized pORF1 were added to 50 mM Tris-HCl (p
H7.5), 10 mM MgCl ₂ , 20 mM dithiothreitol and 1 mM ATP, added to 0.5 ml of a solution, and 5 units of T4 DNA ligase was further added, and the temperature was 14 ° C.
The binding reaction was carried out for 3 days.

E. coli M activated by the above-mentioned method using a solution obtained by extracting the reaction solution with phenol and deproteinizing it.
Transduced with H3,000. Transduced E. coli MH3.
000 was removed to an agar medium (described above) containing L-Broth and 10 mg / ml XG, and cultured at 25 ° C for 2 days. A bacterium that produces a fusion protein containing β-galactosidase having a desired DNA fragment inserted therein decomposes XG to produce a blue colony.
This was selected, and after culturing in the same manner as in Example 1, the plasmid was collected from the cells. The resulting plasmid is called pEA1. env as planned for pEA1
It was confirmed that it had a structure containing the latter half of the gene as in Example 1.

Example 3 Large expression of pEH9 and pEA1: pEH9 and pEH9
Microorganisms containing a plasmid that incorporates a β-galactosidase such as EA1 and the env gene cannot grow when expressed in large amounts due to the toxicity of the fusion protein encoded by the plasmid. The following treatments were performed for the large expression of pEH9 and pEA1.

GM Weinstock et al., Proc. Natl. Ac
Ad. Sci, USA 80 , 4432-4436, 1983, a gene for activating a promoter incorporated in a plasmid is cold-sensitive. coli TK1046 p
Transduction was performed with EH9 and pEA1. OD of the transduced E. coli TK1046 in 25 ml of L-Broth at 30 ° C
After culturing until ₆₀₀ reaches about 0.2-0.3, 1 at 37 ℃
Cultured for hours. The cells were collected, and 1/70 volume of the buffer solution [125 mM Tris-HCl (pH 6.8),
2% SDS, 0.7M 2 mercaptoethanol, 0.0025
% Bromophenol blue), and then suspended at 100 ° C for 3 to
Heated for 5 minutes. After centrifugation (10,000 rpm, 5 minutes), the supernatant was electrophoresed in 7% polyacrylamide containing 0.1% SDS, and the gel was stained with Coomassie blue. As a result, in the case of pEH9, the protein was detected as a main band at a position of a molecular weight of about 156K and in the case of pEA1 at a position of about 149K. These are proteins EH9
And EA1. Since this main band cannot be detected in the cells transduced with only the vector, it is considered to be the band of the desired fusion protein.

Example 4 Preparation of Antibodies to Proteins EH9 and EA1 Containing Part of env Protein: Molecular Weight 156 Detected in Example 3
K and 149K protein bands were cut out and
The gel was ground using a Potter-type glass homogenizer in 2 ml of a solution consisting of MTris-HCl (pH 7.5) and 0.1% SDS. This gel and protein suspension were mixed with an equal amount of Freund's adjuvant (Difco). 2 ml of the mixed suspension was subcutaneously injected (booster) to the back of a rabbit (4 months old male) at 2-week intervals, and the antibody titer in serum was measured every week. The antibody titer was obtained by diluting the serum two-fold and then immunoprecipitating the labeled EH9 or EA1 (immunoprec
It was quantified according to the method of separating it by polyacrylamide gel. Serum showing an antibody titer of 1,000 times or more was obtained by the booster about 3 times.

Example 5 Detection of antibodies in human serum using proteins EH9 and EA1: As in Example 4, E. It was examined as follows whether the fusion protein produced by Escherichia coli can detect the anti-env antibody which is considered to exist in ATLA antibody-positive human serum.

About 0.5 μm of EH9 and EA1 partially purified by acrylamide gel electrophoresis by the method of Example 3
g on a nitrocellulose membrane (Millipore) with a diameter of 0.
It was adsorbed in a spot shape of about 5 cm and dried at room temperature. After shaking the nitrocellulose membrane in 5 ml of bovine serum at room temperature for 1 hour, excess liquid was removed from the nitrocellulose and reacted with 1 ml of serially diluted ATL patient serum at room temperature for 3 hours. Nitrocellulose membrane containing 0.1% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) [50 mM Tris-HCl
(PH 7.5), washed with 0.3M NaCl], and then washed in a conventional manner (HT
owbin et al, Proc. Natl. Acad. Sci., 76 , 4350-435.
4,1979), ¹²⁵ I-labeled anti-human Ig antibody (A
mersham) and exposed to an X-ray film.

As a result, the anti-env antibody of the ATL patient reacts with the fusion protein EH9 or EA1 on the nitrocellulose membrane, and this human antibody molecule is ¹²⁵ I-anti-human I.
Reacted with g-antibody to give black spots on the film. Utilizing this reaction, the env-specific antibody could be measured with a sensitivity 10 times or more that of the conventional antibody titer measurement by the fluorescent antibody method.
Thus, a precipitation reaction radioimmunoassay method using a large amount of EH9 or EA1 protein as an antigen, an ELISA method (a method for detecting an antigen antibody using an enzyme-linked antibody), or a test paper coated with these proteins, etc. It is also possible to analyze a large amount of samples inexpensively and rapidly with higher sensitivity by examining the antibody detection conditions by.

[Brief description of drawings]

FIG. 1 is a flow sheet showing the constitution of the plasmid of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // C07K 14/15 8318-4H 16/08 8318-4H 19/00 8318-4H C12P 21/02 C 9282-4B (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (2)

[Claims] 1. A microorganism comprising a recombinant plasmid in which a 748 bp DNA fragment obtained by HinfI digestion or an AluI digested 551 bp DNA fragment of the env gene of adult T-cell leukemia virus and a gene encoding β-galactosidase are incorporated. 2. The microorganism according to claim 1, wherein the microorganism belongs to Escherichia coli.