JPH07103160B2 - Adult T-cell leukemia virus antigen polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention incorporates a DNA fragment encoding the env gene of adult T-cell leukemia virus (ATLV is also called human T-cell leukemia virus HTLV, but ATLV is used here) or a part thereof. A recombinant plasmid, a microorganism containing the plasmid, an antigenic polypeptide encoded by the env gene of adult T-cell leukemia virus using the microorganism or a part thereof, and a fusion protein of the peptide with an enzyme such as β-galactosidase Regarding manufacturing method.

Adult T-cell leukemia virus
t T cell leukemia (hereinafter abbreviated as ATL) C-type retrours isolated from a patient [Yoshida et al., Proc. Natl. Acad. Sci. 79 , 2031-2036 (198
2)]. ATL patients have a poor prognosis, no effective treatment is available, and half of the patients are often reported to die within 10 months.

In recent years, the presence of an antibody that specifically reacts 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 78 6476-6480 ( 198
1)]. Since then, the presence of this antibody has been confirmed in all ATL patients,
The antigen for this antibody is ATL-associate
d antigen, hereinafter abbreviated as ATLA). this
Antibodies against ATLA (hereinafter abbreviated as anti-ATLA antibodies) are ATL
It has been found to be present in 25% of normal healthy people in the high-frequency zone, and the distribution of anti-ATLA antibody carriers is consistent with the ATL-high frequency area. The main ATLA is this ATLV viral antigen, and the presence of the ATLV genome in the peripheral blood lymphocytes of patients has been proved. Is detected.

ATL and ATLV are closely related, and ATLV is considered to be the causative virus of ATL, but the route of infection is not clear yet. Blood transfusion has been shown to be one of the routes of infection.

Blood transfusions from these people should be avoided because 25% of healthy people in areas of high ATL are positive for anti-ATLA antibodies and these people are very likely carriers of ATLV.

If 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. However, more convenient and rapid detection of anti-ATLA antibody and early detection of ATL are desired.

We have diagnosed ATLV infection, blocked ATLV infection, ATLV
We studied a method to supply ATLA in large quantities and at low cost, which is useful for the treatment of leukemia caused by. As a result, a DNA fragment in the region encoding the antigen peptide gene called the env gene of the ATLV genome was incorporated into vector DNA using the recombinant DNA technique, and the microorganism containing the obtained recombinant DNA was cultured. Or an ATL antigen peptide encoded by a part thereof or the peptide and β
The present invention has been completed by finding that a fusion protein with an enzyme such as galactosidase is produced and accumulated in a significant amount.

One of the env gene products is a glycoprotein with a molecular weight of 62,000 (gp62)
Have already been reported [Hattori et al: Gan (Gan
n) 74 , 790-793 (1983)], and the total DNA of ATLV
The sequence was determined by the present inventors [Japanese Patent Application No. 57-214287, Proc. N.
atl.Acad.Sci.USA 80 , 3618-3622 (1983)] is encoded by the env gene or a part thereof in a microorganism.
This is the first time that the present invention has produced a TLV antigen peptide and a fusion protein of the peptide and an enzyme such as β-galactosidase.

The present invention will be described in detail below.

The present invention relates to a recombinant plasmid incorporating a DNA fragment encoding the env gene of ATLV or a part thereof, a microorganism containing the plasmid, and an ATLV en using the microorganism.
v A method for producing an antigenic polypeptide encoded by a gene or a part thereof is provided.

Construction of the recombinant plasmid of the present invention is performed 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.
. ^{[Kiyoshikira, Proc.Natl.Acad.Sci, USA, 8 - 0} , 3613-3622
(1983)] can be used. The genome of ATLV is composed of LTRs at both ends and at least 4 genes of gag, pol, env and pX. Among these, the protein encoded by the env gene is presumed to be a glycoprotein involved in the infectivity of ATLV, so diagnosis of ATLV infection, prevention of ATLV infection, ATLV infection,
It is expected to be used for the treatment of leukemia caused by the above, and also for the 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.

As the vector DNA, any vector can be used as long as the inserted target DNA can be expressed in a microorganism. Specific preferred plasmids include pORF1 and pORF1
You can raise ORF2. These plasmids are Wein
stock et al.:Proc.Natl.Acad.Sci.USA 80 , 4432-4436,
It is the plasmid described in 1983 and can be prepared by the method described in the literature.

The recombination of the DNA encoding the env gene of ATLV or a part thereof with the vector DNA such as pORF1 or pORF2 is carried out by a general recombinant DNA technique in which both DNAs are digested with a restriction enzyme and then ligated with T4 DNA ligase. Can be done using. 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.

In the case of pATK08 and pORF2 shown as specific examples, as shown in FIG. 1, the env gene of pATK08 was digested with the restriction enzyme HinfI, and the end was blunt-ended with DNA polymerase I (Klenow fragment) to make this fragment The recombinant plasmid pEH9 can be constructed by inserting it into the SmaI site. In addition, pEA1 can be obtained by digesting the env gene of pATK08 with the restriction enzyme AluI and inserting it into the pORF1 site. As shown in FIG. 1, pEH9 incorporates a DNA fragment of 748 base pairs (hereinafter abbreviated as bp) located in the first half of the env gene, and pEA1 contains 551 in the latter half of the env gene.
A bp DNA fragment is incorporated.

The reaction conditions necessary for preparing the above recombinant plasmid are as follows.

Digestion of DNA with restriction enzymes usually requires 0.1 to 100 µg of DNA.
~ 200 mmol (preferably 10-40 mmol) Tris-HCl (pH 6.0-9.5, preferably pH 7.0-8.0), 1-150
18 to 4 using 0.1 to 300 units of restriction enzyme (preferably 1 to 3 units of enzyme per 1 μg of DNA) in 2 to 20 mmol (preferably 5 to 10 mmol) of MgCl _{2 of} millimolar NaCl.
The digestion reaction is performed at 2 ° C (preferably 32 to 38 ° C) for 15 minutes to 24 hours. The reaction is usually stopped at 55 to 75 ° C (preferably
Although heating is performed at 63 to 70 ° C. for 5 to 30 minutes, a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

When binding a DNA fragment, 2 to 200 mmol (preferably 10 to 70 mmol) of Tris-hydrochloric acid (pH 6.0 to 9.5, preferably pH 7.0 to 8.0), 2 to 20 mmol (preferably 5 to 10)
MgCl ₂ 0.1 to 10 mmol mmol) (preferably 0.5 to
2 mmol) ATP, 1-50 mmol (preferably 5-1)
0 mmol of T4 DNA ligase in dithiothreitol.
1 to 37 ° C (preferably 3 to 20 ° C) using 1 to 10 units
The binding reaction is performed for 5 minutes to 72 hours (preferably 2 to 20 hours).

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

ATL encoded by the env gene or a part thereof by transfecting a microorganism with a recombinant plasmid such as pEH9 or pEA1 and culturing the resulting transduced strain
A V antigen polypeptide can be obtained.

Escherichia coli is preferably used as the microorganism, and specifically, a derivative of Escherichia coli K-12 strain MH3.000 [GM Weins
tock et al ., Proc.Natl.Acad.Sci.USA 80 , 4432-4436,
1983] or TK1046 (Id.).

Transduction is performed by the method of SN Cohen et al. [Proc. Natl. Acad. Sci., U
SA 69 , 2110 (1972)]. 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 performed by, for example, culturing this strain in L-Broth, collecting the cells, and then removing 125 mM Tris-H
After suspending in a buffer consisting of Cl (pH6.8), 2% SDS, 0.7M 2-mercaptoethanol and 0.0025% bromophenol blue, heat and centrifuge the supernatant in a 7% polyacrylamide gel containing SDS. Then, the gel is detected by staining with Coomassie blue. It can be confirmed that the polypeptide detected here specifically immunoprecipitates with patient serum, and it is understood that it has the property as an ATLV antigen peptide.

Isolation of plasmids from microorganisms is performed by HC Birnboim et al .: Nucleic Acids Rese
arch) 7 , 1513 (1979).

The pEH9 and pEA1 transduced strains are Escherichia coli CI9 in the American American Type Culture Collection.
ATCC 39591 and Escher ichia coli CI10 ATCC 3959
2 have been deposited internationally.

Examples of the present invention will be shown below.

Example 1. Plasmid pEH9 (plasmid containing the first half of the env gene)
Construction: Plasmid pATK08 was collected from E. coli ATCC 32246 by the method described in Kiyoki et al., Proc. Natl. Acad. Sci. USA, 80 , 3613-3622, 1983. 20 μg of pATK08 is added to 50 mM NaC
l, 6mM Tris-HCl (pH7.5), 6mM MgCl ₂ and 2mM _2- mercaptoethanol dissolved in 200μl of buffer solution, and the restriction enzyme HinfI (Takara Shuzo Co., Ltd. ) Add 60 units,
The reaction was carried out 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 was added to 50 mM Tris-HCl (pH 7.
5), 5mM MgCl ₂ , 2mM dithiothreitol, 0.1mM dATP, 0.1
100 μl of a solution containing mM dTTP, 0.1 mM dGTP and 0.1 mM dCTP
In addition, 3 units of DNA polymerase I (Klenow fragment) [manufactured by Bethesda Research Laboratories (hereinafter referred to as BRL)] was added, and the mixture was reacted at 15 ° C for 1 hour. In this reaction, staggered
The staggard end has been restored to a blunt end. The reaction product was subjected to 1% agarose gel electrophoresis, and a 748 bp DNA fragment was recovered.

On the other hand, the vector plasmid pORF2 was linearized using SmaI by the method described in the same document.

0.5 μg of 748 bp DNA fragment and 5 μg of linearized pORF2
In addition to 0.5 ml of a solution consisting of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM dithiothreitol and 1 mM ATP, 5 units of T4 DNA ligase (Takara Shuzo) was added, and the temperature was 14 ° C.
The binding reaction was carried out 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.
atl.Acad.Sci.USA. 80, E.coli.MH activated 3000 and (Id.) was transduced by 4432-4436,1983]. Transfected E. coli MH 3,000 with L-Broth (medium containing 1 g bactotryptone 10 g, yeast extract 5 g; NaCl 5 g) and 10 m
g / ml 5-bromo-4-chloro-3-indolyl-β-
D-galactoside (5-bromo-4-chloro-3-indol
It was removed to an agar medium containing yl-β-D-galactoside (hereinafter referred to as XG) (L-Broth contains 15 g of agar) and cultured at 25 ° C for 2 days. A bacterium that produces a fusion protein containing β-galactosidase (β-galactosidese) into which a desired DNA fragment has been inserted decomposes XG to form a blue colony. A colony showing a blue color was selected and cultured with L-Broth at 25 ° C for 3 hours. From cultured cells, HC Birnbiom et al .: Nucleic Aci
Plasmid 100 by the method described in ds Research 7 , 1513, 1979.
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 (see Maxam et al.
lbert, Methods in E
nzymology) 65 , 499-560 (1980)], and confirmed by analyzing the DNA inserted into the plasmid and the base sequence in the vicinity thereof.

Example 2. Plasmid pEA1 (plasmid containing the latter half of the env gene)
Configuration: The plasmid pATK08 was collected in the same manner as in Example 1.

20 μg of pATK08 was added to 50 mM NaCl, 6 mM Tris-HCl (pH 7.5), 6
It was dissolved in 200 μl of a buffer solution containing mM MgCl ₂ and 2 mM 2-mercaptoethanol, 60 units of the restriction enzyme AluI was further added, and the mixture was 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 551 bp was recovered.

On the other hand, pORF1 was linearized as in Example 1.

50 μg of 0.5 μg of 551 bp DNA fragment and 5 μg of linearized pORF1
Add 0.5 ml of a solution consisting of mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 20 mM dithiothreitol and 1 mM ATP, and add T4
5 units of DNA ligase was added and the binding reaction was carried out at 14 ° C for 3 days.

E. coli MH3.000 activated by the above method was transduced with a solution obtained by extracting the reaction solution with phenol and removing proteins. The 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 into which a desired DNA fragment has been inserted decomposes XG to form a blue colony. Therefore, this is selected and cultured in the same manner as in Example 1, and The plasmid was collected.
The resulting plasmid is called pEA1. It was confirmed as in Example 1 that pEA1 had a structure containing the latter half of the env gene as expected.

Example 3. Large expression of pEH9 and pEA1: Microorganisms containing a plasmid incorporating a β-galactosidase such as pEH9 and pEA1 and the env gene, due to the toxicity of the fusion protein encoded by the plasmid,
If it is expressed in a large amount, it cannot grow. The following treatments were performed for large expression of pEH9 and pEA1.

GMWeinstock et al ., Proc.Natl.Acad.Sci, USA 80 , 44
According to the method described in 32-4436, 1983, E. coli TK1046, whose gene for activating the promoter incorporated in the plasmid is cold-sensitive, was transduced with pEH9 and pEA1. The transduced E. coli TK1046 was cultured in 25 ml of L-Broth at 30 ° C until the OD ₆₀₀ reached about 0.2 to 0.3, and then at 37 ° C for 1 hour. Collect the cells and
1/70 volume of buffer [125mM Tris-HCl (pH6.8), 2% SD
S, 0.7 M 2 mercaptoethanol, 0.0025% bromophenol blue) and then heated at 100 ° C. for 3 to 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 molecular weight was about 156K, and in the case of pEA1, it was about 149K.
The protein was detected as a main band at the position. These are called 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 the env Protein: The bands of the proteins with molecular weights of 156K and 149K detected in Example 3 were cut out, and 50 mM Tris-HCl (pH 7.5) and 0.1 were added.
The gel was crushed using a Potter-type glass homogenizer in 2 ml of a solution containing% SDS. An equal volume of this gel and protein suspension in Freund's adjuvant (Difco)
Mixed with. Rabbit (male 4 months old) with 2 ml of mixed suspension
Subcutaneous injection (booster) at 2 weeks intervals on the back of
The antibody titer in serum was measured every week. The antibody titer was two-fold dilution of serum and immunoprecipitation of labeled EH9 or EA1 (immunoprec
It was quantified according to the method of separating it by polyacrylamide gel. A serum showing an antibody titer of 1.000 times or more was obtained by about 3 times of booster.

Example 5 Detection of Antibodies in Human Serum Using Proteins EH9 and EA1: As in Example 4, the fusion protein made in E. coli detects anti-env antibody that is considered to be present in ATLA antibody-positive human serum. We examined whether it could be done as follows.

About 0.5 μg of EH9 and EA1 partially purified by acrylamide gel electrophoresis by the method of Example 3 was adsorbed on a nitrocellulose membrane (manufactured by Millipore) in a spot shape with a diameter of about 0.5 cm, and dried at room temperature. After shaking the nitrocellulose membrane in 5 ml of bovine serum at room temperature for 1 hour, the 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 solution containing 0.1% Tween 20 (Wako Pure Chemical Industries, Ltd.) [50 mM T
ris-HCl (pH 7.5), 0.3M NaCl]
in et al , Proc.Natl.Acad.Sci., 76 , 4350-4354,1979)
¹²⁵ I-labeled anti-human Ig antibody (Amersham)
It was reacted with 5 μl and exposed to X-ray film.

As a result, the anti-env antibody possessed by the ATL patient reacted with the fusion protein EH9 or EA1 on the nitrocellulose membrane, and this human antibody molecule reacted with ¹²⁵ I-anti-human Ig antibody to give a black spot on the film. .

Using this reaction, env-specific antibodies could be measured with a sensitivity 10 times higher than that of conventional antibody titer measurement by fluorescent antibody method. Thus, the precipitation reaction radioimmunoassay method using the EH9 or EA1 protein produced in large quantities as an antigen, ELIS
A large amount of sample can be analyzed inexpensively and quickly with higher sensitivity by examining method A (antigen-antibody detection method using enzyme-linked antibody) or test strips coated with these proteins. It is also possible.

[Brief description of drawings]

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

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Claims (5)

[Claims] 1. H of the env gene of adult T-cell leukemia virus
748 bp DNA fragment by infI digestion or 55 by AluI digestion
Antigen polypeptide encoded by 1 bp DNA fragment and β
A fusion protein with galactosidase. 2. The H of the env gene of adult T-cell leukemia virus
748 bp DNA fragment by infI digestion or 55 by AluI digestion
A microorganism having a recombinant plasmid in which a 1 bp DNA fragment and a gene encoding β-galactosidase are incorporated is cultured in a medium, and a 748 bp DNA fragment by HinfI cleavage of the env gene or 551 bp by AluI digestion in the culture. A fusion protein of the antigen polypeptide encoded by the DNA fragment of β-galactosidase is accumulated and produced, and the fusion protein is collected from the culture, by HinfI cleavage of the env gene of adult T-cell leukemia virus. 748bp D
A method for producing a fusion protein of an antigenic polypeptide encoded by a DNA fragment of 551 bp by NA fragment or AluI digestion and β-galactosidase. 3. The method according to claim 2, wherein the microorganism belongs to the genus Escherichia. 4. The method according to claim 3, wherein the microorganism belongs to Escherichia coli. 5. The method according to claim 2, wherein the recombinant plasmid is pEH9 or pEA1.