JP2525054B2 - Adult T-cell leukemia virus infection diagnostic agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to adult T cell leukemia (Adult Tcellleukemia;
Human T cell leukemia virus-I (hereinafter referred to as ATL); HTLV-I
Abbreviated as "), which is a novel HTLV-I infection diagnostic agent using as an antigen a polypeptide having an antigenicity to an antibody of p21 which is a protein located in and inside the membrane.

[Conventional technology]

ATL is a malignant disease that affects adults, and its etiology is due to HTLV-I infection, and it is known that ATL develops at a frequency of about 0.1% among infected persons. This HTLV-I
Blood transfusion, mother-to-child transmission, sexual transmission, and the like are known as infection routes, and it is desired to detect HTLV-I infected individuals at an early stage and to prevent such infections.

Conventionally, as such a diagnostic agent for HTLV-I infection, HT-LV
Detection of antibodies to adult T-cell leukemia-related antigens in plasma or serum when infected with -I
At least one selected from the solubilized proteins of
No. 58-187861), or those using an antigen protein and HTLV-I virus particles obtained by treating the cells with a surfactant (JP-A-59-62527).

However, the diagnostic agent is used in adult T cells such as T cell membranes and nuclei.
It also contains an antigen showing a non-specific reaction other than the cell leukemia-related antigen, and a non-specific immune reaction of such an antigen produces a so-called false-positive sample, which reduces the diagnostic accuracy of HTLV-I infection. there were.

Further, in order to produce a polypeptide having an antigenicity against an antibody against HTLV-I, Escherichia coli recombined with a gene of HTLV-I envelope protein (hereinafter abbreviated as env protein) is grown. Attempts have been made to recover the produced polypeptide.

However, the polypeptide obtained by the above method had low antigenicity to the antibody against HTLV-I. This is presumed to be due to the fact that polypeptides produced in bacteria such as Escherichia coli have no modification such as a sugar chain addition reaction.

On the other hand, a method for producing a polypeptide in which a recombinant virus in which a structural gene of a protein useful as a diagnostic agent is recombined into a polyhedral protein structural gene portion of silkworm nuclear polyhedrosis virus DNA is propagated in a silkworm established culture cell or in a silkworm living body But,
It is known from JP-A-62-208276 and JP-A-61-9288.

However, these publications have no specific description regarding the application of the above method to the production of the polypeptide which is the above-mentioned antigen protein of HTLV-I. To produce a polypeptide which is an antigenic protein of HTLV-I by such a method, the polyhedrin protein structural gene of the silkworm nuclear polyhedrosis virus DNA is recombined with any part of the structural gene of HTLV-I. If it is possible to produce a polypeptide, and further, if a polypeptide is obtained, whether or not the polypeptide has an antigenicity to an antibody of HTLV-I requires further studies. It was

[Problems to be Solved by the Invention]

The object of the present invention is to overcome the above-mentioned problems of the prior art,
A polypeptide having a high antigenicity against an antibody against HTLV-I was developed, and the polypeptide was used as an antigen.
It is an object of the present invention to provide a diagnostic agent which can be diagnosed with extremely high sensitivity and accuracy in the diagnosis of HTLV-I infection.

[Means for solving the problem]

In the diagnosis of HTLV-I infection, the inventors of the present invention have conducted extensive studies to develop a diagnostic agent that can be highly sensitively and accurately diagnosed. As a result, DNA derived from the HTLV-I env protein gene
Specific fragments of the silkworm nuclear polyhedrosis virus (Bombyx N
uclear Polyhedrosis Virus; hereinafter abbreviated as BmNPV)
It was found that such a purpose can be achieved by using, as an antigen, a polypeptide expressed by infecting a silkworm established culture cell or a silkworm larva with a recombinant virus in which a part of the polyhedrin protein structural gene of The present invention has been activated.

That is, the present invention relates to D-derived from the HTLV-I env protein gene.
Among NAs, a fragment of BmNPV is defined by a fragment containing DNA encoding p21 and cleaved within 17 base pairs upstream from the 5'end of the DNA (hereinafter abbreviated as HTLV-I5'-3 'fragment). A part of the somatic protein structural gene was recombined, and then a polypeptide expressed by infecting silkworm established culture cells or silkworm larvae with the recombinant virus was used as an antigen.
The present invention relates to a diagnostic agent for HTLV-I infection.

In the diagnostic agent of the present invention, the polypeptide used as an antigen is a part of polymorphic protein gene of BmNPV, which is recombined with the HTLV-I5'-3 'fragment, and then the recombinant virus is used to establish silkworm established cell lines. Alternatively, it is expressed by infecting silkworm larvae. Hereinafter, the method for producing the polypeptide will be described in detail.

I. Method for Producing Recombinant Virus In the present invention, the method for producing the recombinant virus used in the method for producing the above-mentioned polypeptide is not particularly limited. As a typical production method, lymphocytes were isolated from peripheral blood of ATL patients, and HTLV-I5′-3 ′ was extracted from DNA extracted from the lymphocytes.
The fragment was cut out, and this was inserted into the cloning site of the silkworm expression system vector (pBF vector) to give a recombinant vector. Then, using this vector, HTLV-I5'-3 '
Examples include a method of producing the fragment by recombining it with a part of the polymorphic protein structural gene of BmNPV.

I-1BmNPV In the present invention, BmNPV recombined by the HTLV-I5′-3 ′ fragment is widely known to sericulture companies, and T3 strain is a typical strain isolated by Maeda and Furusawa. Yes, the viral DNA of this strain is ATCC (American
Type Culture Collec-tion) has been deposited as ATCC No. 40188 and is easily available. It can also be isolated from BmNPV infected silkworms by known methods.

The BmNPV DNA can be represented by the restriction map shown in FIG.

Among the BmNPV DNA, in the present invention, HTLV-I5'-
The part recombined by the 3'fragment is a part of the polyhedron protein structural gene of the polyhedron protein gene shown in Fig. 1 excluding the promoter part.

I-2 Method for Obtaining HTLV-I5'-3 'Fragment HTLV-I is a retrovirus having RNA as a gene, and is used as a nucleotide sequence of env gene of DNA synthesized from this gene RNA in infected cells. Seiki et al.
c.Natl.Acad.Sci.USA 80 "(1983) published on page 3621 is known.

In the present invention, the method for obtaining the HTLV-I5'-3 'fragment is not particularly limited. A typical method is a method of obtaining a nuclear HTLV-I5'-3 'fragment from DNA located in lymphocytes in peripheral blood of ATL patients.

To illustrate a typical method, first, lymphocytes are separated from peripheral blood of an ATL patient, DNA is extracted from the lymphocytes, and then cut with a restriction enzyme EcoRI to obtain DNA containing a fragment of about 20 kbp. The DNA was then transformed into the Charon 4A vector Ec
After connecting to the oR I restriction enzyme cleavage site, the phage containing HTLV-I provirus was isolated by screening, and subcloning using the E. coli vector system represented by pUC vector from the provirus and restriction enzyme This is a method of obtaining an HTLV-I5'-3 'fragment by cleavage.

The HTLV-I 5'-3 'fragment contains DNA encoding p21, which is a protein located in and inside the membrane among DNAs derived from the HTLV-I env protein gene (see Fig. 9). In addition, any DNA can be applied as long as it is cleaved within 17 base pairs upstream from the 5'end of the DNA.

The DNA sequence encoding p21 corresponds to the 6116th to 6643th sequences of the HTLV-I env protein gene sequence published by seiki et al., As shown in FIG.
The 1799 base pairs upstream from the 5'end correspond to the 6099th one.

According to the knowledge of the present inventor, even if the HTLV-I 5'-3 'fragment contains a DNA encoding p21, more than 17 base pairs were added upstream from the 5'end of the DNA. If it contains, by the method described below, even if the recombinant virus of BmNPV obtained by using the fragment is grown to produce a polypeptide, the polypeptide cannot be obtained or even if it is obtained. Also has no antigenicity against the antibody against p21 of HTLV-I, and the object of the present invention cannot be achieved.

If a suitable site is shown, a BamHI restriction enzyme cleavage site, which is arranged with GATC from a position separated by 17 base pairs upstream from the 5'end of the DNA encoding p21, can be mentioned.

In the present invention, the HTLV-I5′-3 ′ fragment is pBF
After being recombined into a vector, Bm
Recombinant with a part of NPV polyhedron protein structural gene to be a recombinant virus. Used to obtain recombinant virus
The pBF vector is characterized by the restriction map of FIG. That is, the pBF vector is a vector containing the promoter region of the polyhedron protein gene of BmNPV DNA, the vicinity of the polyhedral protein structural gene, and the gene of the pUC vector which is an E. coli vector.

The pBF vector has a cloning site for XbaI, EcoRI, and StuI restriction enzyme cleavage sites.

Such pBF vectors vary depending on the site containing the polyhedron protein structural gene starting from the nucleotide sequence ATG upstream of the cloning site, and pBF4 to pBF133 as shown in FIG. 3 exist.

In the production of recombinant vectors, any of the above vectors may be used as long as the HTLV-I5'-3 'fragment portion to be inserted and the pBF vector upstream portion have the same reading frame. However, 5'-
In order to construct a recombinant virus capable of efficiently expressing the 3'fragment in the silkworm established culture cells and silkworm larvae, a gene portion encoding a part of the BmNPV DNA-derived polyhedral protein structural gene starting from the nucleotide sequence ATG Contains a lot of
It is preferable to use pBF vectors, for example pBF124, pBF129, pBF133 and other pBF vectors.

I-3- (1) Method for introducing HTLV-I5'-3 'fragment into pBF vector Insertion of HTLV-I5'-3' fragment into pBF vector was carried out by EcoR I, which is present at the cloning site of pBF vector.
What is necessary is just to make use of the Xba I and Stu I restriction enzyme cleavage sites. HTLV-I5'-3 'at the pBF vector cloning site
To insert the fragment, EcoR I, Xba I or Stu I restriction enzyme cleavage sites are ligated to both ends of the fragment by known means such as DNF synthesis or insertion into pUC vector which is a vector system of E. coli, and cleavage. Or EcoR I, 3'at the 5'end of the fragment
Either a Stu I restriction enzyme cleavage site is connected to the end, or an Xba I is connected to the 5 'end of the fragment, and a Stu I restriction enzyme cleavage site is connected to the 3' end of the fragment. Among the above, EcoR I or
A method of connecting Xba I restriction enzyme cleavage sites is preferred.

When an EcoRI restriction enzyme cleavage site is connected to both ends of the 5'-3 'fragment, the fragment is connected to the pBF vector cut with EcoRI restriction enzyme. Also, HTLV-I5'-3 '
Similarly, when Xba I restriction enzyme cleavage sites are ligated to both ends of the fragment, the fragment is ligated to the pBF vector cleaved with Xba I. In this case, at the time of ligation, the pBF vector cleaved with a restriction enzyme in advance was subjected to alkaline phosphatase treatment, and self-ligation of the pBF vector (s
It is preferable to avoid elfligation).

When EcoR I is connected to the 2'end of the HTLV-I 5'-3 'fragment and an Stu I restriction enzyme cleavage site is connected to the 3'end, the fragment is
Connect the BF vector cut with EcoR I, Stu I, and
When Xba I is connected to the 5 ′ end of the LV-I 5′-3 ′ fragment and a Stu I restriction enzyme cleavage site is connected to the 3 ′ end, the fragment should be connected to the pBF vector cut with Xba I and Stu I. Good.

Such a connection method can be performed by a known method using ligase. For example, pBF cleaved with restriction enzymes
The amount of DNA of the 5'-3 'fragment to be inserted is adjusted to be 3 to 8 times the amount of DNA of the vector, and for example, T4D
This is a method of connecting using NA ligase.

Separation and confirmation of the pBF vector into which the above HTLV-I5'-3 'fragment has been inserted can be performed by the following method.

That is, the separation was carried out by adding the above-mentioned connection reaction solution to a competent cell of Escherichia coli represented by JM109 strain (Takara Shuzo Co., Ltd. No.9052) and MV1184 strain (Takara Shuzo Co., Ltd.), and adding E. coli by a known method. After transformation, since the pBF vector contains the ampicillin resistance gene, the liquid after transformation is inoculated into an LB agar medium containing ampicillin, and the appropriate temperature above room temperature, for example, at 37 ° C for 12 hours to 20 hours. It can be carried out by obtaining a single colony that appears after culturing for a period of time as a transformant.

In addition, the confirmation of the pBF vector into which the HTLV-I5'-3 'fragment has been inserted is confirmed by the recombinant vector boiling method or the alkaline lysis method existing in the transformant strain.
mini-preparation (mini pr
) to obtain a recombinant vector suspension, and the recombinant vector suspension prepared in this manner is used for HTLV-I5 ′.
After cutting with any restriction enzyme that can confirm that the 3'fragment has been inserted, such as EcoR I and Xba I, and subjecting the cleaved product to agarose gel electrophoresis and staining with ethidium bromide, a band is expected at the expected position. It can be done by checking whether it exists. In the case of a recombinant vector in which the 5'-3 'fragment with EcoR I or Xba I restriction enzyme cleavage sites on both ends was inserted into the EcoR I or Xba I restriction enzyme cleavage sites of the pBF vector, the upstream of the pBF vector was used. A restriction enzyme, such as BamHI, Xho, which can be used to confirm whether the 5'-3 'fragment to be inserted into the site is inserted in the correct direction.
After cutting with I etc., the cut product is subjected to agarose gel electrophoresis in the same manner, and after staining with ethidium bromide, it may be confirmed at the same time whether or not there is a band at a predicted position.

The recombinant vector isolated as a transformant by the above method is preferably used by increasing the amount of the transformant by growing the transformant. For example, a transformant possessing the recombinant vector is inoculated into an LB liquid medium containing ampicillin, and at an appropriate temperature of room temperature or higher, for example, 37 ° C.
It can be carried out by culturing with shaking for 12 to 20 hours, performing a medium preparation from the culture using an alkaline lysis method, and obtaining a recombinant vector suspension. .

It is preferable to confirm again whether the obtained recombinant vector is the target recombinant vector by the same method as described above.

It is preferable that the obtained recombinant vector suspension is treated with RNase (RNase treatment) and used as a recombinant vector suspension for obtaining a recombinant virus.

I-3- (2) Acquisition of recombinant virus In the present invention, a recombinant BmNPV in which a part of the polyhedrin protein structural gene is recombined with the HTLV-I5'-3 'fragment
Is obtained by simultaneously transfecting BmNPV DNA and the recombinant vector into silkworm-established cells using the calcium precipitation method (co-transfection), and re-elevating the allele between the recombinant vector and BmNPV DNA. be able to.

The above co-transfections are specifically
BmNPV in the presence of 25M calcium chloride and carrier DNA
Mix the DNA and the recombinant vector DNA at a molar ratio of 1: 100, and then add 0.28 M sodium chloride in H
Method of Maeda and Furusawa et al., In which a mixed solution of EPES buffer (pH 7.1) and phosphate buffer is added, and after mixing, the mixed solution is added into Bm cultured cells (Japanese Patent Publication No. 61-9297). It is desirable to follow

After co-transfection, the reaction solution containing the recombinant virus is cultured at a temperature near room temperature, for example, 27 ° C for 5 to 6 days. After the culture, the medium is collected and centrifuged, and the supernatant is used for cloning the recombinant virus. use. The recombinant virus was cloned from the supernatant of the reaction mixture obtained by co-transfection by the plaque assay method [J. Seric Sci.
n. 53 547 (1984)] or by limiting virus isolation by the limiting dilution method. Either method may be used, but it is better to use the limiting dilution method in view of the easiness of the operation method and the small number of separations.

The cloning of the recombinant virus using the limiting dilution method is carried out by diluting the virus solution obtained by co-transfection, and adding 1 × 10 ⁵ to 1 × 10 ⁶ silkworm cells to the virus dilution solution. Infection was carried out by mixing 1: 1 with a number / ml silkworm culture medium, preferably the silkworm-established cell liquid prepared at a concentration of TC-10 medium (see Table 2), and this mixture was microtitered. After injecting into the wells in the tray and culturing at a temperature around room temperature, for example, 27 ° C., and after 2 to 7 days of culturing, the wells in the microtiter tray are microscopically examined and assembled in the shape and morphology of the silkworm cells found in the wells. The presence or absence of a replacement virus is determined. Three types of morphology of silkworm cells found by microscopic examination can be confirmed as shown in FIG.

The silkworm cells shown in FIG. 6 showing the morphology infected with virus, and the medium in the well in which only the cells in which polyhedrin protein is not detected exist are collected, centrifuged, and the supernatant is collected. A recombinant virus solution is obtained by collecting. When BmNPV, which is a wild strain, and a recombinant virus are mixed in the well, it is preferable to collect the medium in the well and repeat limiting dilution to isolate the recombinant virus. .

II. Method for Producing Polypeptide II-1 Silkworm Established Culture Cell In the present invention, the silkworm established culture cell to be infected with the recombinant virus may be any cell as long as it is a silkworm established culture cell capable of proliferating BmNPV.

Cultured silkworm cells that can grow BmNPV include Volkma
nLE, and Goldsmith, PA (1982): Appl.Environ.Mic
robiol., 44, 227-233, (ATCC No. C
RL-8910) and Bm cloned by Maeda et al. From Bm.N.
-Cell lines such as N2, Bm and N4 are known. BmNPV
It is suitable to use Bm.N4 silkworm established culture cells in terms of good growth and easy handling. As the silkworm resin cultured cells used for infection, it is appropriate to use those cultured under known culture conditions, for example, in TC-10 medium containing 10% fetal bovine serum at 27 ° C. for 4 days. Is.

II-2 Method for Infecting Cells and Silkworm Larvae with Recombinant Virus and Propagation Method In the present invention, the polypeptide of interest is prepared by infecting silkworm established culture cells or silkworm larvae with the recombinant virus, and allowing the cells to grow. Expressed. As a method for infecting silkworm-established cultured cells with the recombinant virus, known methods can be used without particular limitation. For example, after placing the prepared culture solution of silkworm established culture cells in a container and depositing the cells on the bottom surface of the container, the old culture solution is removed so that the silkworm established culture cells attached to the bottom surface of the container are not peeled off. It is common to use a method in which the bovine serum as a stabilizer is extracted and added to a silkworm culture medium, and the recombinant virus is added dropwise to the culture. After infecting the recombinant virus, the recombinant virus is grown at a temperature near room temperature, for example, 27 ° C.
For several days.

After culturing, the infected silkworm established culture cell culture was centrifuged, and the precipitated cells were used for confirmation of HTLV-I env protein expression and purification of HTLV-I env protein. It may be used as a recombinant virus solution to infect S.

The method of infecting the silkworm larva with the recombinant virus is not particularly limited. In general, it is preferable to use the fifth-instar silkworm larva as the silkworm larva to be infected. The silkworm larvae are infected by transdermally transfecting a recombinant virus solution having a high virus titer or a recombinant virus solution not subjected to the operation, which is obtained as a supernatant by the infection of the silkworm resin cultured cells.
It can be performed by injecting about 10 to 100 μ. After infecting the recombinant virus, the infected silkworm is bred to grow the recombinant virus, and polyhedrin protein and HTLV-
A fusion protein of I env protein is accumulated in the fat body of silkworm larvae.

The breeding method of the silkworm is not particularly limited, but either mulberry leaf or mulberry leaf homogenate, paste-like sample sterilized and freeze-dried (manufactured by Kyodo Sample Co., Ltd., etc.) soaked with distilled water is used. Give a temperature near room temperature, for example, 27 ℃
A general method of culturing at may be employed.

The breeding period is preferably performed until just before the silkworm larvae die. Although the breeding period varies slightly depending on the virus titer of the recombinant virus solution to be infected, 3 to 5 days after infection can be used as a guide for the breeding period.

A fat pad is taken out from the silkworm larvae described above, and the fat pad is used for confirmation of HTLV-I env protein expression and purification of the HTLV-I env protein.

The method for obtaining the fat pad is to dissect silkworm larvae infected with the recombinant virus by carefully cutting the epidermis so as not to cut the midgut, and after removing organs such as the midgut, accumulate in the lower abdomen with a spatula etc. It is recommended to obtain the fat body by scraping it.

In the present invention, the method for separating the polypeptide from the fat body of the recombinant virus-infected silkworm established culture cells and recombinant virus-infected silkworm larvae obtained by the above-mentioned method is not particularly limited. A preferred method is to suspend the silkworm-established cultured cells or fat pad in a buffer solution, disperse by sonication, add an aqueous urea solution and sonicate again, and then centrifuge to recover the precipitate.

The polypeptide obtained by the above method may be SD if necessary.
It is dissolved in an S aqueous solution, purified by conventional methods such as purification by ion exchange resin and gel filtration, and then used as an antigen of the diagnostic agent of the present invention.

The above-mentioned polypeptide is a part of the polyhedron protein encoded by the polyhedrin gene portion of pBF133 and the like and HTLV-I.
A fusion protein with the p21-containing portion of the HTLV-I env protein, which is a protein located in the membrane and inside the membrane, which is encoded by the 5'-3 'fragment. May be used in combination by further purification by removing polyhedrin protein.

If we specifically show how to remove the polyhedrin protein, we used a proteolytic enzyme that recognizes the amino acid sequence of the peptide part that connects the polyhedrin protein and p21. It is preferable to purify by the purifying means used.

Next, a diagnostic agent for an HTLV-I infected person using the polypeptide obtained by the above-mentioned production method as an antigen will be described.

That is, the diagnostic agent of the present invention uses the above-mentioned polypeptide as an antigen, and p21 that causes an antigen-antibody reaction with the antigen from the test liquid.
The infection with HTLV-I is diagnosed by detecting the antibody against. Therefore, the diagnostic agent of the present invention diagnoses not only ATL patients, but also undiagnosed individuals who carry an antibody against p21 as a result of infection with HTLV-I. In addition, the test solution may be, for example, plasma or serum separated from blood collected according to a conventional method from a subject who wishes to diagnose HTLV-I infection, or a solution obtained by diluting the same with an appropriate buffer. used. In the present invention, the above p21
Detection of an antibody against is not particularly limited as long as the above-mentioned polypeptide is used as an antigen, and a known immunological test method can be adopted. Examples of suitably adopted methods include a method of sensitizing insoluble carrier particles with the above-mentioned polypeptide and detecting an agglutination reaction of the carrier particles caused by an antigen-antibody reaction, an enzyme immunoassay method, and the like. Further, when the produced polypeptide is in a trace amount or is insufficiently purified, it is preferable to detect it by Western blotting. Hereinafter, each of the above detection methods will be described.

First, in the method for detecting the agglutination reaction, the agglutination reaction is a microtiter method for observing the agglutination state of particles in a well of a microtiter plate, or the transmittance of a measurement system that decreases as the agglutination reaction proceeds. It can be detected by a known method for observing and quantifying carrier particle aggregates such as a measuring method. Further, the insoluble carrier particles used are antigen-
Known ones used for the antibody reaction are used without any particular limitation. Examples of those that are preferably used include:
Organic polymer latex and inorganic compound particles obtained by emulsion polymerization of animal erythrocytes such as human, sheep and chicken, polystyrene, styrene-butadiene copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate, polycarbonate, etc. And inorganic organic particles such as glass beads, shirasu porous glass, silica, and alumina. Among them, the composite polymer particles described in JP-A-62-286533 are preferable for carrying out the microtiter method because they have high monoparticle property and few non-specific reactions. The average particle diameter of the insoluble carrier particles is 0.
The thickness of 5 to 3.0 μm, preferably 1.0 to 2.0 μm is used. The method of sensitizing the insoluble carrier particles with the polypeptide is not particularly limited, and a known antigen sensitizing method can be used. Examples of comfortable methods include tannic acid, glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, dichloronitrobenzene, carbodiimides, quinones, and chemical coupling methods using a coupling agent such as chromium chloride, or physical adsorption. Law etc. are mentioned. The amount of polypeptide sensitized per insoluble carrier particle is preferably such that the ratio of polypeptide (mg) / surface area (m ² ) of insoluble carrier particle is in the range of 0.1 to 10 mg / m ² .

Next, the enzyme immunoassay method is a direct method using an enzyme-labeled antibody in which an enzyme is chemically bound to an antibody against p21 in a test solution reacted with the above-described polypeptide sensitized to a solid phase, It can be performed by detecting by an indirect method or the like. The solid phase used may be any known solid phase that is used for physical adsorption of microtiter plates, beads, sticks, test tubes, etc. made of polystyrene, polycarbonate, polypropylene, and polyvinyl, without any limitation. It is particularly preferable to use a commercially available microtiter plate for enzyme immunoassay. To sensitize the solid phase to the polypeptide, the polypeptide is suspended in a 0.01 to 0.1 M phosphate buffer,
For one hour or more. The amount of polypeptide to be sensitized to the solid phase is preferably such that the ratio of polypeptide (mg) / surface area of solid phase (m ² ) is in the range of 0.5 to 50 mg / m ² . The enzyme-labeled antibody to be used is not particularly limited as long as it has reactivity with a human antibody and is chemically bound to the labeling enzyme. For example, commercially available alkaline phosphatase,
Goat anti-human IgG antibody, mouse anti-human IgG antibody labeled with an enzyme such as peroxidase or glucose oxidase,
Goat anti-human IgM antibody, mouse anti-human IgM antibody, etc. can be used. The substrate used for detection may be appropriately used depending on the enzyme labeled in the enzyme-labeled antibody, and for example, when alkaline phosphatase is selected as the enzyme, p-nitrophenyl phosphate or the like may be used. . In the measurement, plasma or serum as a test solution may be added as a stock solution, but it is preferably diluted 100-fold with PBS buffer, normal goat serum or the like.

In addition, the Western blotting method is, for example, Proc.Natl.
It can be carried out according to a known method proposed by Tobin et al. In Acad. Sci. USA 76, 4350 (1979). The detection of the antigen-antibody reaction between the polypeptide and the antibody against p21 in the test liquid can be carried out without any particular limitation in the radioautography, the fluorescent immunoassay and the enzyme immunoassay described in the above literature. However, it is preferable to carry out the direct or indirect method of the enzyme immunoassay from the viewpoint of simplicity and storage stability. Known membranes used for Western blotting can be used without particular limitation for the membrane used for transfer. Examples of materials that are preferably used include nitrocellulose membranes, nylon membranes, polyvinylidene difluoride membranes, diazotized aminobenzyloxymethyl membranes, diazotized aminophenyl thioether membranes, and diethylaminoethyl membranes. It is preferable to use a nitrocellulose membrane.

〔The invention's effect〕

The polypeptide used as an antigen in the diagnostic agent of the present invention corresponds to p21 of HTLV-I env protein and has good antigenicity to an antibody against HTLV-I. Therefore, the diagnostic agent of the present invention enables highly sensitive diagnosis of HTLV-I infected individuals. In addition, since it does not contain other antigens that cause non-specific immune reactions, it does not generate false-positive specimens that result in positive results even if it is not infected with HTLV-I, and Can be diagnosed. Therefore, the diagnostic agent of the present invention can be used for HTLV, for example, by diagnosing a donor in blood transfusion as a subject.
It is possible to detect a person infected with -I and prevent infection with HTLV-I, and its industrial utility value is extremely high.

〔Example〕

Production Example 1 (Acquisition of DNA derived from HTLV-I env protein gene) Lymphocytes were isolated from 10 ml of peripheral blood of ATL patients according to a known method, and the lymphocytes were separated by proteinase K (manufactured by Sigma).
P0390), followed by ethanol precipitation with phenol / chloroform extraction and 1 mg of ATL patient-derived lymphocyte DNA.
I got

10 μg of the DNA was added to EcoR in a solution having the composition shown in Table 4, No. 1.
Cut with restriction enzyme I (Takara Shuzo Co., Ltd. No. 1040)
After ethanol precipitation, it was dissolved in 200 μl of TE buffer.

Then, the solution is subjected to sucrose density gradient centrifugation (sucrose 10-40%
wt / vol, 26000 rpm, 18 hours), and a DNA fragment corresponding to 20 kilobase pairs was obtained by confirmation by agarose gel electrophoresis. Next, 1.0 μg of this DNA fragment was connected to the EcoR I restriction enzyme cleavage site of Sharon 4A vector (Vector DNA, see Yoshiyuki Sakaki Kodansha 1986), and the DNA fragment was inserted into the EcoR I cleavage site of Sharon 4A vector. did. For this connection, T4 DNA ligase (Takara Shuzo Co., Ltd. No. 2011) was used, and the connection reaction was carried out in a solution having the composition shown in Table 5.
It was carried out at 15 ° C for 12 hours. Next, the obtained DNA was subjected to in vitro packaging, and then the treated solution was centrifuged (7000 rpm, 2 hours), and the supernatant was used as a recombinant phage solution for plaque hybridization. The recombinant phage solution was infected with the indicator bacterium LE392 (Takara Shuzo Co., Ltd.) to form plaques, and then ³² P-labeled HTLV-I p
Plaque hybridization was carried out using the olDNA-containing fragment as a probe to isolate phage containing DNA derived from the HTLV-I gene. The recombinant phages obtained are shown in Table 4N.
In the solution with the composition shown in o.1, Hind III (Takara Shuzo Co., Ltd.)
EcoRI (Takara Shuzo Co., Ltd. No. 1040) digested with restriction enzymes, and extracted from the HTLV-I gene-derived DNA env-px-
400 μg of a DNA fragment of about 3.9 kilobase pairs corresponding to the LTR region and containing DNA encoding p21 was obtained. Then, the vector pUC19 for Escherichia coli (No. 3219 manufactured by Takara Shuzo Co., Ltd.) was cleaved with Hind III and EcoR I restriction enzymes under the same conditions, and the above-mentioned DNA fragment was ligated to the cleaved site. The resulting ligation reaction solution contains BamH containing the HTLV-I5'-3 'fragment described below.
Similar to the ligation reaction solution of I-BamH I fragment DNA and pUC19, E. coli JM109 (Takara Shuzo Co., Ltd. No.9052) transformation, mini-preparation, and medium preparation were performed. used. As a result of the above operation, the recombinant vector P in which the DNA fragment derived from the HTLV-I env-px-LTR region gene was inserted into pUC19 in the correct direction.
400 μg of HT3.9Kb / pUC19 was obtained.

200 μg of the above recombinant vector PHT3.9Kb / pUC19 was added to the fourth
In a solution with the composition shown in Table No. 3, Pst I (Takara Shuzo Co., Ltd.)
No.1073) and Hind III (Takara Shuzo Co., Ltd. No. 1060) are used to cleave to obtain a Hind III-Pst I fragment (about 1700 base pairs).

On the other hand, the Escherichia coli vector pUC19 was digested with Sal I (Takara Shuzo Co., Ltd. No. 1080) in a solution having the composition shown in Table 4 No. 1, and mung bean nuclease (Takara Shuzo Co., Ltd.)
No. 2420), and ligated to obtain pUC19 without AccI, SalI, HincII restriction enzyme cleavage sites. Next, the pUC19 was cleaved with Hind III and Pst I in the composition solution shown in Table 4, No. 2, and the above Hi was cleaved at the Hind III and Pst I cleavage sites of the vector.
The nd III and Pst I fragments were ligated.

The ligation reaction was then used for transformation of E. coli JM109, mini-preparation,
400 μg of recombinant pUC19 vector was obtained in which the -Pst I fragment was inserted into the pUC19 vector in the correct orientation. Furthermore, in a solution having the composition shown in Table 4 No. 2, recombinant pUC19 vector was used with Acc I (Takara Shuzo Co., Ltd. No. 1001) and Xba I (Takara Shuzo Co., Ltd. No. 1093). Acc I corresponding to the latter half of the DNA derived from the HTLV-I env protein gene by cutting
A -Xba I fragment (about 1060 base pairs) was obtained.

On the other hand, the recombinant vector PHT3.9Kb / pUC19, 200 μg,
Nco I-Nco was digested with Nco I restriction enzyme (Takara Shuzo Co., Ltd. No. 1160) in a solution having the composition shown in Table 4 No. 5.
An I fragment (about 610 base pairs) was obtained. On the other hand, E. coli vector pU
C18 (Takara Shuzo Co., Ltd. No.3218) was added to Hinc II restriction enzyme (Takara Shuzo Co., Ltd. No. 10) in a solution having the composition shown in Table 4 No. 3.
It was cleaved at 59) and the above Nco I-Nco I fragment was ligated to the Hinc II restriction enzyme site of the vector by fill-in ligation. Next, the ligation reaction solution was added to E. coli JM109.
Used for transformation, mini preparation, and medium preparation of Nco.
400 μg of recombinant pUC18 vector was obtained in which the I-Nco I fragment was inserted into the pUC18 vector in the correct orientation. The vector was digested with XbaI and AccI restriction enzymes in a solution having the composition shown in Table 4 No. 6 to obtain a DNA derived from the HTLV-I env protein gene.
An XbaI-AccI fragment (about 500 base pairs) corresponding to the first half of the above was obtained.

Next, the E. coli vector pUC119 (Takara Shuzo Co., Ltd.)
No. 3319) in a solution having the composition shown in Table 4 No. 6
The vector was digested with an I restriction enzyme (Takara Shuzo Co., Ltd. No. 1093), and the resulting vector was ligated with the above-mentioned Acc I-Xba I fragment and Xba
Connection with the I-Acc I fragment was made. Then, the connection reaction solution was transformed with E. coli JM109, mini-preparation,
Then, it was used in a series of operations following medium preparation, and the DNA derived from the HTLV-I env protein gene was transformed into pUC119.
Recombinant vector inserted in correct direction into vector en
v / pUC119, 200 μg was obtained. The above steps are shown in FIG.

(Production of recombinant vector) Recombinant vector env / pUC119,200 obtained by the above method
μg was dissolved in a solution having the composition shown in Table 4, No. 6, and then Ba
mHI restriction enzyme (No. 1010, manufactured by Takara Shuzo Co., Ltd.) was intermittently added for 9 hours to carry out a cleavage reaction. After cleavage, phenol extraction and ethanol precipitation were carried out successively, the precipitated DNA was dissolved in TE buffer (pH 8.0), and the DNA solution was subjected to agarose gel electrophoresis. And HTLV-I5'-3 '
An agar piece of the band portion corresponding to the BamHI-BamHI fragment containing the fragment was cut out and the fragment was extracted by elution by electrophoresis. Then, the extract is further phenol extracted,
BamHI containing ethanol-precipitated HTLV-I 5'-3 'fragment
-A BamHI fragment was obtained.

On the other hand, 10 μg of the vector pUC19 for E. coli was digested with BamHI restriction enzyme under the same digestion conditions as above. Then, the resulting reaction solution was reacted with 1 μ of an alkaline phosphatase suspension (No. 2120, manufactured by Takara Shuzo Co., Ltd.) at 60 ° C. for 30 minutes. After stopping the alkaline phosphatase reaction, the reaction solution was extracted with phenol and precipitated with ethanol to obtain pUC19 cleaved with BamHI.

BamHI-cut pUC19 thus obtained
BamH I-Bam containing 0.2 μg and the HTLV-I 5′-3 ′ fragment
0.25 μg of the HI fragment was mixed, and a ligation reaction was performed in a solution having the composition shown in Table 5 using T4 DNA ligase at 16 ° C. for 3 hours or more.

Then, 25 μ of the connection reaction solution obtained by the operation was added to 200 μ of a competent cell suspension of Escherichia coli JM109, and the mixture was allowed to stand on ice for 30 minutes. Then, after heat-shocking at 42 ° C. for 2 minutes and further returning to room temperature, 800 μL of LB liquid medium was added and gently shake-cultured at 37 ° C. for 1 hour.

100 μg of the liquid medium containing 100 μg / ml of ampicillin
After inoculating 15 ml / plate of LB agar medium, it was cultured at 37 ° C for 12 hours. 20 single colonies that appeared after culturing were taken out, each containing 30 μg / ml ampicillin, LB liquid medium 15
ml, and cultured at 37 ° C. for 8 hours. 1 ml was collected from each liquid medium, and the plasmid located in E. coli in each medium was extracted by the mini-preparation method. Each of the obtained plasmids is shown in Table 4 No. 4
Kpn I-controlled enzyme (Takara Shuzo Co., Ltd.)
No.1068) was used to carry out the cleavage reaction. After the reaction, each reaction solution was subjected to agarose gel electrophoresis to confirm a plasmid in which the BamHI-BamHI fragment containing the HTLV-I5'-3 'fragment was inserted into pUC19 in the correct direction.

0.2 ml was collected from the liquid medium containing E. coli harboring the confirmed plasmid, and ampicillin 100
After inoculation into 50 ml of LB liquid medium containing μg / ml, the cells were cultured at 37 ° C. for 12 hours.

The plasmid present in E. coli in the obtained liquid medium was extracted by the medium preparation method to obtain 400 μg of recombinant vector BamHI Env (600) / pUC19.

The recombinant vector BamHI Env (600) / pUC19 200 μg
Was dissolved in a solution having the composition shown in Table 4, No. 6, and then Xba I
A restriction enzyme was intermittently added for 9 hours to carry out a cleavage reaction. By subjecting the obtained cleavage product to the agarose gel electrophoresis, Xba I-Xba I containing the HTLV-I5'-3 'fragment was obtained.
A fragment (about 600 bp) 0.15 μg was obtained.

Also, 10 μg of the silkworm expression vector pBF133 was added to Xba I.
It was cut with a restriction enzyme under the same cutting conditions as described above, and then treated with alkaline phosphatase.

1.25 μg of the Xba I-Xba I DNA fragment containing the HTLV-I 5′-3 ′ fragment and pBF133.025 μg cleaved with Xba I were mixed, and a ligation reaction was performed with T4 DNA ligase in the same manner as described above.

Then, Escherichia coli JM109 was transformed using this connection reaction solution and the transformed bacteria were isolated in the same manner as described above. Next, a series of mini-preparation operations were performed on each of the separated cultures, and the plasmid was extracted from each liquid medium.

Then, a digestion reaction with BamHI was performed on each plasmid, and HTLV-I5 ′ was analyzed by agarose gel electrophoresis.
A plasmid was identified in which the Xba I-Xba I DNA containing the 3'fragment was inserted into pBF133 in the correct orientation.

0.2 ml was collected from the liquid medium in which Escherichia coli possessing the confirmed plasmid was present, and ampicillin 30 μg / m
After inoculating 50 ml of LB liquid medium containing l, the cells were cultured at 37 ° C. for 12 hours.

The plasmid existing in E. coli in the liquid medium was extracted by the medium preparation method to obtain 200 μg of recombinant vector BamHI Env (600) / pBF133. This recombinant vector BamHI Env (600) / pBF133 was introduced into E. coli, and the resulting microorganism was deposited as E. coli HTLV-I-Env1 at the Institute of Microbial Technology, Institute of Industrial Science and Technology. The deposit number is Microorganisms Research Institute of Microbiology No. 10072 (FREM P-10072). The above steps are shown in FIG.

(Production of Recombinant Virus) Viral DNA of BmNPV T3 Strain and the Recombinant Vector BamH
Composition liquid I245 μ in Table 1 prepared by mixing I Env (600) / pBF133 in a molar ratio of 1: 100 was mixed with composition liquid II 255 μ in Table 1.

0.5 ml of the resulting suspension was added to 5 ml of BmN4 solution (4 × 10 ⁵ Bmcells / ml) of silkworm-established cultured cells in the medium of TC-10 (Table 2), and the mixture was incubated at 27 ° C. for 20 hours to prepare BamHI Env. (600) / pBF
133 and BmNPV DNA were introduced into silkworm established cells. The obtained culture was further exchanged with TC-10 medium and
C. for 6 days. The culture is then centrifuged (15
The resulting supernatant was used as a reaction solution for cloning a recombinant virus.

^10-6 The cloning reaction solution TC-10 fold locations, 10 ^-7, 10
^-8 to obtain 10 ml of the diluted reaction solution. Relative to the diluted reaction solution, respectively and mixed established silkworm culture cells BmN4 solution ^{(10 5 Bmcells / ml) 10ml} , dispensed the mixture in the microtiter tray 96 by 200 [mu], 27 ° C.
The cells were cultured for 4 days. After culturing for 4 days, the microtiter tray was examined under a microscope to find wells in which silkworm established culture cells in which the cell surface was roughly deformed and showed a virus-infected morphology, and in which polyhedron protein was not detected, The culture was harvested from. The resulting culture was centrifuged (1500
rpm, 10 minutes), and 150 μm of the supernatant was used as a reaction solution for expressing a recombinant virus polypeptide. The above TC-10 medium is prepared by adjusting 900 ml of medium shown in Table 2 to pH 6.30 to 6.35, sterilizing by filtration, and adding 100 ml of fetal bovine serum.

Table 2 culture locations set formed _{NaCl 0.5g KCl 2.87g CaCl 2 · 2H} 2 O 1.32g MgCl 2 · 6H 2 O 2.28g MgCl 2 · 7H 2 O 2.78g Tryptose 2.0g Dextrose 1.1g (glucose) L-glutamine 0.3 ^{g soln a * 100ml soln B **} 100ml soln C *** 1ml NaH 2 PO 4 · 2H 2 O 100ml (0.891g / 100ml) NaHCO 3 100ml (0.35g / 100ml) H 2 O to a total volume of 900ml with _* soln Composition of A L-Arginme 5.79g L-Aspatic acid 3.5g L-Asparagine.H ₂ O 3.98g L-Alanine 2.25g β-Alanine 2.0g L-Glutamic acid 6.0g L-Glutamine 3.0g Glycine 6.5g L-Histidine 25.0g L-Isoleucine 0.5g L-Leucine 0.75g L-Lysine.HCl 6.25g L-Methiouine 0.5g L-Proline 3.5g L-Phenyla anine 1.5g DL-Serune 11.0g L-Threonme 1.75g L-Valine 1.0g The total volume is 1000 ml with H ₂ O ** The composition of soln B L-Cystine 0.25 g L-Tryptophane 1.0 g L-Tyrosine 0.5 g The total volume is 1000 ml with H ₂ O.

** soln C composition Thiamine.HCl 2.0mg Riboflavine 2.0mg D-Ca pantothenate 2.0mg Prydoxine.HCl 2.0mg Para-aminobenzoic acid 2.0mg Folic acid 2.0mg Nicotinic acid 2.0mg Iso-Iuositol 2.0mg Biotin 1.0mg Cholive Cl The total amount is made up to 1000 ml with 20.0 mg H ₂ O (Production of Polypeptide) 100 μl of the above-mentioned polypeptide expression reaction solution was added to 30 ml of BmN4 solution (10 ⁵ Bmcells / ml) of silkworm cultured cells and cultured at 27 ° C. for 5 days. After culturing for 5 days, the culture was collected and centrifuged (1500 rpm, 15 minutes).

Wash the pellet (virus mature cells) with PBS buffer 5
The suspension was suspended in 400 μm of 0 mM Tris-HCl (pH 7.4), sonicated, and then centrifuged (8000 rpm, 20 minutes). 200 μm of the Laemmli buffer was added to the polypeptide obtained as a precipitate, and the suspension was boiled and centrifuged. The supernatant was used as a sample for SDS gel electrophoresis.

As a result of SDS gel electrophoresis, this sample showed that a part of the polyhedron protein encoded by the polyhedrin protein gene part of pBF133 (about 5 Kd) and the HTLV-I5'-3 'fragment encoded HTLV-
A band was detected at a position of about 25 Kd, which corresponds to the total molecular weight of the p21-containing portion (about 20 Kd) of I coat protein (shown in FIG. 4). Incidentally, FIG. 4 shows the expression of HTLV-I env protein in Bm cells confirmed by SDS gel electrophoresis. In FIG. 4, lane1 is a size marker, lane2 is a protein of silkworm cells not infected, and lane3 is a protein of silkworm cells infected with a recombinant virus using BamHI Env (0.6) / pBE133.

Anti-polyhedron protein antibody and HTLV-I p21 as primary antibody
Western blot experiment using a monoclonal antibody against 25Kd polypeptide showed polyhedrin protein and HTLV
It was confirmed to be a fusion protein containing -I p21. or,
At the same time, a Western blotting experiment was performed using normal human serum or ATL patient serum as the primary antibody, and it was confirmed that the antibody has good antigenicity to the antibody against the 25 Kd polypeptide HTLV-I. (Illustrated in FIG. 5). Incidentally, FIG. 5 shows the expression of HTLV-I env protein in Bm cells confirmed by Western blotting. Lane 1 is a size marker, and lane 2 to lane 5 are antigens BamHI Env
The protein of silkworm cells infected with the recombinant virus using (600) / pBF133 was electrophoresed.

Western blotting was performed by a color reaction with peroxidase using adipine and biotin as substrates, using the primary and secondary antibodies shown in Table 3.

Production Example 2 100 μl of the polypeptide-expressing reaction solution obtained in Production Example 1 was added to 30 ml of silkworm established cell culture solution (10 ⁵ Bmcells / ml), and the mixture was cultured at 27 ° C. for 5 days. After culturing for 5 days, the culture was collected and centrifuged (1500 rpm, 15 minutes). Supernatant 0.1ml 5
Percutaneously injected into 100 silkworms on the 1st day of administration, 25
After feeding the mulberry leaf paste pieces at 5 ° C. for 5 days, the mice were dissected, and the fat pads were collected. The fat pad is suspended by adding 10 ml of a PBS buffer, sonicated, and centrifuged (8000 rpm, 20 minutes).
Then, 5 ml of the precipitate was obtained. The precipitate (200 μ) was sonicated again, and a Bio-RAD protein assay was performed.
The amount of polypeptide measured was 60 μg.

After measuring the amount of polypeptide, 50 μL
The suspension was boiled and centrifuged, and the supernatant was used as a sample for SDS gel electrophoresis.

As a result of SDS electrophoresis, a band was detected at a molecular weight of about 25 Kd. By the same method as in Example 4, this 25 Kd polypeptide was a fusion protein containing a polyhedron protein and HTLV-I p21. It was confirmed that the antibody of I had a good antigenicity.

According to a fifth Table 50mM Tris-HCl (pH7.4) 10mM MgCl 2 10mM dithiothreitol (DTT) method of 1 mM ATP Example 1 JP 62-286533 JP Example 1 to produce a composite polymer particles. That is, in a glass flask equipped with a stirrer, methanol 2800cc, ammonia water (25% by weight) 6
After adding 16 cc and 21 cc of an aqueous solution of sodium hydroxide (5 mol /) and maintaining the temperature at 10 ° C., 1428 cc of a methanol solution of tetraethyl silicate (22%) was added with stirring at a dropping rate of 25.5 ccm / hr to react. Thereafter, the silica particles were purified by repeating decantation in a large amount of methanol.

The silica particles thus obtained are allowed to settle, the supernatant is removed, distilled water is added to disperse, and the operation of further sedimentation is repeated twice. After washing the particles, distilled water is added so that the dispersion concentration becomes 10 wt%. , A silica dispersion was obtained.

After replacing the glass flask with a stirrer with nitrogen, 100 ml of the silica dispersion obtained above was added and kept at 40 ° C.
Under a nitrogen atmosphere, 67 mmol of glycerol methacrylate and potassium persulfate were added to 2.9 mmol / under stirring. Then, the temperature was kept at 40 ° C. and polymerization was carried out for 4 hours with stirring. After polymerization, the supernatant was discarded by centrifugation, and the precipitated composite polymer particles were redispersed in distilled water. This operation was repeated 6 times, and the precipitate was washed to obtain purified composite polymer particles.

Next, a 0.02 M PBS buffer solution (pH 7.
The 2% dispersion in 2) was prepared by adding 1M This-HCl containing 1% SDS at a concentration of 10 μg / ml to the polypeptide obtained according to the above Preparation Example 2.
An equal volume of the suspension suspended in (pH 7.4) buffer was mixed. After leaving at room temperature for 1 hour, 0.02M PBS buffer (pH 7.
The particles were washed by centrifugation using 2) to prepare a solution in which the obtained polypeptide-sensitized particles were dispersed at a concentration of 5%.

ATL patient serum, normal human serum, systemic lupus erythematosus patient serum, primary biliary cirrhosis patient serum, and rheumatism patient serum were used as a stock solution with a 2-fold dilution solution according to the multiple dilution method to obtain a phosphate buffer solution (pH 7). .2) was used for dilution, and each diluted solution was added to the well of the microtiter plate in an amount of 25 μm. Next, the prepared sensitized particle solution was added to the well in an amount of 25 μm, and the mixture was stirred for 3 minutes and allowed to stand at room temperature. After 30 minutes, observe the aggregation state of the particles, and for each test solution, the particle ring is clearly large, and the aggregated particles spread in the ring in the form of a film. The sensitivity was evaluated. Then, the liquid to be treated having an antibody titer of 8 or more was diagnosed as positive. Table 6 shows the above results.

Comparative Example 1 MT-2 cells (Gann 75 vol. 989 1981) in RPMI 1640 medium (MAB No. 530-05891) containing 10% calf serum at 37 ° C.
The cells were cultured for 3 days. Then, the culture supernatant, 10,000 rpm,
Centrifuge for 30 minutes, collect the supernatant, and collect the collected supernatant for 10 minutes.
Ultracentrifugation was carried out at 0,000 g for 2 hours to obtain a precipitate. Remove the sediment to 0.
01M Tris-HCl buffer (pH7.4) (100 mM NaCl 10 mM EDTA
, And the resulting supernatant was collected by centrifugation at 3,000 rpm for 1 hour. A 20-65% sucrose density gradient solution was prepared, and the above supernatant was layered thereon and subjected to ultracentrifugation at 30,000 rpm for 18 hours to obtain a soluble cytoplasmic protein solution of MT-2 cells. Then, in the same manner as in Example 1 except that the soluble cell protein was used instead of the polypeptide obtained in Production Example 2,
The diagnosis of HTLV-I infection of various digested liquids was performed. The results are shown in Table 7.

Example 2 The polypeptide obtained according to Production Example 2 was diluted in 1M Tris-HCl (pH7.4) buffer containing 1% SDS so that the OD ₂₈₀ value was 0.4, and 150 ml was added to a cup of an enzyme immunoassay microtiter plate. It was After standing at 4 ° C. overnight, the liquid was drained and the cup was washed with deionized water. To the cup of the thus obtained polypeptide-sensitized microtiter for enzyme immunoassay, 100 μ of a 100-fold diluted solution of each test solution used in Example 1 with PBS buffer was added. After leaving at 37 ° C. for 1 hour, the cup was washed with deionized water, and 100 μl of a phosphate buffer solution of an anti-human IgG antibody-alkaline phosphatase conjugate was added. 37 ° C
After standing at room temperature for 1 hour, the cup was washed with deionized water, and the substrate (p-nitrophenyl phosphate dissolved in 4 mM MgCl ₂ -containing carbonate buffer (pH 9.5) at 4 mg / ml) 100 μ Was added. After standing at 37 ℃ for 1 hour, 1N NaOH 1
The reaction was stopped by adding 00 μ and the 0D ₄₀₅ value was determined. 0D
_{When a} sample having a ₄₀₅ value of 0.07 or more was diagnosed as a positive test liquid, the same diagnostic results as in Example 1 were obtained.

Example 3 SDS-polyacrylamide gel electrophoresis of the polypeptide obtained according to Production Example 1 was performed, and then various test liquids used in Example 1 containing 3% bovine albimine-containing phosphate buffer (pH 7.
Using the 10-fold dilution obtained in 2) as a primary antibody, a Western blot experiment was performed by enzyme immunoassay. A nitrocellulose film is used for the transfer film,
Peroxidase-labeled goat anti-human IgG (v
ectro No.50-0410-06) and peroxidase-labeled goat anti-human IgM (Vector No.50-0410-03) 1: 1
A mixture was used. In addition, 0.05 mol Tris-
20 ml of HCl buffer (pH7.2), 3,3'-diaminobenzidine 4
A coloring solution for peroxidase having a composition of 10 mg of hydrochloride and 10 μ of 30% hydrogen peroxide solution was used. Although the test solution used for the primary antibody was diagnosed as positive although the color was observed and a band was observed, the same diagnostic result as in Example 1 was obtained.

[Brief description of drawings]

Figure 1 is a restriction map of the silkworm nuclear polyhedrosis virus DNA, Figure 2 is a restriction map of the pBF vector, and Figure 3 is pBF.
Types of vector, Fig. 4 shows HTLV protein expression in Bm cells
Figure confirmed by SDS gel electrophoresis, Figure 5 shows H in Bm cells
Western blot analysis confirming TLV-I env protein expression. Fig. 6 shows silkworm cells infected with silkworm nuclear polyhedrosis virus and recombinant virus. Fig. 7 shows DNA derived from HTLV-I env protein gene. Preparation method, FIG. 8 shows the preparation method of the recombinant vector, and FIG. 9 shows the gene and amino acid sequence diagram of the p21 protein portion in the HTLV-I env gene sequence. Hd III …… Hind III, N …… Nco I, Ac …… Acc I, Ec…
… EcoR I, Sp …… Sph I, Sa …… Sal I, Hi II …… Hinc
II, B …… BamHI, X …… Xba I.

Continuation of the front page (56) References JP 61-9288 (JP, A) JP 58-187861 (JP, A) JP 59-62527 (JP, A) J. Immunol. , 137 (9), p. 2941-2951 (1986)

Claims (1)

(57) [Claims] 1. A silkworm of the DNA derived from the adult T-cell leukemia virus coat protein gene, which contains a DNA encoding p21 and is cleaved within 17 base pairs upstream from the 5'end of the DNA. Adult T-cell leukemia whose antigen is a polypeptide expressed by infecting silkworm established culture cells or silkworm larvae with a part of the polyhedrosis protein structural gene of nuclear polyhedrosis virus Viral infection diagnostics.