JPS62296889A - Protein with htlv-i antigen activity and preparation thereof - Google Patents

Abstract

PURPOSE:A microorganism including lac promoter fraction, synthetic codon an plasmid pKI-5 bearing HTLV-I (human T-cell leukemia virus) gene fraction is cultured to produce a protein having HTLV antigen activity. CONSTITUTION:A microorganism, which includes lac promoter fraction, synthetic codon and plasmid pKI-5 bearing gag-env-pX-IV gene fraction of HTLV-I, is cultured. From the culture mixture, an antigen protein reacting with the antibodies to p19, p28 and p40X relation to adult T-cell leukemia. The antigen protein reacts with the antibody to p19, p28 and p40X in the ATLA group and with the serum of the patients with ATL and has a molecular weight of 100K dalton.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention [Field of Industrial Application] Human T-cell leukemia virus (HTLV-I) and adult T.
Cellular leukemia virus (ATLV) has recently been reported to be a homolog of human retroviruses. This H
Antibodies against this virus are present in the serum of patients infected with a virus called TLV-4 or ATLV, and the presence or absence of infection can be determined by testing these antibodies in the serum.

The present invention relates to a method for producing this antigen protein using a novel anti-II protein that reacts with this antibody and a new plasmid.

[Conventional technology]

Adult T-cell leukemia (ATL)-related cell lines include p70°gp68yp53egp46+p40x, p36. p2
8. p24. p21. It is known that there is an ATL-related antigen group (ATLA) consisting of p19+p15 and p14 (Yamamoto, N and Hlnu
ma.

Y,,Int,J,Cancer,30,289-29
3 (1982)).

It is also known that both ATL patients and HTLB-I carriers have specific antibodies against ATLA (Hlnuma, Y., et al., Pro.
c, Natl, Acad.

Set, U, S, A-, 78, 6476-6480 (1
981) ).

On the other hand, recently progress has been made in the development of methods for detecting antibodies against HTLV-I, Rirui, and ATLV, but the antigens used for this are usually HTLV-I or ATLV producing cells. A method for obtaining the virus antigen from the culture supernatant of these cells (Japanese Unexamined Patent Publication No. 58-187861) has also been developed.

The present inventors previously succeeded in obtaining antigenic proteins recognized by monoclonal antibodies against HTLV-I gag gene products plo and p20 (Itamur
a at al,, Gone, 38.57-64+(
1985) ).

[Problems to be Solved by the Invention] When cells are used as antigens, there is a problem in test accuracy because there are many non-specific reactions. The method of obtaining antigen from culture supernatant requires cell culture and subsequent separation and purification.
There was a problem of low production. Therefore, if ATLV patches could be produced using a more direct method, it would be possible to mass-produce highly purified antigens using simple means.

The antigen protein that the present inventors obtained earlier is an antigen protein corresponding to a specific gene part of HTLV-I, and if an antigen protein corresponding to another gene part is obtained, this can be used to target ATIA. This makes antibody detection more reliable and enables analysis from multiple angles.

[Means for solving problems]

The present invention was made based on this viewpoint,
We succeeded in constructing an expression plasmid for the gag-env-pX-IV complex gene product of HTLV-I, and by carrying this in a microorganism, p19゜p of the ATLV antigen group was successfully constructed.
28, and a protein that reacts with antibodies against p40X? This was done after discovering that it is produced by microorganisms.

That is, the present invention provides (1) a pI9.10 that has a molecular weight of 100 Daltons and is associated with adult T-cell leukemia; p28 and p
(2) an antigen protein that reacts with an antibody against 40", and (2) la
c 7' lomota moiety, synthetic codons, and HTLV-
(2) Cultured with a microorganism carrying plasmid pKI-5 containing the gag-env-pX-IV gene portion, pI9. p28 and p
The present invention relates to a method for separating an antigen protein, which is characterized by separating an antigen protein that reacts with an antibody against 40''.

Although the method for synthesizing pKI-5 is not limited, for example, first, the Ec promoter, which is present downstream of the start codon of the expression vector ps1101, which has an SD sequence necessary for partial translation of the 1ac promoter and a start codon, is used.
o Construct ps1201 by inserting a synthetic linker with a Sma I site at the RI site. Next, HTLV-! Cut the gair gene from glovirus with Sma I,
pHY2O2 can be constructed by connecting this to the Sma I site of ps1201. moreover,
To connect the env-pX-IV gene of HTLV-I to pI (Y202), pHY2O2 was treated with Bam HI and Sal I, while any-p of HTLV-I
The env-pX-U3R gene was excised from plasmid pHTLV707 containing the X-U3R gene using Puv II and combined with pH7202 to generate pKr.
-5 can be constructed. This synthetic route is shown in FIG.

As shown in the figure, plasmid pKI-5 consists of the 1ac promoter part, the HTLV-1 gag gene part, and the HTLV-I anv-pX gene part from the left side of the figure. The method for carrying plasmid pKI-5 in microorganisms may be the same as the method for carrying general plasmids or vectors in microorganisms. The microorganisms used are
Any microorganism may be used as long as it can carry plasmid pKI-5, such as Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa.

The method for culturing the microorganism that carries pKI-5 may be the same as the method for culturing the microorganism that plays a role. By adding ampicillin or the like to the medium, bacteria containing the plasmid can be selectively grown. Inglovir-β-D during culture
-Thiogalactopyranoside, adenosine 3/, S/-
The glLg-env-pX-IV complex gene product is induced by transcriptional induction by adding cycli, spider monophosphate, etc., and the culture is continued, and the culture is discontinued when the amount of accumulated antigen protein of the present invention reaches the maximum. . Since this protein is accumulated within the bacterial body, the destruction method for destroying and extracting the bacterial body may be any known method, such as enzyme treatment that destroys cell membranes such as lysozyme, freeze-thaw treatment, ultrasonic treatment, etc. After disrupting the bacterial cells, a highly purified product can be easily obtained by centrifuging, collecting the supernatant, and purifying it by affinity chromatography using an anti-ATLA antibody.

As shown in Example (5), the antigenic protein of the present invention causes a strong antigen-antibody reaction with ATL patient serum, but does not react with healthy human serum. On the other hand, monoclonal antibodies that react with ATLA of p19 and p28 and monoclonal antibodies that react with ATLA of p40x show a reaction.

The molecular weight of this antigen protein is approximately 100 Daltons based on the electrophoresis results in Example (7), and this is also supported by the fact that the molecular weight of the protein calculated from the DNA sequence in Figure 1 is approximately 100 Daltons. Ru. Therefore, this antigen protein is ga
The g gene and pX gene are complex proteins made by expressing not only the env gene but also the env gene.

This antigen protein is clearly new since this 100' antigen protein is not present in cell extracts of MT-2, a leukemia-derived cell line.

[Effect]

Induced BA 17) Antigen protein ij ATLA lll nor 1. Antibodies against p28 and p40” and ATL
Reacts with patient serum.

This protein has kI added with accessory parts necessary for protein synthesis.
It can be produced by microorganisms by using pKI-5 containing the gag-env-pX-IV complex gene portion of TLM-I.

〔Example〕

(1) Construction of Plasmid Revision 202 Plasmid pHY2O2 was previously developed by the present inventors (Itamura at al., Gena +
3L57-64° (1985)). The construction process can be briefly explained as follows. First, it has a 1ae7° lomotor part.

dAAT at Eco RI site of plasmid ps1101
Plasmid ps1201 was constructed by inserting a synthetic oligonucleotide consisting of TcccGGG. Next, ps1
1 of the HTLV-1 gag gene at the Sma'I site of 201. The I Kb Sma I fragment was connected to construct plus de pHY2O2.

(2) Construction of f lasmid pKI-5 Env-p of HTLV-I proviral DNA
The Pvu II fragment of plasmid pHTLV707 containing the X-IV-U3R gene portion was excised, and this fragment was ligated to the Sal I site of plasmid pHY2O2, which had been previously treated with Bam HI, to construct plasmid pKI-5.

The flow of constructing the above plasmid pKI-5 is shown in FIG.

(3) Introduction of pKI-5 into E. coli
E. coli K12545π (Bakkarl, A.
I.

and Zipsar+D,, Nature New
Biol, +243, 238-241 (1973))
was introduced by the calcium chloride method.

(4) Generation of antigenic protein pKI-5'E.
I grew it up to a unit. Isozorovir-β-D-thiogalactobyranoside and adenosine 3/, 5/-cyclic monophosphate were each added to a final concentration of 1 mM to induce production of the gag-env-pX-IV gene product. 100klets at 32℃
I grew it up to a unit. Collect the bacterial cells by centrifugation, 10
) Lis-HCI (pH 8,0), 1mM EDT
A 1o, 2% NaN5 and 104/fnl P
They were suspended in a solution containing MSF at a concentration of 1 × 10 cells/ml.

This bacterial suspension was added to 1 mg7fnl of Norizotium at 0°C.
The cells were incubated for 10 minutes, and then frozen and thawed three times. This is K
After reaction, the mixture was treated with 20 t47fnl DNase IkO at 0° C. for 10 minutes, and then centrifuged to collect the supernatant.

On the other hand, for comparison, ps1201 t is also E, eoli
K12545π was introduced and treated in the same manner to collect the supernatant, which is a bacterial cell extract.

(5) Separation and detection of antigen protein The bacterial cell extract obtained in this way is equivalent to 107 I x 1, and the separately prepared 2 x 10' MT-
Extracts of 2 cells were electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, the proteins in Kurno are electrophoresed and transferred to a nitrocellulose membrane.
Monoclonal antibody F'R19/28 (A), which recognizes this membrane with p19 and p28i, and ATL patient serum (ti
T, regime 640x) (''), p40'''cgR through E-/ri.

-null antibody (C) and healthy adult serum (D).

Protein A (E, Y, Laboratorles, I
) (B, D), or peroxidase-conjugated anti-mouse IgG (Cappal Laborato
ries, Inc.) (A, C) by enzyme immunoassay. The results are shown in Figure 2. In the figure, the first column contains the cell extract of E, coll K12545π, which has ps1201, and the second column contains the cell extract of E, coll, which has pKI-5.
K12545π bacterial cell extract, and the third row is MT-2.
These were obtained using cell extracts.

Molecular weight standards include myosin (200 Daltons), phosphorylase B (92,5 Daltons), 4J) 171?
albumin (68 daltons) and ovalbumin (
Standard solution containing 43r (Dalton) (BsthesdaR
asearch Laboratories, Inc.)
[Effects of the Invention] This invention opens the way to mass production of a protein having ATLV antigenic activity by culturing E. coli, and provides a means by which this protein can be easily obtained with high purity.

Thereby, it is also possible to popularize ATL clinical testing. The antigen protein of the present invention is the invention of the earlier application (Japanese Patent Application No. 1983).
-209512) is related to the gag gene product, whereas 2) is related to the gag-env-pX-IV gene product. Therefore, by using this antigen protein, it is possible to detect most of the known anti-ATT and A antibodies, and it opens the door to analysis from a variety of angles.

[Brief explanation of the drawing]

FIG. 1 shows an example of the method for constructing plasmid pKI-5 used for the synthesis of the antigenic protein of the present invention. Figure 2 shows E, coll K12 containing ps1201.
545π bacterial cell extract, E with pKI-5, eol
i K12545π bacterial cell extract and MT-2 cell extract were electrophoresed, monoclonal antibodies recognizing p19 and p28, ATL patient serum% p40x
This figure shows the results of a reaction with a monoclonal antibody that recognizes 2 and a healthy human serum. Patent applicant Fujirebio Co., Ltd. Agent Patent attorney 1) Masahiro Naka (and 1 other person) Figure 1 amal EcoRl BamHI 5all
Psl+to1g1li°°:: L+q#1iOn ■ jlIZ diagram

Claims (2)

[Claims] (1) An antigenic protein with a molecular weight of 100K Daltons that reacts with antibodies against antigens p19, p28, and p40^X associated with adult T-cell leukemia. (2) 1ac promoter part, synthetic codons and HT
Cultivating a microorganism carrying a plasmid pKI-5 having LV gag, env, and pX-IV gene parts,
Antigen p19, p19 associated with adult T-cell leukemia from culture
A method for obtaining an antigenic protein, which comprises isolating an antigenic protein that reacts with antibodies against 28 and p40^X.