JPH06253883A - Production of hepatitis c virus envelope protein - Google Patents

Abstract

PURPOSE:To obtain a sufficient amount of HCV-Env useful for the diagnosis of hepatitis C, etc., by culturing a bacterial strain belonging to the genus Bacillus and containing a base sequence coding the amino acid sequence of the hepatitis C virus envelope protein. CONSTITUTION:The objective HCV-Env is produced by culturing a bacterial strain belonging to the genus Bacillus (e.g. Bacillus brevis) and containing a base sequence (e.g. the base sequence of formula) coding the amino acid sequence of the hepatitis C virus envelope protein and separating the HCV-Env produced and accumulated in the cultured product.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to biotechnology, and more specifically, the present invention cultures a Bacillus bacterium having a hepatitis C virus envelope protein (HCV-Env) and cultures the same. HCV-Env
H is characterized by generating and accumulating
It relates to a method for producing CV-Env.

[0002]

2. Description of the Related Art 10 to 20% of people who have received blood transfusion so far
It is well known that post-transfusion hepatitis occurs in the. According to an estimate by the Ministry of Health and Welfare (1988), 90% or more of acute hepatitis that occurs after blood transfusion and about 50% of sporadic acute hepatitis are estimated to be non-A non-B hepatitis. About half of the hepatitis thus developed progresses to chronic hepatitis, and 10 to 20% progresses to cirrhosis. In addition, liver cancer may occur. Previously, non-A non-B hepatitis was considered to be hepatitis due to an unknown virus, but recently Houghton et al.
CDNA obtained from infected tissues and serum of hepatitis C patients
By screening the expression products from the A library, a partial cDNA of the non-A non-B hepatitis virus genome
The NA was successfully isolated and named hepatitis C virus (HCV) (European patent EP 0318216).

Furthermore, a part of the hepatitis C virus gene has been cloned by Chiron Corporation of the United States (Science, 244, 359-362 (198).
9)), a method for measuring HCV antibody was developed. Although the current method has been used successfully for the diagnosis of hepatitis C and the examination of blood products for blood transfusion, it has been successful in diagnosing acute hepatitis C and Regarding the determination of the effect of hepatitis C upon treatment, a sufficient effect is not mentioned in the context of the envelope region.

As described above, the blood product for transfusion is tested,
C by removing blood contaminated with hepatitis C virus
In order to prevent hepatitis C, diagnose hepatitis C and acute hepatitis C, and measure the effect of hepatitis C upon treatment, it is required to develop a new antigenic polypeptide with higher accuracy.

[0005]

[Problems to be Solved by the Invention] As a novel antigenic polypeptide, hepatitis C virus (HCV) by Okamoto et al.
Envelope protein (En
v) The gene was cloned (Japan J.E.
xp. Med. 60 , 167, (1990), J. Am. G
general Virology. , 72 , 2697
(1991), Virology. , 188 , 33
1, (1992)). However, these HCV-Env
Was not expressed and produced in a significant amount.
In order to develop a method for measuring an HCV antibody using V-Env, it has been desired to develop a method for efficiently producing HCV-Env. The present invention was made for the purpose of developing an efficient method for producing HCV-Env.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to develop a method for efficiently producing HCV-Env in order to solve the above-mentioned problems, and found that human epidermal growth factor (hEGF) F is located downstream of the base sequence encoding the amino acid sequence (SEQ ID NO: 3 in the sequence listing shown in Table 3 below).
By constructing an expression vector by linking the HCV-Env gene via a nucleotide sequence encoding a recognition amino acid sequence of actor-Xa (protease), introducing the vector into a bacterium of the genus Bacillus, and culturing the Bacillus, It was found that a significant amount of a fusion protein of hEGF and HCV-Env was produced in the culture, and further, HCV-Env was successfully extracted from this fusion protein, and the present invention was completed.

[0007]

[Table 3]

That is, the present invention relates to the production of HCV-Env using a Bacillus bacterium as a host, and more specifically, by culturing Bacillus (Bacillus bacterium) carrying the HCV-Env gene. -The basic technical idea is to efficiently produce HCV-Env by producing and accumulating -Env in a culture and collecting it.

In the present invention, the HCV-Env-encoding gene may be any gene as long as it encodes HCV-Env.
anJ. Exp. Med. , 60, 167, (199
0), J. General Virology. , 7
2, 2697 (1991), Virology. , 1
88, 331, (1992)) and the like. These genes are routinely used (Itakura,
K. J. J. Rossi, and R.M. B. Wall
ace. Annu. Rev. Biochem. , 53,
323 (1984). As the gene encoding the HCV-Env whose amino acid sequence is shown in SEQ ID NO: 4 of the sequence listing shown in Table 4 below, preferably, for example, SEQ ID NO: 2 (see FIG.
The gene having the nucleotide sequence shown in (see) is used.

[0010]

[Table 4]

[0011]

[Table 2]

The vector used in the present invention is a nucleotide sequence encoding the human epidermal growth factor (hEGF) amino acid sequence (SEQ ID NO: 3), for example, a known hEGF nucleotide sequence (Proc. Natl. Acad. Sci. USA). , 8
6 , 3589-3593 (1989)).
Takara, K .; J. J. Rossi, and R.
B. Wallace. Annu. Rev. Bioche
m. , 53 , 323 (1984)) and the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing shown in Table 1 below (see FIG. 1).
Or the hEGF base sequence synthesized based on the hEGF amino acid sequence (Japanese Patent Application No. 4-216605) or a part of the sequence including the N-terminal downstream via a base sequence encoding a recognition amino acid sequence of Factor-Xa (protease) HCV
-Env gene is ligated. Further, in order to be stably retained in the host, a resistance gene against an antibiotic (for example, erythromycin resistance etc.) can be carried.

[0013]

[Table 1]

Any gene can be used as the gene encoding the promoter, SD and signal peptide as long as it functions in the host. Bacillus brevis HPD31
(Note that this strain is the same strain as Bacillus brevis H102 (FERM BP-1087) in the present specification) -derived promoter, SD, and a gene encoding a signal peptide (J. Bacteriol., 17 ).
2 , it is effective to use 1312-1320 (1990). As a method for constructing a plasmid, a conventional method is appropriately used, for example, Molecular Clonin.
g, A Laboratory Manual, Col
d Spring Harbour Laborator
The method described in y (1982) and the like are exemplified.

The microorganism transformed with the expression vector incorporating the HCV-Env gene may be any microorganism capable of expressing the vector and promoter,
Microorganisms of the genus Bacillus, for example Bacillus brevis 47
(JP-A-60-58074, JP-A-62-20158)
3), Bacillus brevis H102 (FERM BP-
1087) and the like, but Bacillus brevis H102 is preferably used. A method for transforming a microorganism may be a known method, for example, the method of Takahashi et al. (J. Bacteriol., 156 , 1130).
(1983)) or the method of Takagi et al. (Agri)
c. Biol. Chem. , 53 , 3099-3100
(1989)) and the like.

The medium used for culturing the obtained transformant may be any medium as long as the transformant can grow and produce the desired foreign gene product.

Examples of the carbon source contained in the medium include glucose, glycerol, starch, dextrin, molasses, urea, organic acids and the like. The nitrogen source contained in the medium includes casein, polypeptone, meat extract,
Organic nitrogen sources such as yeast extract, casamino acid, glycine,
Inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate are used. Alternatively, the culture may be performed using a synthetic medium mainly containing sugar and an inorganic nitrogen source. For auxotrophic bacteria, nutrient substances necessary for their growth may be added to the medium. Examples of the nutritional substance include amino acids, vitamins, nucleic acids, salts and the like.

If necessary for the culture, antibiotics such as penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine, and the like may be added to the medium.
Ampicillin is added. If necessary, an antifoaming agent such as soybean oil and lard oil various surfactants may be added to the medium. The initial pH of the medium is 5.0 to 9.0, and more preferably 6.0 to 8.0. The culture temperature is usually 15 ° C to 42 ° C, more preferably 24 ° C to 37 ° C.
C., and the culture time is usually 16 to 166 hours, more preferably 24 to 96 hours.

According to the present invention, by culturing the transformant under the above-mentioned conditions, a significant amount of HCV-E is added to the culture.
nv is efficiently produced and accumulated as a fusion protein with hEGF.

After the completion of culturing, when the produced fusion protein is in the microbial cells, known methods such as microfluidizer (manufactured by Microfludex Co.), ultrasonic treatment, French press, etc. (basic experimental method for protein and enzyme) , Nankodo,
The fusion protein can be recovered outside the cells by crushing the cells according to pages 1 to 8 (1985)). When it is produced as an inclusion body, it can be solubilized with urea, guanidine hydrochloric acid or the like. When it is produced outside the cells, the cells may be separated from the supernatant by centrifugation, membrane filtration or the like. The fusion protein recovered as described above may be subjected to conventional protein purification methods such as membrane treatment, ammonium sulfate fractionation method, chromatography, etc. (basic experimental method for proteins and enzymes, Nankodo, (1985)).
It can be purified with.

The purified fusion protein may be used as it is, but it is preferable to use it in the form of HCV-Env only after removing hEGF.

The separation of hEGF and HCV-Env was carried out by treating the fusion protein with Factor-Xa and inserting F.
hEGF at the position of the recognition amino acid sequence of actor-Xa
It is possible to obtain HCV-Env by cleaving it into HCV-Env and removing hEGF by affinity chromatography for hEGF. See also Fact
After treatment with or-Xa, HC was purified by a conventional method for protein purification.
V-Env may be purified.

[0023]

According to the method for producing HCV-Env of the present invention, H
CV-Env can be efficiently produced. As a result, C by an antibody detection system using HCV-Env as an antigen
It can greatly contribute to the development of diagnosis, prevention and treatment of hepatitis B.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0025]

Example 1 HCV-Env expression plasmid pHT · h
Construction of EGF / BF / HCV-Env and Preparation of Transformant]

(1) Construction of plasmid pHT110 · hEGF B. brevis HP926 (FERM P-126
64), a plasmid pHT110 prepared from the plasmid pHT926 (Japanese Patent Application No. 4-216605) was digested with ApaLI and BamHI and then treated with alkaline phosphatase (BAP) to obtain a DNA of 3.4 kb.
A fragment was obtained. In addition, it was synthesized by a conventional method (Itacur
a, K. J. J. Rossi. and R.D. B. Wa
llace. Annu. Rev. Biochem. , 5
3 , 323 (1984)), the MWP signal peptide (H. Yamagata et. Al., J. Bac.
teriol. , 169 , 1239 (1987)), and 203b encoding the amino acid sequence of hEGF.
The synthetic DNA of p (FIG. 3) was ligated using T4 ligase to obtain the plasmid pHT110.hEGF (FIG. 4).

(2) Construction of plasmid pHT · hEGF · BF Next, the plasmid pHT110 · hEGF was digested with BglII, and the digested portion was blunted with T4 polymerase.
Conventional method (Itakura, K., JJ Rossi, a
nd R.N. B. Wallace, Annu. Rev. B
iochem. , 53 , 323 (1984)) and the Factor-Xa (protease) shown in FIG.
DNA encoding the amino acid sequence recognized by
F) was ligated using T4 ligase and the plasmid pHT.
hEGF.BF was obtained (Fig. 6).

(3) Plasmid pHT / hEGF / BF
-Construction of HCV-Env and preparation of transformant The nucleotide sequence of the HCV-Env gene shown in SEQ ID NO: 2 in the sequence listing (Japan J. Exp. Med., 60 , (1
990)) based on which a restriction enzyme site was added to 577 bp
The HCV-Env gene (Fig. 2) of
a, K. J. J. Rossi, and R.M. B. Wa
llace. Annu. Rev. Biochem. , 5
3 , 323 (1984)) and plasmid p
SPT18 (Boehringer Mannheim) Pst
A plasmid pHCV · E1 having the HCV-Env gene inserted into the I-EcoRI site was obtained. Then pHCV
-E1 was digested with PstI and the digested portion was blunted with T4 polymerase. Then digested with EcoRI, 580b
An envelope gene fragment of p was obtained. The base sequence (577 bp) shown in FIG.
The V-Env gene (573 bp) has N-terminal and C-terminal added with a restriction enzyme site.

The previously prepared plasmid pHT · hEGF
-BF was digested with StuI-EcoRI, treated with BAP, and the envelope gene fragment obtained above was ligated using T4 ligase. brevis HPD31 was transformed to obtain the desired plasmid pHT / hEGF / BF / HCV.
A clone with -Env was obtained (Fig. 7).

[0030]

Example 2 Cultivation of transformants and production of HCV-Env B. coli harboring pHT / hEGF / BF / HCV-Env. brevis HPD31 was added to erythromycin (10 μg / ml) in 5'PY medium (Agric.
Biol. Chem. , 53 , 2279 (1989)).
The cells were inoculated into and cultured at 30 ° C. for 1 day. Inoculate 1 ml of the preculture liquid into 100 ml of the same composition medium as above,
The culture was shaken for a day. After culturing, collect the cells by centrifugation,
The cells were disrupted with a microfluidizer and then centrifuged to collect the precipitated fraction. The precipitated fraction was added to Tris-Tri.
ton (0.5% Triton X-100, 10m
M EDTA, 20 mM Tris-HCL (pH 8.
After washing in 0) (50 ml x 2), Tris-Urea
/ DTT (8M Urea, 10mM DTT, 20m
It was dissolved in 50 ml of MTris (pH 8.0).

The solubilized fraction containing the fusion protein of hEGF and HCV-Env (hEGF.BF.HCV-Env) was subjected to anion exchange chromatography (Whatman DE53).
Tris.Urea / DTT equilibrated) and adsorbed.

After adsorption, the column was thoroughly washed with Tris Urea / DTT, and then NaCl 0-0.5M
Elution with a gradient of hEGF / BF / HCV-
Env was recovered. Recovered hEGF / BF / HCV-
Env is TBS (100 mM NaCl, 20 mM Tr
Urea is dialyzed against is-HCl (pH 8.0).
Excluded.

After dialysis, the fraction containing hEGF.BF.HCV-Env was applied to an affinity column adsorbed with anti-hEGF antibody to adsorb hEGF.BF.HCV-Env. After washing the column, hEGF.BF.HCV-Env was eluted with 4M sodium iodide (NaI) and collected to obtain purified hEGF.BF.HCV-Env. Next, purified hEGF / BF / HCV-Env
Treated with Factor-Xa at 8 ° C. for 8 hours, cleaved into hEGF and HCV-Env, subjected to the same EGF affinity column as described above to adsorb only hFGF, and unadsorbed fraction (fraction containing HCV-Env) Were collected and cleaved hEGF was removed. These were dialyzed against TBS to obtain purified HCV-Env.

[0034]

Example 3 Amino acid composition and N-terminal and C-terminal amino acid sequences of purified hEGF.BF.HCV-Env and purified HCV-Env The hEGF.BF.HCV-obtained in Example 2
Since Env is not secreted outside the cells, MWP signal peptide (H. Yamagata et. Al.,
J. Bacteriol. , 169 , 1239 (198
It seems that 23 amino acids of 7)) are added and expressed. The primary sequence of the predicted amino acid is shown in FIG.

Purified hEGF / BF / HC obtained in Example 2
After hydrolyzing V-Env with 6N hydrochloric acid for 8 hours, the amino acid composition was measured by the PITC method (PICO-TAG amino acid analysis system manufactured by Waters) (actual measurement value).
The expected result is hEGF / BF / HCV-Env
The amino acid composition (theoretical value) determined from the primary sequence of the amino acid (Fig. 8) is shown in Table 5 below. Both values were almost the same.

[0036]

[Table 5]

Further, the N-terminal amino acid sequence of hEGF / BF / HCV-Env was analyzed by Edman degradation (P. Edma).
n, Arch. Biochem. Biophys. , 2
2 , 475 (1949)), and the results are shown in Table 6 below.
It was shown to. The results were in perfect agreement with the predicted N-terminal amino acid sequence (Fig. 8).

[0038]

[Table 6]

Furthermore, hEGF / BF / HCV-Env
Of the C-terminal amino acid sequence of C. cerevisiae using carboxypeptidase Y (RP Ambler, Method
in Enzymology, 25 , 143 (197).
The results obtained in 2)) are shown in Table 4. This was also in perfect agreement with the predicted C-terminal amino acid sequence of hEGF.BF.HCV-Env (FIG. 8). From the above results, it was shown that the obtained hEGF.BF.HCV-Env is the target protein.

Then, the amino acid composition, N-terminal and C-terminal amino acid sequences of the purified HCV-Env obtained in Example 2 were examined by the same procedure as described above. The measured values of amino acid composition are shown in Table 7 below together with theoretical values obtained from the primary sequence of predicted amino acids. Both values were almost the same.

[0041]

[Table 7]

The N-terminal and C-terminal amino acid sequences are shown in Table 8 below. Both the N-terminal and C-terminal amino acid sequences were in perfect agreement with the expected primary sequence of amino acids (Fig. 9). From the above results, it was shown that the obtained HCV-Env is the target protein.

[0043]

[Table 8]

[0044]

EFFECTS OF THE INVENTION According to the present invention, it becomes possible for the first time to efficiently produce the hepatitis C virus envelope protein. Therefore, the present invention greatly contributes not only to the diagnosis of hepatitis C but also to the development of preventive and therapeutic methods.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of hEGF gene.

FIG. 2 shows the nucleotide sequence of a synthetic HCV-Env gene.

FIG. 3: hEGF containing a part of MWP signal peptide
Shows a synthetic DNA encoding the amino acid sequence of

FIG. 4 is a construction diagram of plasmid pHT110 · EGF.

FIG. 5 shows the base sequence BF.

FIG. 6 is a construction diagram of plasmid pHT / EGF / BF.

FIG. 7: Plasmids pHT / EGF / BF / HCV-E
It is a construction drawing of nv.

FIG. 8 shows the primary sequence of amino acids of hEGF · BF · HCV-Env.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 15/62 // (C12P 21/02 C12R 1:08)

Claims (5)

[Claims] 1. A hepatitis C virus envelope protein is produced and accumulated in a culture by culturing a bacterium of the genus Bacillus having a nucleotide sequence encoding the amino acid sequence of the hepatitis C virus envelope protein. A method for producing a hepatitis C virus envelope protein, which comprises: 2. The hepatitis C virus envelope protein according to claim 1, wherein all or N of the peptides of SEQ ID NO: 3 in the sequence listing.
A hepatitis C virus envelope protein characterized by producing and accumulating as a fusion protein in which a part including a terminus and a hepatitis C virus envelope protein are linked directly or via a spacer and collecting the fusion protein. Manufacturing method. 3. A method for producing a hepatitis C virus envelope protein, wherein the amino acid sequence of the hepatitis C virus envelope protein of claim 1 or 2 is the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing. 4. The hepatitis C virus envelope, wherein the nucleotide sequence encoding the amino acid sequence of the hepatitis C virus envelope protein according to any one of claims 1 to 3 is the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. Protein manufacturing method. 5. The method for producing a hepatitis C virus envelope protein, wherein the Bacillus of claim 1 is Bacillus brevis.