JP3110437B2 - Epidemic non-A non-B hepatitis virus antigen peptide and nucleic acid fragment encoding the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a gene fragment encoding a viral antigen of a virus causing epidemic hepatitis that is neither type A nor type B (epidemic non-A non-B hepatitis virus). The present invention relates to a non-A, non-B, non-B hepatitis virus antigen peptide and a method of using the same.

Background of the Invention and Prior Art It has long been known that there are two types of viral hepatitis, hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis). This was mainly based on the difference in the infection route, and it was confirmed that hepatitis A caused an epidemic by oral infection and hepatitis B was transmitted mainly through blood. The two hepatitis-causing viruses have already been isolated and identified. Hepatitis A virus is a 27 nm diameter RNA virus belonging to the picornavirus [Fineston, SMet
al., Science 182 p1026 (1973)], whereas hepatitis B virus was found to be a DNA virus belonging to the hepadnavirus and having an envelope of 42 nm in diameter.
[Dane, OS, et al., Lancet, Ip695 (1979)] Also, immunoserologic diagnostic methods for these hepatitis viruses have now been established.

With the establishment of a definitive diagnosis method for these two hepatitis viruses, the existence of non-A non-B hepatitis that does not belong to any of them has been revealed [Prince, AM, et al., Lancet.
I p241 (1974)].

Hepatitis after blood transfusion is hepatitis B virus surface antigen (HBsAg)
Although it decreased significantly due to the introduction of the screening method described above, it did not become zero.
No evidence of hepatitis B infection was obtained. For this reason, this hepatitis is generally called non-A non-B hepatitis.

This hepatitis accounts for about 50% of sporadic hepatitis and more than 90% of post-transfusion hepatitis in Japan, as well as chronic hepatitis, cirrhosis and liver cancer.
It is estimated that 50% or more is caused by non-A, non-B hepatitis, which is a major social problem.

Recently, HCV (hepatitis C virus) has been described for this non-A non-B hepatitis virus, which is mainly transmitted via blood.
Although the name has been established, screening methods for antibodies related to HCV have already been developed, but the detailed virological properties are still unknown [Choo et al., Science,
244,359-362 (1989), Kuo et al., Science, 244,362-364.
(1989)].

Separately, it has been found that there is a second viral non-A non-B hepatitis that is prevalent by oral infection in India, Myanmar, Afghanistan, or North Africa [Khuroo, MSAm. J. Med. ., 68 p818-824, (198
0)]. This is generally water-based or epidemic non-A non-B
Hepatitis hepatitis. In Japan, this hepatitis epidemic has not been observed, but it seems that some imports of hepatitis from endemic areas of travelers are seen [Fukuhara et al., Proceedings of the 25th Annual Meeting of the Japanese Society of Hepatology, 151 pages (1989)] ].

The present invention relates to the latter, said epidemic non-A non-B hepatitis virus, which is transmitted mainly through water,
This virus, which is not HAV, HBV or HCV, is referred to herein as a non-A non-B hepatitis virus.

Regarding this non-A non-B hepatitis, the virus itself has not been isolated and identified, and therefore, a method for diagnosing, treating and preventing this hepatitis has not been established. The diagnosis of hepatitis was based on the exclusion diagnosis. That is, the serum of a patient is tested for hepatitis A and B, for which a diagnostic method has been established, and hepatitis is denied. Furthermore, hepatitis is shown as a part of systemic infection. Herpes,
He denied the possibility of cytomegalo and Epstein-Barr virus infection, denied drug-induced, alcoholic, and autoimmune hepatitis and was diagnosed with non-A, non-B hepatitis.

Non-A non-B hepatitis which is the subject of the present invention has its main endemic areas such as India, Nepal, Myanmar and North Africa. It is known that the main route of infection is through drinking water or vegetables, and the symptoms after infection are usually transient infections, i.e., acute hepatitis but not persistent infections. I have. However, mortality is known to be as high as 20% when pregnant women are infected with this virus [Tandon et al., Indian J. Med. Res. 75, 739-7.
44 (1982)], and drinking water pollution often causes pandemics. Recent examples include 1986-1988
Outbreaks of 120,000 cases have been reported in the southwestern Urumqi Autonomous Region of China over the year [Modern Science, July 1987, 62-67, Journal of Infectious Diseases, 64,105-].
111 (1990)]. Therefore, there is a need for people traveling to such endemic areas to develop effective vaccines and diagnostic reagents that can easily detect the state of possession of antibodies, antigens or viruses themselves.

According to previous reports, detection of virus-like particles by immunoelectron microscopy has been reported [Sreenivasan et al., J. Gen. V.
irol., 65, 1995-1007 (1984)], and successful infection experiments in animal models using monkeys [Kane et al., JAMA 252, 3140-3145 (1984)].

The present inventors have also succeeded in infecting cynomolgus monkeys by inoculating a virus solution prepared from feces of an acute-stage patient in an endemic area into the vein, and succeeded in sub-infection. Virus-like particles were also detected in feces and monkey liver cells of infected monkeys [Soe et al., Liver, 9, 135-145 (198).
9)]. Furthermore, it was confirmed that a large amount of virus-like particles were detected in monkey bile [Kyundai Kagaku, July 1987, 62-
67], and succeeded in cloning a viral cDNA fragment, and filed a patent application [Japanese Patent Application No. 2-64629].

It has recently been reported that Genelabs of the United States has cloned the cDNA of non-A non-B hepatitis virus [Reyes et al., Science, 247, 1335-1339 (1990)]. The properties of the constituent proteins have not been clarified yet.

Object of the Invention Under such circumstances, the present inventors have conducted repeated studies for the purpose of cloning the non-A non-B hepatitis causative virus or the virus gene thereof, and as a result, the non-A non-B hepatitis virus The gene encoding the antigen peptide sequence was successfully cloned. Furthermore, the present inventors have confirmed that the peptide obtained by expressing this gene fragment using a gene recombination technique specifically reacts with the serum of non-A, non-B hepatitis patients also at the protein level. The invention has been completed.

That is, the present invention relates to a non-A non-A non-A non-A non-A non-A non-A non-B sample obtained by immunoscreening using a new molecular genetic technique different from the conventional immunoserologic method using a non-A non-B hepatitis-infected cynomolgus monkey bile preparation. It is intended to provide a peptide unique to hepatitis B virus and a gene encoding the same.

Structure and Effect of the Invention The non-A non-hepatitis B virus gene fragment of the present invention comprises
It can be extracted and isolated from bile of cynomolgus monkeys infected with non-hepatitis B virus. First, the bile is diluted with physiological saline, and the tissue pieces are removed by low-speed centrifugation. Next, it is layered on physiological saline containing 20% sucrose, and ultracentrifuged. This allows virus particles to be collected in the sediment. RNA is extracted by treating the virus particles with guanidine thiocyanate, and cDNA is synthesized using the RNA as a template. This cDNA is inserted into a λgt11 vector to create a cDNA library.

The λ phage is infected into E. coli, spread on a bacterial culture plate, and cultured at 42 ° C. for several hours. Then cover with a nitrocellulose filter (NC filter) and incubate for several hours.
Remove the filter and take a replica.

This replica is treated with a blocking solution, washed with Tris-buffered saline (TBS) or the like, and then subjected to immunoscreening. That is, the replica was allowed to react with cynomolgus monkey serum in the non-A non-B hepatitis recovery phase, washed with TBS, etc.,
It is reacted with an enzyme-labeled anti-human IgG, washed, and reacted with a substrate solution to develop a color. A phage corresponding to the developed plaque is selected and subjected to a secondary screening to obtain a reproducible λ.
A phage clone was obtained.

This clone was examined for non-A, non-B hepatitis specificity.

As a result of plaque immunoassay using cynomolgus monkey serum in the non-A non-B hepatitis recovery period and the normal period, a clone highly specific to non-A non-B hepatitis virus infection was obtained. Purification of the phage DNA of this clone [Experimental Medicine Special Issue, Heterogeneous Engineering Omnibus 5 (11),
P31-32 (1987)], digested with EcoRI, and subjected to polyacrylamide gel electrophoresis to confirm a 0.22 Kbp insert (HT3-3). This is called the expression vector pUEX2
(Amersham) and subcloned (HT3-3
A) It was confirmed that the E. coli expression product reacted with the cynomolgus monkey serum described above.

Purification of the liver and chromosomal DNA of normal life and non-A, non-B hepatitis acute cynomolgus, after agarose gel electrophoresis, Southern hybridization was carried out using a ³² P-labeled HT3-3. HT3-3 did not react with any DNA, and thus HT3-3 was found not to be chromosome-derived DNA.

The DNA sequence of HT3-3A of the present invention was determined by the dideoxy method. As a result, HT3-3A was a cDNA fragment of a total of 216 bp derived from the non-A, non-B hepatitis virus gene, and its nucleotide sequence was as shown in FIG. This DNA sequence was read back to amino acids, and a frame matching the expression frame of λgs11 and pUEX2 was defined as an open reading frame. This base sequence and amino acid sequence are stored in a database (Gene
A search by tyx-CD software development 1990) revealed no known viruses, bacteria or other homology.

From this amino acid sequence, the hydrophilicity / hydrophobicity pattern of the peptide encoded by HT3-3A was analyzed based on the method of HOPP & WOOD et al. As a result, results as shown in FIG. 3 were obtained, and it was confirmed that three particularly hydrophilic regions were present in this peptide region.

As described above, the cDNA fragment obtained by the present invention is a non-A non-B
The fact that it encodes a peptide region having strong hydrophilicity among hepatitis hepatitis virus antigens was considered to be very significant from an immunological point of view. In addition, since such a hydrophilic peptide is easy to handle, it is very useful from the viewpoint of practicality.

In the present invention, as a preferred example of the non-A non-B hepatitis virus antigen peptide, not only a peptide consisting of 72 amino acids as shown in FIG. 2 is disclosed, but also a non-A non-B hepatitis virus It is thought to be closely related to antigenicity. Also disclosed are the three highly hydrophilic or non-A non-B hepatitis virus antigen epitopes containing them as described above. Such an antigenic epitope is the amino acid sequence of (A) to (C) below.

In addition, since the antigen epitopes (A) and (B) are extremely close to each other in the amino acid sequence of FIG. 2, the following sequences including (A) and (B) are also considered to be important epitopes. .

As shown in the analysis pattern of FIG. 3, it is confirmed that the peptide region of the above amino acid sequence is a peptide having particularly strong hydrophilicity. Furthermore, in order to confirm the relevance to non-A non-B hepatitis, antigens obtained by incorporating and expressing this gene fragment in an Escherichia coli expression system were used for the sera of many hepatitis patients and normal persons. HT3
A Western blot assay for -3A was performed. As a result, antibody-positive individuals were detected in non-A non-B hepatitis patients at a higher rate than normal, hepatitis B, and other groups of hepatitis, and the non-A non-B Specificity for hepatitis B has been demonstrated.

The gene sequence of the present invention can be expressed by using an appropriate expression system and used for an antibody test of non-A non-B hepatitis virus. It can also be used to detect non-A non-B hepatitis virus or related antigens in liver tissue of non-A non-B hepatitis infected patients.

Furthermore, the non-A, non-B hepatitis virus-related antigen obtained in the present invention is extremely useful for preparing a vaccine for preventing infection.

In addition, the gene sequence itself is non-A non-B hepatitis DNA
It is extremely useful for the development of probe diagnostic kits.

Such a nucleic acid fragment encoding the non-A non-B hepatitis virus antigen peptide of the present invention, the non-A non-B hepatitis virus antigen peptide, and various detections of the non-A non-B hepatitis virus-related antigens and antibodies using the same. The method is believed to be extremely useful, especially in the detection of non-A non-B hepatitis virus in Japan.

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

Examples (1) Preparation of non-A, non-B hepatitis virus: A virus solution prepared from the stool of the acute phase of a non-A, non-B hepatitis patient was inoculated into a cynomolgus monkey vein to establish infection. In addition, a passage infection using feces of infected monkeys was established (Liver 9, p135-145 (1989)).

When the bile of the infected monkey was observed with an electron microscope,
Virus particles with a diameter of about 27 nm were detected (Hyundai Chemistry 1989
July, p62-67). Virus particles were purified from about 2 ml of this bile, and the following experiment was used.

(2) Cloning of non-A non-B hepatitis virus gene fragment: The virus particles obtained in (1) were treated with 5.5M guanidine thiocyanate to extract nucleic acids derived from the virus genome. cDNA synthesis was performed using cDNA synthesis system plus (Amersham), and the synthesized cDNA was cloned into a λgt11 vector using a cDNA cloning system λgt11 (Amersham). As a result of in vitro packaging, a library of 5.7 × 10 ⁴ plaque forming units (PFU) was obtained.

(3) Screening of non-A non-B hepatitis virus-related clones using cynomolgus monkey serum at the recovery stage of non-A non-B hepatitis: (A) Preparation of a replica filter for antibody screening One sheet from the cDNA library constructed in (2) LB plate [1.5% Agar, 1% Bacto-tryptone, 0.5% Bacto
Bacterial culture plate containing yeast extract, 1% NaCl pH7.5, 50ug / ml ampicillin (Eiken; 82mmφ)]
1000 PFU of phage were taken and E. coli Y1090 was separately infected with phage without insert at 37 ° C. for 15 minutes. Top Agar 4ml (0.7% Agar, 1% Bacto-tryptone,
0.5% Bacto-yeast extract, 1% NaCl, pH7.5, 50ug / m
lampicillin) and cultured at 42 ° C for 3 hours.
Then, a nitrocellulose filter (NC filter: S & S, C, C) impregnated with 10 mM IPTG (Sigma)
(ode BA85, 82 mmφ), and the culture was continued at 37 ° C. After 3 hours, remove the NC filter from the plate,
Washed with TBS containing 20% Tween 20 (TBS-T), immersed in blocking solution (TBS-T solution containing 5% skim milk),
Left overnight at ° C.

(B) Antibody screening Replica filter soaked overnight in blocking solution
After washing with TBS-T, the cells were immersed in cynomolgus monkey serum from the normal phase and the non-A non-B hepatitis recovery phase diluted 50-fold with a blocking solution, and reacted at 37 ° C with shaking. One hour later, TBS-
At T, the replica filter was washed three times for 10 minutes each time, and then immersed in a blocking solution containing a 1000-fold diluted peroxidase-labeled anti-human IgG goat antibody (manufactured by Biomakor), and shaken at 37 ° C. Reacted. After 1 hour, 3 times with TBS-T for 10 minutes each time, then with TBS
After washing for 10 minutes, the coloring solution [0.6 mg / ml 4-chloro-1-naph
thol (manufactured by Bio-Rad), 20% MeOH, 0.06% H ₂ O ₂ ] to develop color. Phages corresponding to plaques developed on the NC filter were selected and subjected to secondary screening. That is, each phage selected in the primary screening was separately infected with Escherichia coli Y1090 together with a phage having no insert fragment, and spread again on an LB plate of an 82 mm petri dish (manufactured by Eiken Co., Ltd.) to prepare a replica filter. These were subjected to antibody screening by the above-mentioned method to obtain four clones (HT3-1, 2, 3, 5) of phages which reacted with reproducible cynomolgus monkey serum in the non-A non-B hepatitis recovery period. The phage DNAs of these clones were purified [see Experimental Medicine Extra Edition, Gene Engineering Summary 5 (11), pp. 31-32 (1987)], and the restriction enzyme EcoR
After digestion with I, subject to polyacrylamide gel electrophoresis,
1 Kbp (HT3-1), 0.3 kbp (HT3-2), 0.22 kbp (HT3-3,
The inserted fragment of 5) was confirmed.

(4) Southern blot analysis using HT3-1,2,3,5: Southern blot analysis using HT3-1,2,3,5 was performed as follows. Chromosomal DNA was purified from normal and non-A non-B acute hepatitis liver of cynomolgus monkeys,
After cutting with I, run on a 1% agarose gel by electrophoresis,
Transferred to a nylon filter. Using this filter as a probe, each clone labeled with [ ^32- P] by the multi-prime method was subjected to Southern hybridization (No. 4).
Figure). Each probe did not react with chromosomal DNA of normal and non-A non-B acute hepatitis cynomolgus monkeys.
T3-1, 2, 3, and 5 are not clones derived from the chromosome DNA of cynomolgus monkeys, but are considered to be derived from exogenous nucleic acids such as viruses.

(5) Subcloning of HT3-1,2,3,5: (3) Purification of the phage DNA of the clone obtained in (B) [Experimental Medicine Extra Edition, Genetic Engineering Summary 5 (1)
1), p31-32 (1987)], cut with the restriction enzyme EcoRI, inserted into the EcoRI site of pUEX2 vector (manufactured by Amersham), subcloned, and subcloned HT3-1B, 2E, 3A, respectively. 5A was obtained.

(6) Nucleic acid base sequence and amino acid sequence of HT3-1B, 2E, 3A, 5A: (A) Determination of base sequence of HT3-1B, 2E, 3A, 5A clone Using plasmid DNA obtained in (5) as a template , [Α-
^The reaction was performed by the dideoxy method using ³⁵ S] dCTP (1000 Ci / mmol) and Sequenase V2.0 kit (manufactured by USB). Using a 6% polyacrylamide-8M urea gel, the mixture was electrophoresed at 2200 V for 3 hours and exposed for 16 hours.

(B) Obtained base sequence and predicted amino acid sequence As a result of the above, HT3-1B, 2E, 3A, and 5A overlap with each other and one continuous sequence was obtained. FIG. 1 shows the results of decoding the HT3-3A sequence and the predicted amino acid sequence, respectively.
As shown in FIG.

The predicted hydrophilicity / hydrophobicity profile of the amino acid sequence is shown in FIG.

As a result of searching the obtained base sequence and amino acid sequence in a database (described above), there were no viruses, bacteria or other substances showing high homology.

(7) Examination of the specificity of HT3-3A against non-A non-B hepatitis: (A) Preparation of Escherichia coli lysate pUEX without plasmid and insert obtained in (5)
Escherichia coli HB101 strains were separately infected with each of 2 to prepare recombinants. This Escherichia coli was cultured at 30 ° C. in an LB medium, and the temperature was induced at 42 ° C. in the logarithmic growth phase to induce expression,
Two hours later, cells were collected. Bacteria are pelleted by coarse centrifugation, 1 mM
TBS-T containing PMSF and glass beads were added, and vigorously stirred for 15 minutes to disrupt the cells. This was centrifuged at 3,000 rpm for 10 minutes, and the precipitate was suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM EDTA to obtain an E. coli lysate.

(B) Western blot assay (7) The E. coli lysate obtained in (B) was subjected to SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and subjected to a coloring reaction from a blocking reaction according to (3). Was. However, anti-human IgM goat antibody was used as the secondary antibody for the acute phase patient serum. The results of the Western blot assay are shown in Tables 1, 2 and 3.

Table 1 Non-A, non-B type reaction with hepatitis infection cynomolgus time serum serum HT3-3A pUEX2 1. infection after 0 days + - 17 days + - 21 days ++ - 28 ++ - 42 days + - 56 days + - 2. 0 days after infection--21 days ++-28 days ++-35 days ++- 42 days + -Table 2 Serum with sera from non-A non-B hepatitis patients in Myanmar HT3-3A pUEX2 patients A acute phase--recovery phase 1 ± -recovery phase 2 + -patient B acute phase +-recovery phase 1 + -recovery phase 2 ± -patient C acute phase ±-recovery phase 1 + -recovery phase 2 + -patient D acute phase +-recovery phase 1 +- convalescent 2 - - Table 3 normal cynomolgus, normal human, B-type hepatitis patients, reactions with other hepatitis patient sera sera HT3-3A pUEX2 normal cynomolgus -1 - - - 2 - - normal human -1 - - - 2 - - B hepatitis -1 - - - 2 - - - 3 - - other hepatitis -1 - - - 2 - - HT3-3A from White reacts very well with non-A, non-B hepatitis infection cynomolgus time serum and Kyanma non-A, non-B hepatitis patient serum, pUEX2 from lysates without insert did not react with these sera. Nor did it react with normal cynomolgus monkey serum, normal human serum, hepatitis B patient serum, or other proteins derived from HT3-3A serum from hepatitis patients.

As described above, the HT3-3A clone of the present invention is non-A non-B
It was shown to be hepatitis-specific.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of HT3-3A cloned in the present invention. FIG. 2 shows the entire amino acid sequence encoded by HT3-3A cloned in the present invention. FIG. 3 shows the hydrophilicity / hydrophobicity profile of the peptide encoded by HT3-3A based on the amino acid sequence. FIG. 4 is a schematic diagram of Southern hybridization between HT3-1, 2, 3, 5 cloned in the present invention and cynomolgus monkey chromosomal DNA.

────────────────────────────────────────────────── ─── of the front page continued (51) Int.Cl. ⁷ identification mark FI G01N 33/569 G01N 33/569 L 33/576 33/576 Z // (C12P 21/02 C12R 1:19) examiner Sachiko Yamamura (56) References JP-A-3-266987 (JP, A) European Patent Application Publication 318216 (EP, A1) Proc. Japan. Acad. , Vol. 65, p. 219-223 (1989) Hyundai Kagaku, July issue, p. 62-67 (1989) (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 15/00 C07K 7/06 C07K 7/08 C12P 21/02 C12Q 1/68 G01N 33/576

Claims (9)

(57) [Claims] An epidemic non-A non-B comprising the following amino acid sequence:
A nucleic acid fragment encoding a hepatitis virus antigen peptide. 2. The nucleic acid fragment according to the above (1), comprising the following nucleotide sequence. 3. An epidemic non-A comprising the following amino acid sequence:
A nucleic acid fragment encoding a peptide having a non-hepatitis B virus-specific epitope. 4. The nucleic acid fragment according to the above (3), comprising the following nucleotide sequence. 5. An epidemic non-A non-B comprising the following amino acid sequence:
Hepatitis virus antigen peptide. 6. An epidemic non-A comprising the following amino acid sequence:
A peptide having a non-hepatitis B virus-specific epitope. 7. The peptide according to (6), which is obtained by incorporating the nucleic acid fragment according to (3) or (4) into an appropriate expression vector and expressing this in a host cell. . 8. A method for immunologically detecting an epidemic non-A non-B hepatitis virus-related antibody, comprising using the peptide according to (6) or (7). 9. A nucleic acid probe for detecting an epidemic non-A non-B hepatitis virus gene, which comprises a nucleic acid fragment represented by the following nucleotide sequence or a sequence complementary to the nucleotide sequence: .