JPH03103180A - Nucleic acid fragment to code non-a, non-b type hepatitis virus antigen and use thereof - Google Patents

Abstract

NEW MATERIAL:A nucleic acid fragment to code non-A; non-B type hepatitis virus antigen peptide. EXAMPLE:A nucleic acid fragment to code an amino acid sequence shown by the formula. USE:For detecting non-A, non-B type hepatitis virus. PREPARATION:Plasma having high GPT value is concentrated about 1,000 times, whole nucleic acids are purified, DNA derived from human is decomposed, RNA is purified, cDNA is synthesized, inserted into lambdagt11 vector to prepare cDNA library. Escherichia coli is infected with lambda phage, cultured and replica is subjected to immunoscreening to give a clone.

Description

[Detailed Description of the Invention] - 1 The present invention provides a gene fragment encoding a viral antigen of a virus that causes serotype hepatitis that is neither A nor B (non-A IPB hepatitis virus), and a non-A, non-B hepatitis virus. Regarding antigenic peptides and their usage.It has long been known that there are two types of viral hepatitis: hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis).This is mainly due to the route of infection. Based on differences,
It has been confirmed that hepatitis A causes epidemics through oral infection, and hepatitis B is primarily transmitted through blood. These two hepatitis-causing viruses have already been isolated and identified, and the hepatitis A virus is an RNA virus with a diameter of 27 nm that belongs to the bicornavirus [Fine
ston S. M. et al. , Science
182 pl026 (1973)], while hepatitis B virus has a diameter of 42nI1, which belongs to the hepadnaviruses.
It was determined that it was a DNA virus with an envelope of [Dane, 0. S. , et at
．． , Lancet, I p695 (1970)]
Furthermore, immunoserological diagnostic methods for these hepatitis viruses have now been established. As definitive diagnostic methods for these two hepatitis viruses have been established, the existence of non-A, non-B hepatitis that does not belong to either of them has become clear [Prince A. M. ,e
tal. , Lancet. I p241 (1
974)]. Post-transfusion hepatitis has decreased significantly with the introduction of screening methods for hepatitis B virus surface antigen (HBsAg), but it has not been eliminated, and patients who have hepatitis do not show evidence of hepatitis A or B infection. was not 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, and it is estimated that more than 50% of chronic hepatitis, liver cirrhosis, and liver cancer are caused by non-A, non-B hepatitis. It has become a major social problem.

Separately, it has become clear that there is a second type of viral non-A, non-B hepatitis that is prevalent through oral infection in India, Burma, Afghanistan, and North Africa [
Khuroo, M. S. ^m. J. Med
．． , 68 p818-824, (1980) Ko. This is commonly referred to as waterborne or epidemic non-A, non-B hepatitis. Although there is no outbreak of this hepatitis in our country, there seems to be some cases of hepatitis being imported from endemic areas by travelers [f! Hara et al., Proceedings of the 25th Annual Meeting of the Japanese Society of Hepatology, p. 151 (1989)]. The present invention relates to the former serotype non-A, non-B hepatitis virus that is mainly transmitted through blood,
This virus is referred to herein as a non-A, non-B hepatitis virus. The virus itself for this non-A, non-B hepatitis has not been isolated and identified, and therefore no diagnostic, therapeutic, or preventive methods for this hepatitis have been established. Furthermore, the only way to diagnose this hepatitis was to make a diagnosis of exclusion. That is, the patient's serum is tested for hepatitis A and B, for which diagnostic methods have been established, to deny these hepatitis cases, and to show hepatitis symptoms as part of systemic infection. The possibility of herpes, cytomegalo, or Epstein-Barr virus infection was ruled out, and drug-induced, alcohol-induced, or autoimmune hepatitis was ruled out, and the patient was diagnosed with non-A, non-B hepatitis. It is known that the virus that causes hepatitis is infectious.
This was proven by an American research group in 1988 through infection experiments using chimpanzees [Tabor, E. ．．
et al. , Lancet. I p463 (197
8)]. Despite many efforts around the world, the virus that causes the disease remains unknown even after more than 10 years. Using the blood and liver tissue of patient-infected chimpanzees as materials, type A and B types such as agar gel precipitation reaction, immunoelectric countercurrent method, radioimmunoassay, fluorescent antibody method, and electron microscopy method are used.
Almost every approach used in hepatitis research has searched for the virus and its associated antigen-antibody systems, but no certainty has yet been obtained. The history of research into non-A, non-B hepatitis viruses can be said to be a history of expectations and disappointments. Many candidates for viruses or antigen-antibody systems have emerged, but they have been rejected one after another [Prince, A. M. , An
n. Rev. Microbiol. ．． 37, P2L
7. (1983)]. A recent example is the retrovirus theory by Seto et al. [Seto, B. et
al. : Lancet. Ip
941-943 (1984)], according to them,
Reverse transcriptase activity was detected in serum and blood preparations that have been proven to cause non-A, non-B hepatitis in chimpanzees, and SigI! In density gradient centrifugation, this enzyme: 1. 14g/m
In other words, it had a floating density similar to that of retroviruses. Subsequently, Prince et al. inoculated primary cultured chimpanzee liver cells with patient serum and reported that retrovirus-like particles were observed [ Pr
ince, A. M. et al. Lancet.
I: pl071-1075 (1984)]. However, reverse transcriptase activity was denied by Hollinger et al. [Hollinger at al.
, Lancet, I p41 (1986)].
Furthermore, the virus particles observed by Prince et al. were ruled out as myxovirus contamination. Problems that make research on non-A, non-B hepatitis difficult include the low virus concentration in the serum of 102 to 103, and the presence of antibodies in chimpanzees that have been reinfected with the same inoculum. The problem is that the only experimental models for infection are chimpanzees and marmosets. Recently, there was a report that Chiron Corporation of the United States had captured the cDN^ of a non-A, non-B hepatitis virus [Ch.
oo, Q et al. , Science, 244
, P359-362 (1989), Kuo, G.
et al. , Science, 244.
p362-364 (1989)], the properties of the virus itself, sequence information, and properties of the virus structure and proteins have not yet been clarified.

In general, diagnostic methods for different viruses are completely different due to differences in their immunoserological and molecular genetic properties. In addition, different strains have some different immunoserological properties, so using the same diagnostic method will result in differences in detection sensitivity due to differences between strains, and in vaccines, differences in immunogenicity and ability to protect against infection. It is generally known that in molecular genetic diagnostic methods, such as DNA probe diagnosis, hybridization between a probe and a viral nucleic acid is not practical unless the homology at the nucleic acid level is extremely high. In other words, due to differences at the nucleic acid level between forests,
There may be cases in which DNA hybridization does not occur and DNA probe diagnosis cannot be performed effectively. Hepatitis B, which is a well-known and well-analysed serotype of hepatitis, has major subtypes of the hepatitis B virus in each region, such as Europe, America and Southeast Asia, that is, the prevalent strains characteristic of that region. Since it is known that there are subtypes), non-A
Even among non-B hepatitis viruses, there may be regionally specific virus species or strains.

Therefore, in order to establish diagnostic and preventive methods for non-A, non-B hepatitis viruses that are prevalent in specific regions, especially Japan, it is necessary to identify the major non-A, non-B hepatitis virus strains in Japan. ．． Menmi Tatsudan 1 Under these circumstances, the present inventors conducted repeated research with the aim of cloning the causative virus of non-A, non-B hepatitis or its viral genes. We succeeded in cloning the gene encoding the antigenic peptide sequence of non-B hepatitis virus. That is, the present inventors finally detected non-A, non-B hepatitis by using immunoscreening method that incorporates a new molecular genetic method different from the conventional immunoserological method using GPT-high plasma from blood donors. We have cloned the gene encoding a peptide unique to the virus. Furthermore, it was confirmed that the expression product obtained by expressing this gene fragment using genetic recombination technology specifically reacts with the serum of patients with non-A, non-B hepatitis at the protein level. It's arrived. Japan) When cloning nucleic acid fragments as the object of the present invention, livers of Japanese people infected with non-A, non-B hepatitis and livers of chimpanzees infected with non-A, non-B hepatitis were used as research materials. It is conceivable that the virus-specific cDNA could be selected by extracting the mRNA and synthesizing the cDNA using the virus, and then subtracting it with the chromosomal DNA. However, it is extremely difficult to secure sufficient quantities of good experimental materials for this purpose. Another possible research material would be plasma from non-A, non-B hepatitis infected individuals or infected chimpanzees. Non-A, non-B in humans
The existence of carriers of hepatitis has been confirmed, and the higher the donor's GPT value during blood transfusion, the higher the incidence of non-A, non-B hepatitis after transfusion, so it is assumed that plasma with high GPT values is more likely to be a carrier. ing. Therefore, we pooled high GPT plasma from Japanese blood donors, which is available in relatively large quantities, and used it as research material. In addition, plasma from a chimpanzee that has developed non-A, non-B hepatitis after being inoculated with the serum of a Japanese patient with non-A, non-B hepatitis can also be used, but there are currently some problems due to the availability of chimpanzees. As mentioned above, the concentration of non-A, non-B hepatitis virus in plasma is estimated to be only about 102 to 103, so extraction of viral nucleic acid and synthesis of cDNA requires 10
It is necessary to concentrate the virus approximately 00 times. However, human plasma is a protein solution of around 7%, and it is impossible to simply concentrate it, so it is difficult to remove viruses while removing protein. It needs to be 4m. Precipitation with a precipitant such as polyethylene glycol (PIEG), which we used, is a mild method that is relatively easy to perform, is suitable for processing large amounts of plasma, and is less likely to inactivate the virus. ．． Other methods include beretting using ultracentrifugation,
Salting out by adding salts such as ammonium sulfate, ultrafiltration, gel chromatography, etc. can be used. 100 like this
Plasma concentrated approximately 0 times is treated with guanidinium thiocyanate, extracted with phenol/chloroform, and all nucleic acids in the concentrated plasma are purified by ethanol precipitation. Next, human-derived DNA that is contaminated with DNA degrading enzymes
RNA is purified by phenol/chloroform extraction and ethanol precipitation. cDNA is synthesized from the purified RNA and inserted into the λgtll vector to create a cDNA library. Infect Escherichia coli with the λ phage, spread on a bacterial culture plate, and incubate at 42°C for several hours. After that, cover with a nitrocellulose filter (NC filter) and culture for several hours.
Peel off the NO filter and take a replica. This replica is treated with Proking solution, washed with PBS, etc., and then subjected to immunoscreening. That is, the replica is reacted with human or chimpanzee serum in the convalescent or acute stage of non-A, non-B hepatitis, washed with PBS, etc., reacted with enzyme-labeled anti-human IgG or IgM, and after washing, reacted with a substrate solution. Develop color. Secondary screening was performed by selecting phages that corresponded to the colored Blaak, and reproducible clones were obtained. We investigated the specificity of this clone for non-A, non-B hepatitis. Plaque immunoassay was performed using IgG from chimpanzees in the convalescent stage of non-all-B hepatitis, first-stage carrier, and normal stage, and the results showed that hepatitis was non-A, non-B. We were able to obtain highly specific clones during the first carrier stage. This clone was subcloned and analyzed by agarose gel electrophoresis. 2
An 8Kbp insert fragment (C825) was confirmed. Chromosomal DNA was purified from chimpanzee livers of normal phase and acute phase of non-A, non-B hepatitis, and leukocytes of normal people, and after performing agarose electrophoresis, 32p-labeled C825 was purified.
Southern hybridization was performed using the clone, and C825 did not react with any DNA, and it was therefore determined that C825 was not chromosome-derived DNA. In addition, a pool of Japanese plasma with high GOT and GPT values (non-A
(Non-B hepatitis infectivity has been confirmed in chimpanzee infection experiments), RNA was extracted from chimpanzee plasma passaged from the American NIH-derived non-All-B hepatitis strain F strain, and Japanese normal human plasma, and cDNA was extracted. A PCR reaction was carried out using part of the base sequence of the C825 clone as a primer [SAiki et al. Science 239.
p487- (1988)]. Similarly, DNA was extracted from normal human liver and PC was used using the same primers.
An R reaction was performed. As a result, the C825 base sequence was detected only from the pool of Japanese plasma with high GOT and GPT values. This suggests that the non-A, non-B hepatitis virus that we captured that contains the C825 nucleotide sequence is significantly different in its nucleic acid sequence from the non-A, non-B hepatitis virus of the F strain derived from the US NIH. ．． The DNA sequence of the 0825 clone of the present invention was determined by the dideoxy method. As a result, C825 clone was non-A
A total of 269 bP cDNA derived from non-B hepatitis virus genes
A fragment, and its base sequence was as shown in Figure 3. When we reread this DNA sequence into amino acids, we found that only one frame had a stop codon in the middle, which was part of the open reading frame. This nucleotide sequence and amino acid sequence are stored in a database (Ge
netyx-CD Software Development 1989), there were no viruses, bacteria, or other homologous substances known to date. From this amino acid sequence, based on the method of HOPP & WOOD et al., we determined the hydrophilic peptide encoded by C825.
The hydrophobic pattern was analyzed. As a result, the results shown in Figure 5 were obtained, and it was confirmed that this peptide region was a highly hydrophilic peptide as a whole. moreover,
It was confirmed that within this peptide consisting of 89 amino acids, there were four regions with particularly strong hydrophilicity. In this way, the CDNAIT piece obtained in the present invention is non-A
The fact that it encoded a highly hydrophilic peptide region among non-B hepatitis virus antigens seemed to be very significant from an immunological standpoint. In addition, such hydrophilic bebutides are easy to handle, making them extremely useful from a practical standpoint. The present invention not only discloses a peptide consisting of 89 amino acids as shown in FIG. The present invention also discloses the four highly hydrophilic non-A, non-B hepatitis virus antigen evitopes mentioned above, which are thought to be closely related to sex. Such antigen evitobes are as follows (A) to (D).
) is the amino acid sequence of (A) Glu Thr Ala Lys Arg A
rg Leu Ala Arg Gly, (B) Th
r Thr Arg His Asp Ser Pro
Asp Ala, (C) Thr Arg Val
Glu Ser Glu Asn Lys Val V
al, (D) Pro Leu^rg Ala Glu
Glu Asp Glu As shown in the analysis pattern in Figure 5, the peptide region of the above amino acid sequence is
It is confirmed that the peptide is particularly hydrophilic.

To further confirm the relationship with non-A IPB hepatitis, we performed plaque immunoassay and dot immunoassay for C825 using serum from a large number of hepatitis patients and normal individuals. As a result, a higher proportion of non-A, non-B hepatitis patients were detected as antibody-positive compared to normal people, hepatitis B, and other hepatitis groups, and immunoassay showed that non-A, non-B hepatitis was detected at the protein level.
Specificity for type hepatitis was proven. The gene sequence of the present invention can be expressed using an appropriate expression system and used for antibody testing of non-A, non-B hepatitis viruses, or can be used to immunize animals with the expressed protein to generate antibodies. It is also possible to detect non-A, non-B hepatitis virus in the liver tissues of patients infected with non-A, non-B hepatitis. Furthermore, the non-A, non-B hepatitis virus obtained in the present invention is extremely useful for producing vaccines for preventing infection. In addition, the gene sequence itself is the DNA of non-A, non-B hepatitis.
It is extremely useful for developing probe diagnostic kits. Such nucleic acid fragments encoding non-A, non-B hepatitis virus antigen peptides, non-A, non-B hepatitis virus antigen peptides, and various methods for detecting non-A, IP, hepatitis B viruses using these of the present invention are particularly useful. Non-A in Japan
It is considered to be extremely useful in detecting non-B hepatitis viruses. The present invention will be explained in more detail below with reference to Examples. t{Bs antigen negative GP donated by the Japanese Red Cross Society.
Human boule plasma (approximately 8.2111) with a T value of 100 or higher was concentrated 1000 times using the following method. First, human boule plasma was roughly centrifuged to remove insoluble matter. 1710 for this
volume of 5M sodium chloride solution, then l/10 volume of 40
% (W/W) Polyethylene glycol liquid (PEG600
0, manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 7500) was added at 4°C with stirring. After standing for one hour, centrifugation was performed at 7,000 rpm for 20 minutes to remove the supernatant, and the sediment was mixed with TNE solution (10 + oM Tris-
HCI. pH7.4, 1+++MEDTA, 140
mM NaCI) was added and redissolved. This solution was layered on top of a centrifuge tube in which 20%, 15%, 10%, and 5% sucrose TNH solutions were layered in stages, and the solution was heated at 4°C and 80,000 ml.
Ultracentrifuged at xG for 12 hours. After several minutes, the supernatant was removed and the precipitate was dissolved in 8 ml of Gffr. G.P.
It was made into a 1000-fold concentrate of T-high human pool plasma. First, 5 times the amount of guanidium thiocyanate solution (4M guanidium 11 thiocyanate, 50+*M Tris-11c non-117.6,
10mM EDTA, 0.1M 2-mercaptoethanol, 2% sarcosyl) was added, and after stirring, phenol/
Total nucleic acids in concentrated plasma were purified by chloroform extraction and ethanol precipitation using glycogen as a carrier.
Next, in order to degrade the human-derived DNA present in this total nucleic acid, RNase-free DNase 1.1 was added in the presence of 2mM vanadyl ribonucleotide complex.
5KU/ml (manufactured by Hönger/Mannheim), 5
0+M Tris-■CI pH7.4, 1mM ED
In 400JA of a mixture of TA, 10mM MgCla,
Processed at 37°C for 30 minutes. Then 25hM EDT
The reaction was stopped by adding 16IA of solution A and 8A of 10% SDS solution, and RNA was purified by phenol/chloroform extraction and ethanol precipitation. Furthermore, in order to remove a large amount of glycogen present in this RNA and impurities that may be present in trace amounts, QIAGRN pack-100 (
The purification operation was performed using DIAGEN (manufactured by DIAGEN). cD
NA''--1- All of the RNA purified by the above method was converted into cDNA using cDNA Synthesis System Brass (manufactured by Amersham).
We held a gathering. Next, the synthesized cDNA was cloned using cDNA cloning λgtll (manufactured by Amersham).
cloned into l1 vector, in vlt
Results of ropa 7 caging: t. A library of 2xto6 plaque forming units (PFU) was obtained. (4>Non-A, non-BW hepatitis (NANIl[I) convalescent phase and carrier phase:” - NANBH
Since the primary antibody used for screening the slow cDNA library was chimpanzee plasma from the NAN BI1 convalescent stage and the first carrier stage, a high nonspecific reaction was expected. Therefore, in order to suppress this non-specific reaction, E. coli Y used for the absorption operation of chimpanzee plasma for screening.
1090 lysates were prepared. That is, from a single colony, LB medium [1%
Bacto-trytone (manufactured by Difco), 065
% Bacto-yeast extract (manufactured by Difco, 1% NaCl, PH 7, 5) was added to 29 L of E. coli Y1090 culture solution 20+al which had been cultured overnight at 37°C.
It was added to II medium and further cultured at 37°C overnight. This culture solution was transferred to a centrifuge tube and centrifuged at 9,000 rpm for 10 minutes at 4°C, and the supernatant was removed to obtain a precipitate. 1g of this sediment
4+l of RIPA solution <1% Sodium Deoxycholate, 1% Triton X-100, 0.3 MNa
Cl, 0.1% SDS, 0. IM Tris-HCI
Solubilize by adding p[{7.5, 1mM PMSF),
This was further centrifuged at 9,000 rpm for 10 minutes at 4°C, and the supernatant was used as E. coli lysate. B. One LB plate was prepared from a cDNA library constructed from RNA in human pool plasma concentrate with high GPT levels] [1.5% Agar (manufactured by Nissui Pharmaceutical Co., Ltd.), 1% Bacillus]
to-tryptone, 0,5% Bacto-yea
extract, 1% NaCl pH 7.5, 5
A bacterial culture plate containing 0 μg/+ol ambicillin (manufactured by Nunc; 23c + *
0 for 15 minutes at 37°C and
40ml (0.7%Agar, 1%Bactotr
yptone10. 5% Bacto-yeast
xtract+1% NaCl, p[17.5, 50μ9
/ml ambicillin) and cultured at 42°C for 4 to 5 hours. Thereafter, a nitrocellulose filter (NC filter: manufactured by S&S, Code BA85, 2) impregnated with 10mM IPTG (manufactured by Sigma)
3 cm x 23 cm), and culture was continued at 37°C. After 3 hours, remove the NC filter from the plate.
Wash with PBS, soak in blocking solution (PBS solution containing 5% skim milk, 0.05% NaN3),
Shake overnight at ℃.
After washing with BS, NANB [{
Chimpanzee pool plasma from convalescent stage and carrier 1 stage (plasma for screening) [NANBH Chimpanzee pool plasma from convalescent stage and carrier 1 stage was diluted 5 times with PBS, added with 1/20 volume of E. coli lysate, and incubated at 4°C overnight. Absorption operation for specific reaction was carried out and further diluted 2 times with PBS. ] and reacted with shaking at room temperature. 2
After an hour, PBS-T (0.05% Tween20
After washing the replica filter three times for 15 minutes each time with a PBS solution containing PBS solution containing peroxidase-labeled anti-human IgG and IgM goat antibodies (
The cells were immersed in an incubation buffer (PBS solution containing 1% bovine serum albumin) containing Fab, manufactured by MBL, and reacted at 37°C with shaking. 1 hour later
PBS-T for 15 minutes each time, 4 times in total, then PB
After washing with S for 5 minutes, color developing solution [0. 02%DA
B (manufactured by Sigma), 0.1% NiCh 6j120,
0.005% 11202] to develop color. A secondary screening was performed by selecting a phase corresponding to the blur that developed on the NC filter. That is, 200 PFU of each phage selected in the primary screening was separately incubated with E. coli Y109 together with 200 PFU of a phage without an insert.
0 was infected and re-plated onto an LB plate in a 90III1 Petri dish (manufactured by Becton Dickinson) to prepare a replica filter. These were subjected to antibody screening using the method described above, and three phage clones (C8-2, CIO-1, C16-1) that reacted with chimpanzee plasma in the NANBH recovery phase and carrier phase with good reproducibility were obtained. When using chimpanzee IgG fraction for body reaction, 5
It was diluted with PBS to a concentration of 0 μ9/1 giant, added with 1/20 volume of E. coli lysate, and subjected to absorption treatment for non-specific reaction at 4'C overnight before use. As a result of plaque assay, 2 clones (CIO-1.C
16-1> also reacted with antibodies in normal chimpanzee serum, and its specificity for NANIII was low. However, C8-2 reacted at a high rate with the chimpanzee serum or IgG fraction of stage 1 NANBH carriers, but did not react at all with the serum or IgG fraction of normal chimpanzees. The results are shown in 61.

Table 1 Regarding the three types of clones obtained in (4), (4).C 2
Replica filters were prepared in the same manner as in the next screening, and IgG fractions were purified using NANBH recovery phase, carrier 1 phase, and normal chimpanzee serum or ammonium sulfate precipitation and DEAE-Cellulofine ram (Seikagaku Corporation). A plaque assay was performed. The method is similar to that for antibody screening, except that the primary anti-normal 0/8 2/8 NANBH convalescent l/64/6 NANB [
l Carrier stage 1 3/4 2/4 Based on these results, is C8-2 a clone that is particularly highly specific for NANIII carrier stage 1 chimpanzee serum? Yes. This C8-2 phage DNA was purified [Experimental Medicine Special Issue, Genetic Engineering Collection ■ (11>, P31-32 (
After cutting with the restriction enzyme EcoRI (manufactured by Toyobo), it was inserted into the EcoR1 site of the pUc118 vector, and subcloning was performed [Douglas 1987).
llanahan, J. Mol. Biol. Out,
See P557-580 (1983)]. When this subcloned plasmid pC825 was digested with EcoRr and developed on a 2% agarose gel by electrophoresis, an insert fragment (C825) of approximately 0.28 Kbp was confirmed (
Figure 1). Southern blot analysis using C825 was performed as described below. Normal and U.S. NIH-derived F strain infection of chimpanzees with NANB [{Acute phase (83 days after inoculation with NANBH virus)
! 1) liver, and chromosomal DNA from normal human white blood cells.
A was purified, 20μ9 of each was cut with EcoRI, developed on a 1% agarose gel by electrophoresis, and transferred to an NC filter. This filter was processed using the multi-prime method [ 9
Southern hybridization was performed using a C825 probe labeled with [2p] (Fig. 2). As can be seen from this figure, when autoradiographed for one week, the C825 probe reacts with the c8-2 clone before subcloning, but reacts with normal and NANIIH acute phase chimpanzee chromosomal DNA or normal human chromosomal DNA.
It did not react with A. This suggests that c825 is not a clone derived from human chromosomal DNA, but is derived from a foreign nucleic acid such as a virus. Using plasmid DNA incorporating the C825 gene fragment as a template, [ a -32P] dCTP (800
Ci/m mol) was used in the reaction, Kleno
The polymerase reaction using w fragment was performed using Takara Shuzo's 7DEAZA Sequencing Kit 1~. Using 8% polyacrylamide 8M urea gel, electrophoresis was carried out at 1800V''C' for 4 hours and exposed for 16 hours. The results are shown in Figures 3 and 4, respectively. The hydrophilicity/hydrophobicity profile of the predicted amino acid sequence of C825 is shown in Figure 5. The obtained nucleotide sequence and amino acid sequence were searched in the database (described above). As a result, there were no viruses, bacteria, or other substances that showed high homology.The materials used to obtain the C825 clone were pooled human plasma with high levels of Gffr and GPT, and plasma from chimpanzees that had become chronic after being inoculated with the F strain of NANBH derived from the American NIH. (Infectivity has been confirmed) Regarding chromosomal DNA derived from normal human plasma and human liver,
The C825 base sequence was detected using PCR reaction. First, for each plasma, Lsl was precipitated using a 5M sodium chloride solution and a 40% (W/W) polyethylene glycol solution in the same manner as in section (1), and a 500-guanidium thiocyanate solution was added to the precipitate. was added, and total nucleic acids were purified by phenol/chloroform extraction and ethanol precipitation. This was mixed with 50mM Tris-■C
l p[(8.3, 6mM MgCI2,
40mM KCI, 1mM DTr, 1m
M dNTPs, 1. 3KU/ml RNasin,
30mg/ml random fryer, 4KU/ml
In a 20J mixed solution of reverse transcriptase (manufactured by BRL) at 37°C.
, and was allowed to react for 1 hour and 30 minutes. Take this reaction solution IA and
0mM Tris-HCI plr8.3, 50mMK
C.I. l, 5mM MgCI2, 0.01% (w/v)
Gelatin, 100 nM dNTPs, 250 nM primer (positions shown in Figure 6) 20 U/gzl Ta
q in a 50A mixed solution of polymerase at 94°C;
30 seconds, 55°C; 30 seconds, 72°C; Birkin Elmer
Used a thermal cycler manufactured by Cetus. Regarding chromosomal DNA derived from human liver, 10μ9 was subjected to PCR reaction under the same conditions as above in the reaction solution with the above composition. After the PCB reaction, 5JA of each sample was taken and electrophoresed on a 2% agarose gel. It was developed and transferred to an NC filter. Hybridization of this filter was performed using a [32P]-labeled C825 oligo probe (the position is shown in Figure 6). As a result, the C825 base sequence was obtained from Gffr, GPT, which served as the material.
Although high levels were detected in human pool plasma, normal human plasma, chimpanzee plasma of the F strain derived from the US NIH, and chromosome D
It was not detected in NA (Fig. 7). In the same manner as in (4), the specificity of the C825 clone for non-A, non-B hepatitis was examined by Braak assay using Japanese hepatitis patient serum. The results are shown in Table 2 below. Table 2 Patient serum acute non-A non-B hepatitis chronic non-A non-B hepatitis acute hepatitis B chronic hepatitis acute hepatitis A positive/sample 5/39 1 2/3 1 0/2 0 1/20 0/ 20 Positive rate (%) 12.8 38.7 0.0 5.0 0.0 Escherichia coli Y1089 containing ambicillin (Ap) (50μ
g/■l) cultured in LB, λgtll phage and C
8-2 phage at 37℃1 at a multiple infectivity (moi) of 10.
Adsorb for 5 minutes, form a colony on the LB plate (containing ^p), pick up the colony with a toothpick, transfer to the LB plate at 30℃ and 42"C, and culture. Grows at 30℃ but not at 42℃. Colonies were selected.λ
Let the lysogens derived from g t 11 and λC8-2 be Lλ11 and LλC8-2, respectively. These lysogens were cultured at 30°C in 1+alL B medium (containing Ap), and after inducing the expression of lactose oberon by adding 1 M IPTG, the cells were harvested. The bacterial cells were dissolved in 1 ml RIPA solution. Then, 1 g of glass beads were added, crushed with a vortex mixer, and the supernatant was collected and used as a lysogen extract in the dot assay. 5 μl of the prepared lysogen extract was spotted on a nitrocellulose filter. After drying, block The reaction to color reaction was carried out in the same manner as in (4).As a result, L
To determine as positive a serum that reacts with the λC8-2 extract but does not react with the Lλ11 extract, see Figure 8. The results of this judgment are shown in Table 3. Table 3 Serum positivity/sample positivity rate (%) Normal people 0/17 0.0 Non-A, non-B hepatitis patients 15/29 51.7 Hepatitis B patients 1/9 11.0 Other hepatitis patients
○/8 0,○As shown above, the positive rate is extremely high at 51.7% in the non-A, non-B hepatitis patient group, whereas in normal people, hepatitis B patients, and other patients, the positive rate is very high at 51.7%. Positive rate for hepatitis is 0.0%, 11. The C825 clone of the present invention was shown to be specific for non-A, non-B hepatitis.

[Brief explanation of drawings]

Figure 1 shows PC825 cloned in the present invention.
Fig. 2 is a schematic diagram of the EcoRI insert fragment after 2% agarose electrophoresis development. FIG. 2 is a schematic diagram of Southern hybridization of C825 cloned in the present invention with human and chimpanzee chromosomal DNA. FIG. 3 shows the base sequence of C825 cloned in the present invention. FIG. 4 shows the entire amino acid sequence encoded by C825 cloned in the present invention. Figure 5 shows the hydrophilicity/hydrophobicity profile of the peptide encoded by C825 based on the amino acid sequence. FIG. 6 shows the positions of the primer and oligoprobe in the C825 base sequence used in the PCR reaction in Example (8).

Claims (14)

[Claims] (1) A nucleic acid fragment encoding a non-A, non-B hepatitis virus antigen peptide. (2) The non-A, non-B hepatitis virus peptide is a peptide containing at least one amino acid sequence selected from the group consisting of the following amino acid sequences (A) to (D). Nucleic acid sequences. (A) [There is a gene sequence] (B) [There is a gene sequence] (C) [There is a gene sequence] (D) [There is a gene sequence] (3) Said item (2) containing at least one nucleic acid sequence selected from the group consisting of the following nucleic acid sequences (A) to (D):
Nucleic acid sequences described in Section. (A) [There is a gene sequence] (B) [There is a gene sequence] (C) [There is a gene sequence] (D) [There is a gene sequence] (4) The nucleic acid fragment according to item (2) above, wherein the non-A, non-B hepatitis virus antigen peptide is a peptide comprising the following amino acid sequence. [There is a gene sequence] (5) The nucleic acid fragment according to item (4) above, which includes the following base sequence. [There is a gene sequence] [There is a gene sequence] (6) A non-A, non-B hepatitis virus antigen peptide containing at least one amino acid sequence selected from the group consisting of the following amino acid sequences (A) to (D). (A) [There is a gene sequence] (B) [There is a gene sequence] (C) [There is a gene sequence] (D) [There is a gene sequence] (7) The non-A, non-B hepatitis virus antigen peptide according to item (6) above, comprising the following amino acid sequence. [There is a gene sequence] (8) The non-A, non-B hepatitis virus antigen peptide according to item (6) above, wherein the peptide is a chemically synthesized peptide. (9) The peptide is a peptide obtained by incorporating the nucleic acid fragment of item (1) into an appropriate expression vector and expressing it in a host cell. Hepatitis B virus antigen peptide. (10) A nucleic acid probe for detecting a non-A, non-B hepatitis virus gene, which is composed of a nucleic acid fragment of at least 10 bases included in the following base sequence. [There is a gene sequence] (11) A method for detecting non-A, non-B hepatitis virus, which comprises hybridizing the nucleic acid probe of item (10) with DNA of a target sample. (12) An anti-non-A, non-B hepatitis virus antibody prepared using the peptide described in item (6) above as an antigen. (13) The antibody according to item (12) above, wherein the peptide is the peptide according to item (7) above. (14) An immunological detection method for non-A, non-B hepatitis virus, which comprises using the antibody described in item (12) above.