JPH04121193A - Fused peptide of hepatitis virus of non-a non-b type and production thereof - Google Patents

Abstract

NEW MATERIAL:A fused peptide between peptide A shown by formula I or a peptide equal to the peptide in terms of antigenicity and peptide B shown by formula II or a peptide equal to the peptide. USE:A reagent for measuring an antibody of hepatitis of non-A non-B type. PREPARATION:Gene fractions encoding two peptides EC825 and CH15 are fused so as to give a single reading frame. The fractions are manifested with Escherichia coli as fused protein with beta gal.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel fusion peptide of an antigen related to a virus that causes serotype hepatitis that is neither A nor B (non-A, non-B hepatitis virus), a method for producing the same, and an anti-serotype hepatitis virus using the same. A non-B
This invention relates to a method for measuring hepatitis virus antibodies.

It has been known for a long time that there are two types of viral hepatitis: hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis).This is mainly based on the difference in the route of infection.
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 rv belonging to the picornavirus [Finest
on, S.

14, et al, 5science 182 p.
1026 (1973)], while the hepatitis B virus
It was determined to be a DNA virus with an envelope of 42 nm in diameter that belongs to the hepadnavirus family.
[Dane, 0.5. , et al.

Lancet, I p695 (1970) Currently, immunoserological diagnostic methods for these hepatitis viruses have 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.

M., et al., Lancet, I p241
(1974) 1゜Post-transfusion hepatitis was significantly reduced by the introduction of a screening method for hepatitis B virus surface antigen (HBsAg), but it did not go to zero; No evidence of hepatitis 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, 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.

Apart from this, India, Myanmar, Afghanistan,
Alternatively, it has become clear that there is a second type of viral non-A, non-B hepatitis that is prevalent through oral infection in countries such as Africa [Khuroo, M, S, At J, Me
d,, 68 p818-824. (1980)],
This is commonly referred to as waterborne, or epidemic non-A, non-B hepatitis.

Although this hepatitis epidemic has not been seen in Japan, there seems to be some importation of hepatitis from endemic areas by travelers. ].

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 treatment or prevention methods have been established for this hepatitis, and the only way to diagnose this hepatitis is by 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 Ehrstein-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 5197 times 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)], However, despite many efforts around the world, the actual state of the causative virus is still unknown, even after more than 10 years.

Recently, there was a report that the US company Chiron had captured the cDNA of a non-A, non-B hepatitis virus [Ch.
oo, Q et al, 5cIence, 244
．． P2S5-362 (1989), Kuo, G.
et il, 5science, 244. p362
-364 (1989), European Published Patent Publication EP 318
216^], the properties of the virus itself and the properties of the virus constituent proteins have not yet been clarified.

-Differences in viruses, differences in immunoserological characteristics, and differences in molecular genetic characteristics make diagnostic methods very important. In addition, differences in strains are partially due to immunoserological properties, so when using the same diagnostic method, differences in detection sensitivity between strains will occur, and in vaccines, differences in immunogenicity and ability to protect against infection will occur.

In molecular genetic diagnostic methods, such as DNA probe cutting, it is generally known that hybridization between a probe and a viral nucleic acid is not practical unless the homology at the nucleic acid level is very high. That is, there may be cases in which DNA hybridization does not occur due to differences in the nucleic acid level between strains, making DNA probe diagnosis ineffective.

Hepatitis B, which is a well-known and well-analysed serotype of hepatitis, has major hepatitis virus subtypes in each region, such as Europe, America, and Southeast Asia. Since it is known that there are
Even among non-B hepatitis viruses, it is thought that there are viruses or virus strains that are unique to a region.

So far, antibody measurement reagents for non-A, non-B hepatitis include:
There is a commercially available product (OatsHCVAbELI SA test) developed by Chiron-Ortho, in which a specific peptide of non-A, non-B hepatitis virus is expressed in yeast as a fusion protein with SOD (human superoxide desmutase). However, it has been reported that this kit cannot detect all the sera of patients with non-A IF and B hepatitis that are prevalent in Japan (No. 26).
Annual General Meeting of the Japanese Society of Hepatology, 1990).

This result is thought to reflect differences in antigenicity between the strains prevalent in the United States and those prevalent in Japan.

Therefore, a method for diagnosing non-AIPB hepatitis viruses that are prevalent in certain regions, such as Japan in particular.

In order to establish a prevention method, it is necessary to identify the major non-A, non-B hepatitis virus strains in Japan.

Under these circumstances, the present inventors conducted repeated research aimed at 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 hepatitis B virus.

Furthermore, the present inventors fused these two different cloned non-A, non-B hepatitis virus gene fragments into a single reading frame and attempted to express them in E. coli. We succeeded in obtaining a fusion protein that has high reactivity with non-A, non-B hepatitis-related antibodies. That is, the present invention provides these gene fragments, peptides translated from them, and methods for using the same.

When cloning the nucleic acid fragments used in the present invention, the livers of Japanese people infected with non-A, non-B hepatitis and the livers of chimpanzees infected with non-A, non-B hepatitis were used as research materials. Extract and synthesize cDNA,
It is conceivable that virus-specific cDNAs may be selected from among them by subtraxyllaning with chromosomal DNA. However, it is extremely difficult to secure sufficient quantities of good experimental materials necessary for this purpose.

Another possible research material would be plasma from non-A, non-B hepatitis infected individuals or infected chimpanzees.

The existence of carriers of non-A, non-B hepatitis has been confirmed in humans, and the higher the donor's GPT value during blood transfusion, the higher the incidence of non-A, non-B hepatitis after transfusion. It is estimated that the frequency is high. 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, the serum of Japanese patients with non-A, non-B hepatitis was inoculated.
Plasma from chimpanzees that have developed hepatitis can also be used, but some problems remain due to the current 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, and it is necessary to concentrate the virus while removing protein. Precipitate formation using a precipitant such as polyethylene glycol (PEG), which we used, is a mild method that can be performed relatively easily, is suitable for processing large amounts of plasma, and is less likely to inactivate the virus.

Other methods that can be used include pelleting using ultracentrifugation, salting out by adding salts such as ammonium sulfate, ultrafiltration, and gel chromatography.

Plasma concentrated approximately 1000 times in this way is treated with guanidium thiocyanate, extracted with phenol/chloroform, and purified with ethanol precipitation to purify all the nucleic acids in the concentrated plasma. Human-derived DNA is degraded and RNA is purified by phenol/chloroform extraction and ethanol precipitation.

cDNA is synthesized from the purified RNA and inserted into a λgtll vector to create a cDNA library.

E. coli is infected with the λ phage, spread on a bacterial culture plate, and cultured at 42°C for several hours. After that, cover with a nitrocellulose filter (NC filter) and culture for several hours.
Peel off the NC filter and take a replica.

This replica is treated with a blocking solution, washed with PBS, etc., and then subjected to immunoscreening.

That is, the replica was reacted with non-A, non-B hepatitis convalescent or acute phase human or chimpanzee serum, and PBS
After washing, react with enzyme-labeled anti-human IgG or IgM, and after washing, react with a substrate solution to develop color. Select phages corresponding to colored plaques and perform secondary screening to obtain reproducible clones. Ta.

These clones were subcloned and a 0.44 Kbp insert fragment (EC825
)It was confirmed. The base sequence of EC825 was determined by the dideoxy method. As a result, the base sequence of EC825 is as shown in Figure 1, and EC825 is C825 (
Contains the base sequence of Japanese Patent Application No. 1-238848), and further contains 5
It was confirmed that the clone had an extension of 171 bp on the '' side. On the other hand, the present inventors have already obtained a gene fragment (CH3S) specific to non-A, non-B hepatitis using a similar method. The base sequence was as shown in FIG. 3, and was a completely different base sequence from EC825. This difference in base sequence means that both are specific to non-A, non-B hepatitis viruses, and yet have different epitopes.

In order to increase the detection rate of antibodies in the serum of patients with non-A, non-B hepatitis, it is possible to mix peptides specific to several non-A, non-B hepatitis viruses. Producing and refining it from scratch also poses problems in terms of the amount of work involved.

Under these circumstances, the present inventors developed gene fragments encoding two peptides (EC825, CH3S) that are specific to non-A, non-B hepatitis viruses and have different epitopes in a single readout. We succeeded in fusion in frame and expressed this in Escherichia coli as a fusion protein with βgal.

The βgU1-EC825-CE (15 fusion protein of the present invention was proven to retain both antigenicity by 5DS-PAGE and Western blot, and its detection can be used as a reagent for measuring antibodies for non-A, non-B hepatitis. It is expected that the rate will improve.

Also, great effects can be obtained in industrial applications.

The gene sequence of the present invention can be expressed using other suitable expression systems and used for antibody testing of non-A, non-B hepatitis viruses. It is also possible to immunize animals with the expressed protein to produce antibodies, and use this to detect non-A, non-B hepatitis viruses in liver tissue.

Furthermore, the non-A, non-B hepatitis virus obtained in the present invention is
It is extremely useful for producing vaccines to prevent 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, non-B hepatitis viruses using these of the present invention are particularly useful. It is considered to be extremely useful in detecting non-A, non-B hepatitis viruses in Japan.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

A GPT specimen donated by the Japanese Red Cross Society, negative for HBs antigen.
Human boule plasma (approximately 8.211) having a value of 100 or more was concentrated 1000 times by the following method. First, pooled human plasma was roughly centrifuged to remove insoluble matter. To this was added 1710 volumes of 5M sodium chloride solution, then 1/10 volume of 40% (
W/W) Polyethylene glycol liquid (PE06000,
(manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 7500) was added at 4°C with stirring. - hours, centrifuged at 7000 rpm for 20 minutes to remove the supernatant, and the sediment was mixed with TNE solution (10mM Tris-HCl
, pH 7, 4, 1 FtDT^, 140mM NaC
Add 1), re-dissolve it, and add the Kono solution to 20% of the original dye.
15%, 10% and 5% TNE solutions were layered on top of a centrifuge tube in stages, and the mixture was incubated at 4°C and 80,000 x G.
After centrifugation for 12 hours, the supernatant was removed, and the precipitate was dissolved in 8 ml+7) PBS to obtain a 1000-fold concentrate of human pooled plasma with high GffT and G1'T values.

(2) Gffr, RNAa enrichment from GPT high human pool plasma concentrate First, add 5 times the amount of guanidium thiocyanate solution (4M guanidium thiocyanate, 50i + M Tris-HCI pH 7, 6
, 105M EDT^, 0.1M 2-mercaptoethanol, 2% sarcosyl) were added, stirred, extracted with phenol/chloroform, and the total nucleic acids in the concentrated plasma were purified by ethanol precipitation using glycogen as a carrier. In order to degrade human-derived DNA present in nucleic acids, RNase-free DNase 1.1 was added in the presence of a 2-part vanadyl ribonucleotide complex.
5KO/ml (Boehringer/Manheim), 50
■M Tris-HCI pH7,4,1■M EDT
^, 37°C in a 400Δ mixture of 105M MgCh
, and treated for 30 minutes. After that, 250*14 EDT^
Solution 16A and 10% SDS solution 8A were added to stop the reaction, and R was removed by phenol/chloroform extraction and ethanol precipitation.
NA was purified. Furthermore, in order to remove a large amount of glycogen present in this RNA and impurities that are thought to exist in trace amounts, Q■^GEN pack-100 (DIA
Purification operation was performed using GEM Co., Ltd.).

C-1- All of the RNA purified by the above method was converted into cDNA using cDNA Synthesis System Plus (manufactured by Amajam).
After the synthesis, the synthesized cDNA^ was cloned into λgtl by cDNA cloning λgtll (manufactured by Amajam).
1 vector. In vitro packaging resulted in a library of 1.2 x 10' plaque forming units (PFU).

Since the primary antibody used for screening the cDN^ library was NANBH convalescent and carrier phase chimpanzee plasma, a high nonspecific reaction was expected. Therefore, in order to suppress this non-specific reaction, E. coli Y1090, which is used for the absorption operation of chimpanzee plasma for screening,
A lysate was prepared. That is, from a single colony, LB medium containing ampicillin 50μ9/1 [1% Bacto
-tryptone (manufactured by Difco), 0.5% Bac
to-yeast extract (manufactured by Difco),
201 pieces of Escherichia coli Y1090 culture cultured in 1% NaCl, pi (7,5) at 37°C overnight were added to 21i LB medium and further cultured overnight at 37°C. This culture was transferred to a centrifuge tube, Centrifugation was performed at 9,000 rpm for 10 minutes at 4°C, and the supernatant was removed to obtain a precipitate.41 per 1 g of this precipitate.
of RIPA solution (1% sodium deoxycholate, 1%
Triton X-100, 0,3M NaC1,0,1
%SDS, 0. IM Tris-HCI p) [7,5
.

1-2 ■ G (rr, RNA in GPT-high human pool plasma concentrate)
From the cDNA library constructed from
acto-tryptone, 0.5% Bacto
-ye & st eXtraCt, 1% NaCl pH 7,
Take 10,000 PFU of phages per bacterial culture plate (manufactured by Nunc; 23 cm x 23 cm) containing 5,50 m/ml ampicillin, and use E. coli Y109.
0 for 15 minutes at 37°C and
40m1 (Ogo%^gM, 1% Bacto-tryp
tone, 0.5% Bacto-yeaSte
xtract, 1% NaCl, PI (7, 5, 50μ
9/1 ampicillin) and cultured at 42°C for 4 to 5 hours. Then, a nitrocellulose filter (IC filter: manufactured by SlS, Code B^85.23) was soaked with 105M IPTG (manufactured by Sigma).
cmX23CI+), and further culture was continued at 37°C.

After 3 hours, remove the NC filter from the plate and add PBS.
Wash with Blocking solution (5% skim milk, 0.5%).
(PBS solution containing 05% 1aN3) and shaken overnight at 4°C.

C, S 1-25 Replica filter soaked in blocking solution overnight
After washing with BS, NANBH convalescent and carrier phase chimpanzee pool plasma diluted 10 times with PBS (screening plasma) The lysate was added, and absorption operation for non-specific reaction was performed overnight at 4°C, and the mixture was further diluted 2 times with PBS. ] and allowed to react while cooling at room temperature. 2
After an hour, the replica filter was washed three times with PBS-T (PBS solution containing 0.05% Tween 20) for 15 minutes each time, and peroxidase-labeled anti-human IgG and IgM goat antibodies diluted 1000 times each ( M.B.
Soak in incubation buffer (PBS solution containing 1% bovine serum albumin) containing Fab, manufactured by Company L,
The reaction was carried out at 37°C with shaking. 1 hour later, PBS
- T for 15 minutes each time for a total of 4 times, then PBS for 5 times.
After washing for a minute, coloring solution [0.02% DAB (manufactured by Sigma), 0.1% N1Ch.6R20.0-QO5%HJz
It was immersed in water to develop color. Phage corresponding to the plaques developed on the NC filter were selected and subjected to secondary screening.

That is, each phage 200 selected in the second screening
200 PFU of phage without insert of PFLl separately
It was also infected with Escherichia coli Y1090, and 90 μsial (
A replica filter was prepared by re-plating on an LB plate (manufactured by Becton Dickinson). These were subjected to antibody screening using the method described above, and one phage clone that reacted with chimpanzee plasma in the NANBH convalescent and carrier stages with good reproducibility was obtained. Purify this phage DNA [Experimental Medicine Special Issue, Genetic Engineers General Aircraft fl (11)
, P31-32 (1987), restriction enzyme Ec
After cutting with oRI (manufactured by Toyobo Co., Ltd.), it was inserted into the EcoRr site of the pUc118 vector, and subcloning was performed [
Douglas Hanahan, J. Mo1. Bi
ol, 1J)jz, p557-580<1983)
Reference, this subcloned plasmid pEC8
25 was digested with EcoRI and developed on a 2x agarose gel by electrophoresis, an approximately 0.44 Kbp insert fragment (E
C825) was confirmed.

Plasmid II incorporating the FtC825 gene fragment
Using NA as a template, [a-”P]dCTP (80
0 Cj/m mo+> was used in the reaction. Kleno
The polylase reaction using w fragment was performed using Takara Shuzo's 7DEAZA sequencing kit. 8
% polyacrylamide-8M urea gel.
Electrophoresis was performed at 1800 V for 16 hours and exposed.

The results of decoding the base sequence obtained above and the amino acid sequence predicted therefrom are shown in FIGS. 1 and 2, respectively. As a result, EC825 was found to be a fragment containing the full length of C825 and further extended by 171 bases on the 5' side.

-S≧ A plasmid was constructed to express a fusion protein of EC825 and C)115 in E. coli. First, EC825
To add restriction enzyme recognition sites, EcoRI and Bag old to the gene fragment, PCR (polymerase chain
ain reaction). A 5-fold amount of guanidium thiocyanate solution (414 guanidium thiocyanate, 25
mM sodium citrate, 0.5% sarcosyl, 0.1
M2-mercaptoethanol) was added, stirred, and extracted with phenol/chloroform/isoamyl alcohol.
Total nucleic acids in plasma were purified by propatool precipitation using glycogen as a carrier. This is 5(1++
M Tris4C1p[+8.3, 6mM MgC
h, 40mM KCI, 1mM DTT, 1mM
dNTPs, 1.3 KU/ml RNasin, 30
The reaction was carried out at 37°C for 1 hour and 30 minutes in a 20Δ mixed solution of Maro g/+gl random primer and 4KU/■l reverse transcriptase (manufactured by BRL). Take 5 μg of this reaction solution 105
M Tris-HCI pH8,3, 50w+M
KCI, 1.5mM MgCb, 0.01% (w/
V) Gelatin, 1100n dNTPs, 250nM primer (the base sequence is shown in Figure 5) 20U/l
+l Taqpolymerase mixture 50Δ,
94℃: 30 seconds, 55℃: 30 seconds, 72℃; 1 minute = 1
I used a Perkin Elmer Cetus thermal cycler to carry out the reaction for 35 cycles).
Gene fragments amplified by PCB are treated with phenol/
After extraction and purification with chloroform, this was extracted with the restriction enzyme EcoR.
It was cut with I and B to expose the respective restriction enzyme cleavage sites. In a similar manner, C) 5 of the 115 gene fragment
A gene fragment was obtained with a restriction enzyme cleavage site of Bag) [Sal I on the 1,3” side. On the other hand, plasmid pUEX2 (manufactured by Amajam) was digested with the restriction enzyme EcoR.
After cutting with I and 5ail, it was extracted and purified with phenol/chloroform. Each of the above three gene fragments (1100n) obtained in this manner was added to a T ligase reaction solution (66m
M Tris-HCIpFI7.6.6.6mM Mg
T at Cl2゜10■M DDT, l■M ATP)
The reaction was carried out at 4° C. for 4 hours using 2 units of 4 ligase. E. coli) IBIOI was transformed with this reaction solution according to the method described in "Gene Manipulation Experimental Methods" by Higashikata Takagi, p. 161, and "metabolism" was obtained from colonies grown on agar medium containing 50 μg/iil of ampicillin. A plasmid was prepared according to the method described in Volume 17, Volume 4 [Lipase J p81-89 (1980). As a result pUEX
To obtain plasmid pUENS-35 in which the fusion gene of EC825 and Cold5 was inserted between EcoRI-3alI of 2, see Figure 6.

E. coli carrying plasmid pUENs-35 was grown in LB medium 1.
0ml (0.5% yeast extract, 1x Bactodrypton,
After inoculating the cells in 0.5% NaCl) and culturing at 30°C overnight, the turbidity was adjusted to 80 using a Klett meter, and the cells were incubated at 30°C.
It was incubated for 90 minutes.

The culture temperature was raised to 42°C, and after culturing for an additional 2 hours, the cells were collected by centrifugation. 1m14 TBST including PMSP
(50■MTris-HCI pH7,9,150
mM NaCl 1,0,5% Tween 20)
Suspend the bacterial cells in
The bacterial cells were destroyed by stirring vigorously for 15 minutes. After separating the bacterial cell disruption solution and glass beads, an insoluble fraction was obtained by centrifugation. This was added to 11 TE (50mMT
ris-41CI pH 7, 5, 10mMEDT^), and the suspension was subjected to the following 5DS-PAGE,
The presence or absence of antigen production was examined by Western blotting. The samples were electrophoresed on an 8% 5DS-polyacrylamide gel and then stained with 0.25xCBB. A similarly electrophoresed sample was transferred to a nitrocellulose filter by TFI, and the filter was reacted with 5% skim milk for 1 hour at room temperature, and then reacted with non-A, non-B hepatitis patient serum for 1 hour. After washing the filter with TBST, anti-human I
It was reacted with a gG peroxidase-conjugated antibody (manufactured by Bio-Rad) at room temperature for 1 hour. After washing with TBST, color was developed with hydrogen peroxide and diaminobenzidine. As a result pUEN
In the E. coli extract sample containing s-35, the molecular weight was 1.
Two broad Dalton bands were observed at 20 and 130. The band at 0 molecular weight 130 was the EC8 of the present invention.
The molecular weight was almost the same as that estimated from the 25-C old 5 fusion gene. Further, the band with a molecular weight of 120 was estimated to be the EC825 (or C825) peptide site cleaved by E. coli protease from the results of Western blotting.

On the other hand, in the negative control E. coli extract sample containing only pUEX2, which does not contain the fusion gene of EC825-C)115, no band that reacted with non-A, non-B hepatitis patient serum was observed (see Figure 7). .

1% lysozyme was added to the bacterial cells whose expression was induced using the method described above.
After disrupting with Triton x100, 3 g of inclusion bodies were obtained by centrifugation. This is 100111 (7
) Buffer (8M urea, 20@M tris-H
The suspension was suspended in CI 300 SonoX Mace, 2-4DTT), disrupted with a polytron, further stirred at room temperature for 1 hour, and then centrifuged at 8000 rpm for 30 minutes to remove the insoluble fraction. Fusion protein EC825-C in centrifugation supernatant
1 (15 - t = 7 acrylic s3o.

After purification by gel filtration (5x100++m), PHA was prepared by fixing it on sheep blood cells with tannic acid by a conventional method.

Using the obtained PHA, specific antibodies contained in the serum of non-AIIEB hepatitis patients were measured. As a result, C100 antigen (Oat HCV-Ab ELISA test), synthetic peptide (KCL antibody assay kit Patent application 2-1621)
41) and EC825 antigen (ELISA method), 9 out of 42 sera (214 %).

From this result, it was considered that by making the two peptides into a fusion protein, a new epitope specific to non-A, non-B hepatitis appeared. That is, the fusion protein EC825-C old 5 of the present invention contributes to improving the detection rate of non-A, non-B hepatitis patient serum, and is extremely useful as an antigen for antibody measurement.

[Brief explanation of the drawing]

FIG. 1 shows the base sequence of EC825 cloned in the present invention. Figure 2 shows the predicted amino acid sequence from EC825. Figure 3 shows the C used for constructing the fusion gene in the present invention.
Figure 4 shows the amino acid sequence predicted from CI'[15. Figure 5 shows the base sequence of the primer used for PCH. Figure 6 shows the nucleotide sequence of plasmid pUENS. Fig. 7 is a schematic diagram showing the results of 5DS-PAGE and Western blotting.

Claims (7)

[Claims] (1) A fusion peptide of the following peptide A encoded in the genome of a non-A, non-B hepatitis virus or a peptide antigenically equivalent to this peptide, and the following peptide B or a peptide equivalent to this peptide. Peptide A: [There is a gene sequence. ] [There is a gene sequence. ] Peptide B: [There is a gene sequence. ] (2) The fusion peptide according to item (1) above, consisting of the following amino acid sequence. [There is a gene sequence. ] [There is a gene sequence. ] (3) A fusion peptide characterized in that the non-A, non-B hepatitis-related fusion peptide described in item (1) is further fused to a carrier protein. (4) The above-mentioned (3) wherein the carrier protein is β-gal.
) The fusion peptide described in section 2. (5) Incorporating a fusion gene fragment of gene fragment A or an equivalent gene fragment, which is part of the non-A, non-B hepatitis virus cDNA, and gene fragment B or an equivalent gene fragment into an appropriate expression vector; A method for producing a fusion peptide, which comprises culturing cells transformed with this recombinant vector to express the fusion peptide described in item (1) above, and recovering the same. Gene fragment A: [There is a gene sequence. ] [There is a gene sequence. ] Gene fragment B: [There is a gene sequence. ] [There is a gene sequence. ] (6) The method for producing a fusion peptide according to item (5) above, wherein the fusion gene fragment has the following base sequence. [There is a gene sequence. ] [There is a gene sequence. ] (7) A method for measuring anti-non-A, non-B hepatitis virus antibodies, which comprises using the peptide according to any one of items (1) to (4) above as an antigen.