KR0139087B1 - Nanbv diagnostics and vaccines - Google Patents

Abstract

The present invention provides gene (cDNA) fragments encoding non-A, non-B hepatitis antigen proteins, recombinant plasmids having the gene fragments, and host cells transformed with the recombinant plasmids. The present invention also relates to capsid, envelope and nonstructural protein region antigen polypeptides obtained from transformants, and provides methods and vaccines for detecting non-A, non-B hepatitis viruses with higher accuracy. .

Description

Non-A Non-B Hepatitis Diagnosis Methods and Vaccines

1 is a schematic diagram of a method for constructing a complementary gene (cDNA) fragment encoding a novel non-A non-B hepatitis antigen protein according to the present invention.

2 is a schematic representation of the plasmid vector pTZ18U.

Figure 3 is a schematic diagram of cloning the novel gene fragment of Figure 1 into the plasmid vector pTZ18U of Figure 2.

4 shows the nucleotide sequence of the novel gene fragment of FIG. 1 and the amino acid sequence encoded by this gene.

5 is a diagram comparing the nucleotide sequence of the new gene fragment with the nucleotide sequence of the US (US) non-A non-B type gene and the amino acid coded therefrom.

6 is a schematic representation of the expression plasmid vector pT7-7.

7 is a diagram showing a method of generating a restriction enzyme site by PCR (polymerase chain reaction, polymerase chain reaction) method of the constructed gene fragment of FIG. 1 and inserting it into the expression plasmid vector pT7-7 of FIG. to be.

8 is a schematic representation of the control flask vector pGP1-2.

Figure 9 shows the electrophoretic analysis on polyacrylamide gels of the expressed new non-A non-B hepatitis antigen protein.

10 shows the results of elution fractions of novel non-A non-B hepatitis antigen proteins, expressed gene products during protein purification.

FIG. 11 shows antibody measurements in non-A non-B hepatitis, hepatocellular carcinoma and liver cirrhosis patients blood using purified novel non-A non-B hepatitis antigen proteins.

FIG. 12 shows the locations of specific cDNA clones of viruses isolated from non-A and non-B hepatitis patients and the specific cDNA clones selectively subcloned therefrom.

13 is a diagram of a plasmid expression vector pUEX2.

14 is a diagram of a plasmid expression vector pMALcR1.

FIG. 15 is a schematic of subcloning the DA703 gene (cDNA) fragment into the plasmid vector of FIG.

Figure 16 is a schematic of subcloning the DA356 gene (cDNA) fragment into the plasmid vector of Figure 14.

FIG. 17 is a schematic diagram of subcloning the DA168 gene (cDNA) fragment into the plasmid vector of FIG. 14.

Figure 18 is a schematic of subcloning the DA99 gene (cDNA) fragment into the plasmid vector of Figure 14.

19 is a diagram showing the base sequence of (a) DA703 and hepatitis virus amino acid sequence encoded therefrom.

(b) shows the nucleotide sequence of DA356 and the hepatitis virus amino acid sequence encoded therefrom.

(c) shows the nucleotide sequence of DA168 and the hepatitis virus amino acid sequence encoded therefrom.

(d) shows the nucleotide sequence of DA99 and the hepatitis virus amino acid sequence encoded therefrom.

20 is a diagram showing the purification result of polypeptide D1 by ion exchange resin using CM Sepharose.

21 is a diagram showing purification of polypeptide D3 using gel filtration chromatography.

22 is a diagram showing purification of polypeptide D4 using gel filtration chromatography.

FIG. 23 is a diagram showing SDS-polyacrylamide electrophoresis and western blotting results of purified polypeptides D1, D2, D3 and D4.

24 is a table showing the measurement of non-A, non-B type agitis virus antibody by enzyme immunoassay using purified polypeptides D1, D3, D4 antigens.

25 is a table showing E. coli transformants prepared in the present invention and deposited in KFCC.

The present invention constructs and clones a new complementary gene (cDNA) encoding a non-A non-B hepatitis virus antigen and clones the non-A non-A ratio using a novel protein antigen produced using the gene. A method for immunological detection of non-Hepatitis B vaccines and non-A Non-B hepatitis related antibodies.

Non-A non-B hepatitis is mainly caused by blood transfusions and is 6 to 10 times higher in the rate of transition to chronic hepatitis, cirrhosis, and liver cancer than hepatitis A or B. [Sakamoto et al. ; Cancer Res., 48, 7294-7297, 1988]. The main causative agent of non-A non-B hepatitis was discovered in 1988 by Choo, Chiron of the United States, allowing the study and diagnosis of this name. Studies of non-A non-B hepatitis virus that have been conducted to date indicate that the virus is an RNA virus composed of approximately 9.4 x 10 ³ nucleotides, and the genes for structural and nonstructural proteins have been cloned. The non-A non-B hepatitis virus from which the gene was derived was named HCV (hepatitis C virus), and the cloned complementary gene (cDNA) of the cloned virus was introduced into an appropriate plasmid vector and expressed in yeast. Has been proposed to use the obtained HCV antigen protein as a detection or vaccine for antibodies against non-A non-B hepatitis virus in the blood (see European Patent No. 0318216A1).

However, other researchers in Japan have cloned genes of non-A non-B hepatitis virus that differ significantly by 20-30% [Takamizawa et al. ; J. virol., 65 (3), 1105-1113, 1991] Genetic information or antigens encoded for non-A hepatitis virus infected by carriers in a given region or country unit The proteins are homologous to one another but have been found to be highly heterologous to non-hepatitis viruses infected in other regions or countries. [Yoshihiro et al. ; Nucleic Acids Res. 17 (24), 10367-10372, 1988 and Nobuyuki et al. ; Proc. Japan. Acad. 66, B (9), 219-223, 1989].

Diagnosis of non-A non-B hepatitis vaccines or hepatitis virus antibodies is based on the use of antigen-antibody reactions. When used as a diagnostic reagent or vaccine raw material, the accuracy of the diagnosis is low or the efficacy of the vaccine is poor.

In order to overcome these problems, non-A non-B hepatitis antigen proteins used for diagnosis and vaccination have been identified as non-A non-B type homologous to the characteristics of the region or country. The fragrance protein encoded by the hepatitis virus gene should be used.

Therefore, the present inventors intended to use genetically engineered antigenic protein as a raw material for diagnostic reagents and vaccines by using genetic information of non-A hepatitis virus infected to Koreans in consideration of regional characteristics. For this purpose, the antigenic protein encoded by the non-A non-B hepatitis virus gene from the blood of Korean carriers should be used.

Therefore, the present inventors intended to use genetically engineered antigenic protein as a raw material for diagnostic reagents and vaccines using genetic information of non-A hepatitis virus infected to Koreans in consideration of regional characteristics. To this end, Korean carriers have cloned the non-A non-B hepatitis virus gene from the blood to capture the capsid region and envelope of the non-A non-B hepatitis virus. A new gene encoding the region was constructed, and the nucleotide sequence and the amino acid sequence encoded therefrom were determined and analyzed. As a result, more than 93% of the gene nucleotide sequence was obtained for non-A hepatitis B virus carriers in Korea. Although the amino acid sequence showed more than 95% homogeneity, the heterogeneity between 20-28% of the gene sequences and 15-20% of the US non-A non-B virus, HCV. A novel nucleotide sequence was found to indicate heterogeneity between amino acid sequences of the phases. Based on this, the novel capsid and envelope antigen proteins produced by the genetic engineering method using the complementary genes (cDNA) of the above-described region are non-A hepatitis B virus antibodies. It is used as a diagnostic reagent and vaccine.

Hereinafter, the present invention will be described in detail. For the purpose of improving the specificity or heterogeneity of the sequence of the genetic information encoding the non-A non-B hepatitis antigen according to the region as described above, RNA derived from non-A hepatitis B carriers provides genetic information that ultimately determines non-A non-B antigen proteins in the form of a strand. Reference-Figure 1

The present invention provides novel complementary gene (cDNA) fragments encoding the capsid and envelope antigen proteins of non-A hepatitis B virus and their nucleotide sequences and amino acid sequences encoded therefrom. To provide. [Reference- FIGS. 4 (a) and 4 (b)]

A double stranded complementary gene (cDNA) is constructed from the viral RNA described above, cloned into a plasmid vector suitable for introduction into E. coli host cells, and the nucleotide sequence of the gene fragment is determined.

Reverse transcriptase is used for the construction of complementary genes (cDNA) from RNA, and amplification of cDNA (complementary DNA) by polymerase chain reaction (PCR) using primers, one of the latest advanced genetic engineering techniques. [See FIG. 1]. It is already known that cDNA synthesis method using PCR has a higher accuracy or efficiency than other methods.

In the present invention, capsid complement gene (cDNA) clones K1, K2 and K3 and envelope complement gene (cDNA) clone E1 using the above-described method from the RNA of Korean non-A hepatitis virus carriers. , E2 and E3 were prepared, and the homogeneity between the nucleotide sequences between the same clones or the amino acids encoded therefrom was found to be at least 93% and at least 95%, of which the capsid complement gene (cDNA) clone K1 and the envelope were found. The base sequence and amino acid sequence of the complementary gene (cDNA) clone E1 are shown in FIGS. 4 (a) and 4 (b).

The constructed double-stranded complementary gene (cDNA) fragment was expressed in the vector pTZ18U of the plasmid for cloning [United Stated Biochemical co. : Recombinant plasmids pTZ18U-Cap and pTZ18U-Env were prepared by making Gene Scribe-Z ^™ kit] and [FIG. 2] blunt-end using restriction enzyme smal and conjugating at this position. The schematic method is shown schematically in the figure.

The base sequence of a specific gene (DNA, deoxyribohexane) finally determines the protein, and the novel complementary gene (cDNA) of which the novel capsid and envelope antigen proteins of the present invention are also constructed. Determined by base sequence.

In the present invention, the new gene fragments of the capsid and envelope were constructed by the method of sequencing of the dedeoxy (Dideoxy) sequencing method [see Figure 4 (a) and 4 ( b)], 20-25% between base sequences and 16-20 between amino acid sequences as a result of comparing the corresponding regions of the US (HC) HCV genes reported by chiron Co., Ltd. % Difference (see FIG. 5 (a), 5 (b), 5 (c), 5 (d)).

The present invention uses novel novel complementary gene (cDNA) fragments whose sequencing has been determined using the following novel method: plasmid vector pT7-7 [S-Tabor et al. : Recombinant plasmids pT7-Cap and pT7-Env inserted in PNAS, USA 82, 1074-1078, 1988], [Reference- FIG. 6] and E. coli DH5α strains which are suitable host cells thereof [Ref.-J. Bacteriol., 159, 1036, 1986].

In the present invention, a novel method for artificially generating a restriction enzyme site capable of conjugation into a plasmid vector pT7-7 expressing Escherichia coli as a host cell was introduced into the constructed gene fragment. The host-vector system for is based on the complementary gene (cDNA) expression of a novel non-A non-B hepatitis virus constructed in a suitable transformation vector. The generation of specific restriction enzyme sites in the gene fragments is possible by designing a primer containing a nucleotide sequence of the corresponding restriction enzyme site and amplifying it using a PCR method. Recombinant plasmids pT7-Cap and pT7-Env were prepared by conjugating the gene fragment containing the plasmid vector to the corresponding restriction enzyme site of the plasmid vector, and the recombinant plasmid prepared in FIG. Transformants were prepared by introducing the calcium chloride method.

E. coli DH5α transformed with plasmid pT7-Cap was named E.coli YCS-C and E. coli YCS-C transformed with plasmid pT7-Env and YCS-E were dated June 21, 1991 The association was deposited with accession numbers KFCC-10733 and KFCC-10734, respectively.

The present invention provides a novel capsid and envelope antigen protein expressed from an E. coli K38 transformant comprising a recombinant plasmid carrying a genetic fragment encoding a non-A hepatitis antigen protein. And amino acid sequences deduced from the new gene fragments.

E. coli K38 strain of the recombinant plasmid prepared [see FIG. 7] [see -J. Bacteriol., 159, 1038, 1988], for the expression of new gene fragments in the transformation with the plasmid vector pGP1-2 [Reference-S. Tabor et al. ; PNAS, USA 82, 1074-1078, 1988, [reference-FIG. 8], contains the RNA polymerase gene of bacterial phage T7 downstream of the P _L promoter of lambda phage (phage λ).

Thus, the inserted gene in the recombinant plasmid can induce expression by the activity of the T7 RNA polymerase by inactivating CI 857, a temperature sensitive repressor, by temperature rise under the P _L promoter. Tabor et al. ; Proc. Natl. Acad. Sci, USA 82, 1074-1078, 1988].

Selection of the transformants is carried out in a medium containing ampicillin and kanamycin according to the characteristics of the plasmid vector used, and Rifampicin is used to selectively inactivate other RNA polymerases by E. coli, thereby reliving only new genes under the T7 promoter. It can be specifically overexpressed by T7 RNA polymerase that is not inactivated by fahsin.

Figure 9 shows an example of polyacrylamide gel electrophoresis analysis for the gene product derived and expressed by the method described above.

In order to easily identify and quantify this overexpressed gene product in the protein purification process, cells cultured with 35S-methionine specifically labeled with the radioactive isotope 35S-methionine were expressed and radiolabeled. The lysate can be easily purified by adding the capsid and envelope antigen proteins to a large number of cells that are overexpressed to track this label during purification to facilitate purification of the gene product.

The present invention provides a purification method for a gene product expressed in E. coli K38 convertor to be used as an antigen in a method for measuring an antibody of non-A non-B hepatitis virus.

Purified expression protein can be compared with the presence or absence of the desired protein by amino acid sequencing, etc. and the protein expressed by the conventional technique, and the amino acid sequence shown in FIGS. 4 (a) and (b) expressed in the transformant. The new gene product having the present invention is purified by various methods after the transformant is destroyed by a small number of destruction devices, and the expression protein is spilled into the culture medium.

Purification methods include chromatography, ion exchange resin, affinity chromatography, and centrifugation. In the present invention, methods such as centrifugation and chromatography are used for elution of protein and fractionation of the eluted protein. Is confirmed by radioactive isotopic labeling or quantification, and concentrated to provide amino acid sequence analysis.

The present invention provides the following novel methods for developing immunodiagnostic methods for measuring non-A non-B tongue hepatitis virus antibodies.

First, the novel non-A non-B hepatitis virus genes of FIGS. 4A and 4B provide antigens used in immunodiagnosis. Conventional methods use a gene of non-A non-B hepatitis virus called HCV (US) as a means of antigen production. As described above, the non-A hepatitis virus is separated by 20-30% of the sequence of genes that make antigens according to regions. It is reported that the amino acid composition of the recombined protein differs by about 10-20%, and according to the type of the virus, antibodies produced in the patient's blood may be different, and the accuracy of immunodiagnosis depends on the type of virus provided as antigen. do. Therefore, in the present invention, in order to improve the low accuracy in the conventional immunodiagnostic method and obtain a more accurate diagnosis result due to the difference in the nucleotide sequence and amino acid composition, the ratios of FIGS. 4 (a) and (b) A novel gene constructed from a virus isolated from blood of a non-A hepatitis B patient is provided as an antigenic protein source for immunodiagnosis.

Second, the gene encoding the capsid protein in the structural protein region of the novel non-A non-B hepatitis virus of FIGS. 4 (a) and (b) was expressed in a suitable E. coli HB101. This capsid protein is purified and provided as a raw material of an immunodiagnostic method as an antigen having the antigenicity of the viral capsid portion. Conventional immunodiagnostic methods used to measure non-A non-B hepatitis virus antibodies mainly use the polymerase portion of the non-structural protein portion of HHCV (US) as an antigen. When a virus invades the body, antibodies to the virus initially form antibodies to the polymerase in the nonstructural protein, but mainly to the capsid protein of the virus. In the case of non-A hepatitis virus, antibodies to capsid proteins are formed earlier, and the antibody is determined for the presence of non-A non-B hepatitis virus. It is an important indicator of Therefore, in the present invention, the capsid encoded by the gene using the novel capsid gene constructed in FIG. 4 as a method for developing the immunodiagnosis method of the antibody against non-A non-B hepatitis virus ( The capsid) protein was produced and purified in Escherichia coli by genetic recombination, and used as an antigen of immunodiagnosis, showing 10-20% higher accuracy than conventional immunodiagnosis.

In addition, the purified capsid antigen is adsorbed in a suitable container to form an immobilized solid phase and react with an antibody against a capsid antigen present in the blood to form an antigen-antibody complex. Another antibody is used to quantitatively or qualitatively determine the amount of the antibody in the antigen-antibody complex, which reacts with and binds to a human antibody in the antigen-antibody complex, which contains an enzyme or radioisotope. Labeling allows measurements. In case of using an enzyme, the substrate is reacted with an appropriate concentration. In the case of using a radioisotope, the radioactivity is measured by measuring radioactivity to determine the amount of antibody bound to the antigen-fixed phase.

A more detailed description of the use of enzymes is that a purified protein with non-A non-B hepatitis virus antigen in microflakes (Nunc, 2x8 strips), for example, at a concentration of 1 μg / ml per well After 150μl adsorption, the antigen is fixed. Specimens such as blood containing non-A non-B hepatitis virus antibody are reacted with the control group. At this time, the antibody present in the sample during the reaction forms an antigen-antibody complex by binding to the viral antigen immobilized on the microplate.

The unbound material is washed with a suitable washing solution, and then reacted with the antigen-antibody complex by adding a goat anti-human immunoglobulin antibody and an enzyme-enzyme conjugate to which the enzyme is coupled. After the reaction, the substrate solution reacts with the enzyme. Induces an enzyme-substrate reaction.

After the enzyme-substrate reaction, the reaction stop solution was added to each well to stop the enzyme reaction, and then the absorbance of each well was measured by a photometer to measure the amount of antibody bound to the antigen-fixed solid.

As a result of the above-mentioned original invention, the base sequence of the complementary gene of the non-A non-B virus which was cloned from the blood of non-A non-B hepatitis patients in Korea is known. It shows 20% to 30% heterogeneity with hepatitis virus complement gene (cDNA) [ref-European Patent 0318216 A1], and it uses non-A non-A non-A using polypeptide derived from this complement gene (cDNA). Since it was confirmed that hepatitis B carriers can be diagnosed accurately, the inventors of the present invention used cDNA library production method to more clearly identify non-A non-B hepatitis virus subtypes present in Korea. Recombination of the virus's complementary gene (cDNA) from non-A hepatitis B blood has been attempted.

This additional invention includes a capsid region and an envelope region from the blood of non-A non-B hepatitis patients in Korea and includes non-A non-B hepatitis blood. A novel complementary gene (cDNA) fragment that is reactive with and a portion of the nonstructural protein region, and a novel complementary gene (cDNA) fragment that is reactive with non-A non-B hepatitis patients blood Clone and determine the sequence of amino acids whose novel complementary genes (cDNAs) are encoded from them.

As a result of analyzing the nucleotide sequence and amino acid sequence of the complementary genes (cDNAs) encoding the structural protein region obtained in the present invention, it showed high homogeneity with the cDNAs K1 and E1 of the original invention. On the other hand, a novel complementary gene (cDNA) encoding a non-structural protein region exhibits high heterogeneity with a known hepatitis C virus complementary gene (cDNA) [see European Patent 0318216 A1] and its nucleotide sequence and amino acid sequence. Was found.

Therefore, in this additional invention, the structural protein region of another novel non-A non-B virus which shows high homogeneity in the nucleotide sequence and amino acid sequence of the above-mentioned K1, E1 complementary gene (cDNA) Novel non-A non-homologous heterogeneous genes encoding the complementary gene (cDNA) encoding the non-structural protein region of known hepatitis C virus [c-ENA 0318216 A1]. To obtain complementary gene (cDNA) fragments encoding the viral non-structural protein region, and also to obtain a non-A non-A polypeptide of a novel polypeptide obtained by genetically expressing and purifying these novel complementary genes (cDNAs). Provided for use in reagents and vaccines for diagnosing hepatitis B virus antibodies.

Detailed description of the present invention is based on the above-mentioned descriptions, and various cDNA fragments encoding antigens specific for Korean non-A hepatitis B patients are described. It provides a method to find from cDNA library of non-hepatitis B patients. After centrifugation of the plasma sample of Korean chronic non-A non-B hepatitis patients to remove the precipitate, the supernatant was again centrifuged at high speed to obtain a precipitate. The RNA solution layer was separated from the precipitate by RNAzol extraction. The RNA solution layer was added with isopropanol and centrifuged to separate RNA precipitate. RNA isolated from cDNA synthesis kit using Promega reverse transcriptase was used as a template, random primers were used to make cDNA, and the cDNA library was conjugated to the EcoRI site of λgt11 phage DNA. made. Λgt11-cDNA phage library of Korean non-A non-B hepatitis patients was infected with E. coli Y1090, mixed with an appropriate amount of LB-top-agar solution, and plated on NZCYM-agar plate. It was. The plated applicator was hardened and then cultured in an incubator to form plaque. These plaques were transferred to nitrocellulose filters, blocked with blocking solution, and immunoscreened with serum from non-A non-B hepatitis patients in Korea. As a result of immunoscreening, six types of λgt11-cDNA clones (DA107, DA135, DA100, DA174, DA150, and DA78) that showed specific immune responses to serum of non-A hepatitis B patients in Korea Was separated.

In the present invention, λgt11- showing positive response as a result of immunoscreening with serum of non-A non-B hepatitis patients from λgt11-cDNA library of Korean non-A non-B hepatitis patients. cDNA clones were amplified in Escherichia coli Y1090 cells, and the synthesized phage DNA was isolated by phenol / chloroform extraction and ethanol precipitation. The recombinant phage DNA was isolated and digested with restriction enzyme EcoRI, and each cDNA fragment was subjected to agarose gel electrophoresis. The E. coli DH5α cells were transformed after conjugation to the EcoRI site of the pTZ18U plasmid vector. PTZ18U plasmid DNA conjugated with cDNA specific for non-A non-B tongue hepatitis patients is isolated from each transformed cell, and then subjected to a dideoxy sequencing method. ) Was used to determine the base sequence of the cDNA. Refer to the base sequence of the non-A hepatitis virus genome and the amino acid sequence encoded therefrom for the determined base sequence and amino acid sequence. Nat1. Acad. Sci. USA, vol. 87, p9524-9528, 1990; Gene, vol. 103, p 163-169, 1991; J. Virology, p1105-1113, Mar., 1991, etc.] Corresponding locations were determined and regions of the six cDNA clones of DA107, DA135, DA100, DA174, DA150, and DA78 in FIG. 12 were shown as rectangles. Reference-Figure 12].

The present invention provides selective subcloned cDNAs from specific cDNAs of non-A non-B hepatitis virus. From the nucleotide sequence of DA107 containing the capsid and unveloped cDNA clone structural protein region shown in FIG. 12, the capsid cDNA DA703 and cDNA DA356 were understood in a PCR method using a self-primer plasmid expression vector pUEX2 (Amersham, USA) [ Reference-FIG. 13] and pMALcR1 (US NEB Co., Ltd.) [Ref.-FIG. 14], the recombinant expression plasmids pDA703 and pDA356 were constructed (see FIG. 15, FIG. 16). In addition, selective clones DA85, DA122, DA168 and DA99 were obtained by amplification by a PCR method using DA primers containing a portion of NS2, NS3 and NS4 of the non-structural protein (NS) region of the cDNA clone. The dotted rectangle is shown in FIG. 12 (see FIG. 12). Among the clones, the plasmid expression vector pMALcR1 [see Fig. 14] was expressed by subcloning and Western blotting showed that the recombinant plasmids pDA168 and pDA99 of the DA168 and DA99 clones showed high reactivity. It is shown in the figure.

The present invention provides the nucleotide sequences of the cDNAs DA703, DA356, DA168, and DA99 on the recombinant plasmids pDA703, pDA356, pDA168 and pDA99 constructed and amino acid sequences encoded therefrom (see FIG. 19 (a), (b), (b) c) and (d)].

The present invention is a result of the temperature rise of the E. coli HB101 strain transformed with the recombinant recombinant plasmid pDA703 and the E. coli DH5α strains transformed with pDA356, pDA168 and pDA99, or by induction and expression by IPTG and pDA703, pDA168, Gene products expressed in the pDA99 transformants showed high reactivity while significantly lower reactivity for the pDA356 transformant.

E. coli HB101 transformed with plasmid pDA703 was named E. coli YCS-S, and E. coli DH5α transformed with plasmids pDA356, pDA168 and pDA99 was named E. coli YCS-V, E. coli YCS-T and YCS-F, respectively. 25.

The present invention provides a method for preparing non-A non-B tongue-based hepatitis-specific antigen polypeptides D1, D2, D3, and D4 expressed by E. coli transformants YCS-S, -V, -T, and -F. to provide.

The cells of the polypeptide transformant were recovered and subjected to ultrasonic grinding, and the crushed cells were centrifuged to obtain an insoluble fraction containing polypeptide D1. The fusion site was cut with cyanogen bromide, solubilized with urea-containing buffer, and then cation exchange resin. Polypeptide D1 can be purified as follows. (See FIGS. 20 and 23)

For polypeptides D2, D3, and D4, the soluble fraction of the host cell's ultrasonic lysate is taken first to efficiently obtain each polypeptide fusion protein as an amylose-affinity chromatograph and increase its purity as an anion exchange resin, followed by specific proteolysis. The fusion site is cleaved as an enzyme. The cleaved fusion protein purely separated recombinant non-A non-B hepatitis-specific antigen polypeptides D2, D3, and D4 by gel filtration chromatography. 23 degrees]

Accordingly, the present invention provides a non-A non-B type compared to the above expression process, wherein the expression and purification consists of the entire amino acid sequence of D1, D2, D3 and D4 polypeptides (see FIG. 19) or a part thereof. Hepatitis-specific antigen polypeptides are provided.

In the present invention, the capsid protein in the structural protein region of the novel non-A non-B hepatitis virus of FIG. 19 (a) of the novel non-A non-B hepatitis virus A gene encoding the gene (cDNA) and a gene encoding the nonstructural protein region of FIGS. 19 (c) and (d) (cDNA) are cloned into an expression vector, and then transformed into E. coli, and the transformed E. coli is transformed. A novel method of purifying some polypeptide fragments in capsid proteins and nonstructural proteins obtained by culturing and using these mixtures as a raw material for immunodiagnostic methods. In patients with non-A hepatitis B, only antibodies to non-structural protein sites are produced, or only antibodies to non-structural protein sites exist for a period of time, depending on the individual's immunological differences. Therefore, when the mixture of the capsid protein and the non-structural protein portion fragments is used as a raw material for immunodiagnosis, only the capsid protein is used as a raw material for immunodiagnosis, and thus the accuracy is higher.

In fact, when only capsid protein was used as a raw material and when a mixture of capsid protein and non-structural protein site fragment was used as a raw material, the enzyme immunoassay was performed in 32 patients with liver disease patients. There was a sample that only reacted with the enzyme immunoassay using the structural protein site and the capsid mixture as a raw material. More detailed analysis revealed that the sample contained only antibodies to the non-structural protein of the virus. An example showing a better accuracy of the diagnostic method using a mixture of capsid and nonstructural protein site fragments than the diagnostic method was identified.

The present invention provides E. coli transformants that produce antigens of the non-A non-B tongue hepatitis virus structural and nonstructural protein regions described above, and these E. coli transformants have been deposited in KFCC. [Reference-Figure 25]

In general, the first consideration when making a vaccine against a virus is that it should be a vaccine that uses antigens to make neutralizing antibodies with the ability to destroy the virus in the body. To this end, the method of attenuating or inactivating the entire virus and using it as a vaccine has been widely used in the manufacture of vaccines against viruses. In addition, a method of selectively separating or preparing the antigenic portion that makes neutralizing antibodies has been used as a vaccine. . This method can be used for the production of non-A non-B hepatitis vaccines, and its structure and characteristics are similar to those of non-A non-B hepatitis virus. In the case of flaviviruses, it has been reported that the envelope protein of the three structural protein sites of the virus produces the most important neutralizing antibody [Roehing The Viruses; the Togaviridae and Flaviviridaes, Plenum Press, 279-328, 1988]. According to the present invention, the novel envelope gene (cDNA) of FIGS. 4 (b) and 19 (b) was prepared for the production of non-A non-B hepatitis vaccine. ) Was used to prepare and purify the envelope antigen protein and use it as a vaccine.

In the present invention, the envelope genes of FIGS. 4 (b) and 19 (b), which encode the envelope antigen protein, are expressed in a suitable E. coli or animal cell, and then purified. After destroying the cells, proteins such as ion exchange resins, affinity chromatography, gel filtration, ultracentrifugation, etc., are purified to have a purity that meets the specifications of the vaccine.

Immunologically confirmed that the purified protein has the same antigenicity of hepatitis virus, the vaccine is then combined with aluminum hydroxide or other suitable adjuvant. At this time, the amount of antigen as a vaccine is up to 1μg-80μg / ml protein and the type of vaccine is prepared in liquid form. Inoculation period of vaccine can be boosted from one to four times in order to increase antibody production of neutralizing antibody, and the interval of inoculation is about 2 to 6 months. The route of administration of the vaccine is intramuscular injection and subcutaneous injection.

Example 1

Isolation of Non-A Hepatitis B Virus RNA

Denatured solution (4M guanidium) in precipitates obtained by diluting the plasma of non-A non-B hepatitis patients with PBS (phosphate buffered saline) in equal proportion and centrifuging at 100,000xg for 8 hours. Thiocyanate, 25 mM sodium citrate: ph 7.0, 0.5% sarcosyl, 0.1 M vercaptoethanol) is added to the suspension and transferred to a polypropylene tube.

0.1 ml of 2M sodium acetate (ph4.0), 1 ml of phenol, and 0.2 ml of a 49: 1 ratio mixture of chloroform-isoamyl alcohol were mixed in this tube, and then mixed. Centrifuge for 20 minutes. Take the RNA liquid phase present in the upper part of the centrifuged tube, add the same amount of isopropanol, and leave it at -20 ° C for 2 hours, and then centrifuge at 10,000xg for 20 minutes to obtain RNA precipitate. The RNA precipitate was washed with 75% ethanol, recovered by centrifugation, dried in vacuo and dissolved in 0.5% SDS solution.

Example 2

Construction of Complementary Gene (cDNA) Fragments from Non-A Hepatitis B Virus RNA

CDNA wkrwpsms from RNA of non-A hepatitis B virus [Ref.- 1] A cDNA synthesis kit of US BRL using a reverse transcriptase of avian myeloblastosis virus (AMV) and a method thereof were performed. . In this experiment, 24 oligomers of 5'-CCCCATGAGGTCGGCGAAGCCGCA-3 'were used for capsid region cDNA production as antisense primers for cDNA preparation, and 5' for envelope region cDNA production. 27 oligomers of -CCAGTTCATCATCATATCCCAAGCCAT-3 'were used.

The concentration of each primer used was 50 pmole.

Example 3

Cloning and sequencing of the constructed new gene (cDNA)

In order to amplify the amount of cDNA produced, 24 oligomers of 5'-CTGCCTGATAGGGTGCTTGCCGACT-3 'were used as antisense primers in a capsid-based cDNA mixture prepared with 50 pmole of sense primer. In Example 2, after adding 50 pmole of the same antisense primer used for capsid region cDNA preparation, Taq DNA polymerase, buffer solution of GeneAmp PCR (polymerase chain reaction) kit of Perkin Elmer Cetus, USA and its CDNA amplification was performed according to the method.

Meanwhile, in the envelope region cDNA amplification experiment, 23 oligomer 50 pmole of 5'-TTGGGTAAGGTCATCGATACCCT-3 'was used as a sense primer, and the antisense primer was used in Example 2 50 pmole of the same antisense primer used for envelope region cDNA preparation was used. At this time, PCR conditions were amplified by repeating the procedure of denaturation at 94 ° C. for 1 minute, binding at 55 ° C. for 2 minutes, and ring reaction at 3 ° C. for 3 minutes at 36 times. The cDNAs thus amplified were separated by low melting agarose gel electrophoresis, filled-in with Klenow enzyme, and phosphorylated with polynuclentide kinase.

The phosphorylated cDNA fragment was blunt-end conjugated to the restriction enzyme Smal position of the plasmid vector pTZ18U [see FIG. 2] of Escherichia coli to prepare recombinant plasmids pTZ18U-Cap and pTZ18U-Env. Degree]. On the other hand, the base sequence of the produced new complementary gene (cDNA) was determined as a dideoxy nucleotide analysis method using universal and reverse primers. It is shown in (b).

Example 4

Expression and Identification of Non-A Hepatitis B Virus New Gene (cDNA)

Conjugation site at the end of cDNA for cloning the new complementary gene (cDNA) fragments of the capsid and envelope regions of which sequencing has been determined to E. coli plasmid PT7-7 (see FIG. 6). Referred to in Example 3 using artificially designed capsids and envelope primers containing restriction enzyme NdeI sites or EcoRI sequence sites at the 5 'end for the purpose of artificially generating PCR method was used to generate capsid and envelope region complementary gene (cDNA) fragments having restriction enzyme sites of NdeI and EcoRI at 5 'and 3', respectively, and then the restriction enzyme NdeI on PT7, a vector for E. coli expression, was expressed. Recombinant plasmids pT7-Cap and pT7-Env were prepared by conjugation at the EcoRI position (see FIG. 7). This recombinant plasmid was co-hosted with E. coli host K38 (Hfr) strain [p. Bacteriol., 159, 1038, 1985], and then transformants were selected from medium containing ampicillin and kanamycin, and the transformants were transformed into 2X YT medium containing ampicillin and kanamycin (2% tryptone, 1%). yeast extract, 0.6% NaCl, 0.2% Glycerol, 50mM KH ₂ PO ₄ , ph7.2) and incubated at 30 ° C. When the number of cultured cells reached absorbance (A600) of 1.5, the temperature was raised to 42 ° C and Rifampicin was added. Incubated.

When used as a tracer to confirm expression of the capsid and envelope genes cloned under the T7 promoter, the cells were incubated with a radioisotope ³⁵ S-methionine, which was 20 μg / ml of thiamine ( thiamine) and 0.01% of 18 amino acids (cysteine and methionine are not added) incubated for 30 minutes at 60 ° C for 60 minutes, raised to 42 ° C, incubated for 20 minutes with rifampicin, and then lowered to 30 ° C. After incubation for 10 minutes, ³⁵ μSi-methionine labeled with ¹⁰ μCi of radioisotope was added. Meanwhile, the gene product expressed in the transformed Escherichia coli was confirmed after separating the lysate of the transformant by 15% polyacrylamide gel electrophoresis [Reference-Article 9].

Example 5

Purification of Expressed New Gene Products

5 g (wt, wet weight) of 2 liter culture cells were dissolved in 60 ml of buffer A (10 mM potassium phosphate, ph7.1, 1 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF) and labeled with 35S-methionine. 5 mg of the cell lysate (wt.) And lysozyme (1 mg / g cell wt.) Were added and the cells were sonicated on ice. The sonicated sample was centrifuged at 15,000 × g for 30 minutes, the supernatant was collected, filtered through a 0.2 μm Nalgene-filter to remove impurities, and the filtered sample was applied to a DEAE-cellulose column. The solution was washed with Buffer A until no protein was released, and 600 ml of KCI density gradient from 0 to 500 mM was added through Buffer A and eluted at a rate of 15 ml / hr. After confirming each of the eluted fractions by radioactivity, protein quantitation and inducibility, the fractions of radioactive protein fractions around 170 mM KCI were collected and diluted with 100 mM KCI buffer A, 100 mM KCI and 5% glycerol. The solution was applied to a phosphocellulose column (2.5 × 15 cm) diluted with buffer A containing (glycerol) and equilibrated with buffer A containing 100 mM KCI and 5% glycerol. This was prepared by dissolving 600 ml of 100-600 mM KCI density gradient with buffer solution A containing 6% glycerol at a rate of 15 ml / hr, and measuring the radioactivity of each of the eluted fractions [ref. After making a 350mM KCI density gradient with elution activity, the solution was eluted at a rate of 15ml / hr, and the radioactivity of each eluted fractions was measured (see FIG. 10). Fractions of 350 mM KCI density gradient were collected and concentrated to 4 ml using the ultrafiltration method (YM10 diaflo ultrafiltration).

Example 6

Determination of Non-A Non-B Hepatitis Virus Antibodies by Enzyme Immunoassay

Enzyme-immunoassay using purified protein was used to determine the presence of non-A non-B hepatitis virus antibody against 19 samples of sera of non-A non-B hepatitis patients.

Purified viral protein was added to 0.1N NaHCO3 solution to a concentration of 100ng-1μg / ml, and 150μl was dispensed per well of microplates (Nunc, Immunoplate, 2X8 strips) and reacted for 16-24 hours in a 4 ° C chamber. After the reaction, the filtrate in the well was removed by suction, and each well was washed three times with 200 μl of a washing solution containing 0.05% Tween-20 to minimize non-specific reactions. The antibody present in the sample was measured as follows using the microplate prepared as above.

100 μl of the serum sample diluted 1:10 to 1: 100 with 0.01 M PBS (ph7.2) was added to the corresponding well of the microplate and reacted at 37 ° C. for 1 hour to form an antigen-antibody complex.

After the reaction, the unbound sample filtrate was aspirated, and each well was washed three times with 200 μl of a washing solution containing 0.05% Tween-20. 100 μl of goat anti-human immunoglobulin-peroxidase conjugate was added to each well and reacted at 37 ° C. for 1 hour to bind the antigen-antibody complex.

After the reaction, the wells were washed three times with 200 μl of Tween-20 washing solution, and then 1 tablet of substrate substrate containing 12.8 mg of Ortho Phenylene Diamine 2HCl was dissolved in 5 ml of substrate buffer containing 0.02% of 35% hydrogen peroxide solution. 100 μl of each well was added and the enzyme-substrate reaction was carried out at room temperature for 30 minutes, and 100 μl of 1N-H ₂ SO ₄ solution was added per well to stop the reaction, and then the absorbance was measured using a photometer.

Example 7

Preparation of λgt11-cDNA Library from Non-A Non-B Hepatitis Plasma

An amount of RNAzol (RNAzol) was added to the precipitate obtained by diluting the plasma of non-A non-B hepatitis patients with PBS (Phosphate Buffered Saline) buffer solution at the same ratio and centrifuging at 160,000 xg for 3 hours. , CM Tinna Sci.). 0.1 volume of chloroform is added to the above solution and then suspended and transferred to a polypropylene tube. Centrifuge the tube in this solution for 15 minutes at 4 ℃ and 12,000 xg, take supernatant, add the same amount of isopropanol, store for 2 hours at -20 ℃, and centrifuge for 15 minutes at 4 ℃, 12,000 xg. A precipitate was obtained. The RNA precipitate was washed twice with 75% ethanol, collected by centrifugation, dried in vacuo, and dissolved in distilled water treated with 0.1% diethylpyrocarbonate (DEPC). The RNA solution thus prepared was stored at -70 ° C until use.

About 500ng of the isolated RNA was synthesized according to the method using a cDNA synthesis kit of promega (promega) using a reverse transcriptase of avian myeloblastosis virus (AMV). In this experiment, random primers (hexadeoxy ribonucleotides) were used as primers for cDNA preparation.

The constructed cDNA is conjugated with an EcoRI adapter with T4-DNA conjugate, and phosphorylated with T4 polynucleotide anoxidase. EcoRI adapters that were not conjugated to cDNA in the cDNA solution were removed using a spin column, and the cDNA was conjugated with a T4-DNA conjugate at the EcoRI position of λgt11 vector DNA. Recombinant DNA was packaged in vitro with phage infected E. coli cell extract (Promega). Thus, the λgt11-cDNA library prepared from the plasma of non-A non-B hepatitis patients contained on average 10 ⁶ to 10 ⁷ pfu of recombinant phage.

Example 8

Immune Screening of cDNA Libraries of Non-A Non-B Hepatitis Patients and Isolation of cDNA Clones Specific to Serum of Non-A Non-B Hepatitis Patients

Immun screening was attempted using the serum of non-A non-B hepatitis patients from the cDNA library prepared in Example 7. Approximately 20,000 to 30,000 pfu phage of the λgt11-cDNA library of non-A hepatitis B patients was added to 200 μl of E. coli Y1090 cell culture cultured in 5 ml NZCYM culture medium containing 0.02% maltose for 8 hours. Then adsorbed at 37 ° C. for 20 minutes. The solution of E. coli was mixed with 7.5 ml of LB coated agarose solution (49 ° C.) and plated onto a 15 cm diameter NZCYM-agar plate.

Leave the coated agarose at room temperature until solidified and incubate at 42 ° C for 3.5 hours to form a plaque. Subsequently, the NZCYM-agar plate was covered with a nitrocellulose filter moistened with 10 mM IPTG, and the plate covered with the filter was further incubated at 37 ° C for 6 hours. Label on the nitrocellulose filter and store this plate at -20 ° C for 5 minutes before transferring the nitrocellulose filter onto 3MM paper.

The nitrocellulose thus prepared is washed twice with 30ml TBST (50mM Tris-cl ph7.9, 150mM NaCl, 0.05% Tween) (5 minutes each) and blocked for 30 minutes at room temperature with 15ml blocking solution (1% BSA TBST). Let's do it. After diluting the serum of patients with non-A non-B hepatitis by 1,000-fold, 10 ml solution of E. coli extract at 1 mg / ml was reacted at room temperature for 30 minutes, and then nitrocellulose was reacted at 4 ° C. Reaction at 1 hour. After washing the filter three times with TBST solution, 10 ml of 7,500-fold diluted goat anti human immunoglobulin-alkaline phosphatase conjugate was reacted at room temperature for 1 hour, and washed three times with TBST solution.

Next, the filter was changed in a 10 ml solution containing NBT (nitro blue tetrazolium): BCZP (5-bromo-4-chloro-indolyl-phosphate): alkaline phosphatase buffer = 33 μl: 16.5 μl: 5 μl. . The plaques at the positive sites were stored in phage buffer and stored at 4 ° C. Secondary immunoscreening was performed using the above method to form 500-800 plaques from the isolated phage solution. As a result, a single phage showing antigen-specific responses to serum of non-A non-B hepatitis patients. Plaques were separated. All 6 × 10 ⁵ plaques were immunoscreened to isolate 6 phage plaque flocks that were positive for the sera of non-A non-B hepatitis patients.

Example 9

Sequence Analysis of Non-A Hepatitis B Virus cDNA

CDNAs of six λgt11-cDNA clones specific for non-A non-B hepatitis patients isolated in Example 8 were subcloned into pTZ18U plasmid vectors, and the nucleotide sequences were analyzed by dideoxy chain termination. The site was determined. [Reference-Figure 12]

Example 10

Construction of Selective Subcloning and Recombinant Expression Plasmids of Specific cDNAs from Non-A Non-B Hepatitis CDNA

Thirty oligomers of 5'-GCG GTT AAC GGG AGG TCT CGT AGA CCG TGC A-3 'were identified from the base sequence of the DA107 clone containing the structural protein region and the envelope region of the cDNA clone. [Ref.-Fig. 15] Meanwhile, 28 oligomers of '-GCG GAA TTC ACG TGC GGC TTC GCC GAC C-3' and the antisense primer 5'-GCG CTG CAG CTA CCA AGC CAT Thirty oligomers of GCG ATG ACC-3 'were used to amplify by PCR method (see FIG. 16).

At this time, PCR conditions were repeated for 1 minute denaturation at 94 ℃, 2 minutes binding at 55 ℃ and 3 minutes cyclization reaction at 72 ℃ 45 times, the amount of oligomer used was 50pmole, respectively.

The amplified cDNAs were separated by low melting agarose gel electrophoresis, and as shown in FIG. 15, the amplified capsid region cDNA DA703 was digested with restriction enzyme HpaI / EcoRI and then pUEX2 plasmid [Reference-13]. Recombinant expression plasmid pDA703 was constructed by subcloning the HpaI / EcoRI restriction enzyme position of the vector (see FIG. 14). In addition, the amplified envelope region cDNA DA356 was digested with the restriction enzyme EcoRI, PstI and subcloned into the EcoRI / PstI restriction enzyme position of pMALcR1 [ref. 14] to prepare a recombinant expression plasmid pDA356 [ref. 16]. ].

Meanwhile, in order to select a clone specific for an immune response from a DA174 clone including a portion of NS2, NS3 and NS4, which are non-structural protein (NS) regions among cDNA clones, a nucleotide sequence in the DA174 DNA fragment was used as a PCR method. Four kinds of primers were made and PCR was performed. At this time, PCR conditions were denatured for 1 minute at 94 ° C., 2 minutes for 2 minutes at 55 ° C., and cyclic reaction for 3 minutes at 72 ° C., and the amount of oligomer used was 50 pmole, respectively.

Sense primers for selective cloning of DA168 and DA99 cDNA, which were highly reactive when Western blotting with sera from non-A non-B hepatitis patients, showed 5'-ATG GAA TTC ACC ATG for DA168. CGG TCT CCG GTC TTC AC-3 '32 oligomers were used for the DA99 for the 5'-CCA GAA TGA ATT CAC CCT CAC CCA CCC ACC CCA-3' 33 oligomers.

As the antisense primer, 33 oligomers of 5'-CCT AGT CGA CCG TAT AGC CCG TCA TCA GAG CGT-3 'were used for DA168, and 5'-ATG GAA TTC CAC ATG TGC TTC GCC CA for DA99. 29 oligomers of −3 ′ were used to amplify the PCR conditions described above.

As shown in FIGS. 17 and 18, the amplified NS3 partial region cDNA DA168 is located at the restriction enzyme EcoRI / SAl I position, and the amplified NS4 partial region cDNA DA99 is located at the restriction enzyme EcoRI position. Recombinant expression plasmids pDA168 and pDA99 were constructed by subcloning into the EcoRI / Sal I or EcoRI / EcoRI positions of FIG.

Example 11

Nucleotide and Amino Acid Sequence Determination of Structural Protein Region cDNA DA703, DA356 and Non-Structural Protein Region cDNA DA168 and DA99

CDNA DA703 [reference- FIG. 19 (a)], DA356 [reference- FIG. 19 (b), DA168 [reference- FIG. 19 (c)] from the recombinant plasmids pDA703, pDA356, pDA168 and pDA99 prepared in Example 10 ] And DA99 [see-Figure 19 (d)] were determined by dideoxy chain termination method, and amino acid sequence encoded therefrom was also determined.

Example 12

Expression and Purification of Polypeptide D1 from E. Coli Transformant of Recombinant Expression Plasmid pDA703

E. coli HB101 strain (E. coli YCS-S) transformed with the expression polyamide pDA703 was incubated at 30 ° C. in LB medium (1% bactotripton, 0.5% yeast extract, 1% NaCl) containing 50 μg / ml of ampicillin, and then absorbed. When (A600) became 0.5, the temperature was raised to 42 ° C. and incubated for 16 hours. Each time the culture medium was taken and the cell lysate was subjected to SDS polyacrylamide gel electrophoresis and Western blotting, and it was confirmed that the capsid fusion protein was expressed at the 36 kilodalton position.

In order to purify DA703-polypeptide D1, an antigenic protein of the capsid moiety, the selected transformant was incubated at 30 ° C. for 12 hours in a fermenter using 2 liters of culture agar, followed by raising the culture temperature to 42 ° C. After inducing the expression, the culture was centrifuged at 10,000 xg for 30 minutes to recover the cells and washed twice with physiological saline (NaCl 0.85%). After washing, the cells were suspended in 0.2 liter of 50mM tris buffer (ph 8.0), pulverized the cells with ultrasonic grinding (400 watts, 3 min 3 times) and centrifuged to remove soluble proteins in the supernatant and infiltrated insoluble. Inclusion bodies, proteins, were recovered. In order to remove the membrane proteins, DNA, lipids, etc. of the recovered cells, lysozyme, DNA degrading enzyme, sodium deoxycholate, Triton X-100, etc. were treated to obtain a clean inclusion body. The washed inclusion body was dissolved in 70% formic acid to cleave methionine present at the N-terminus and C-terminus of polypeptide D1 on the β-galactosidase-polypeptide D1 fusion protein, and then the methionine site of the fusion protein as cyanogen bromide. After the cleavage reaction was completed, the reaction solution was concentrated under reduced pressure to powder the cleaved protein, and insoluble cleavage proteins were denatured as 1 liter of 50 mM Tris buffer (pH 8.9) to which 6M urea and 10 mM dithiothreitol (DTT) were added. After solubilization, the mixture was adsorbed onto 200 ml of CM Sepharose CL-6B (Pharmacia, sweden) resin, which was a cation exchange resin, and eluted with a gradient of NaCl to obtain a fraction of the cleaved DA703 polypeptide D1 (see FIG. 20).

Each fraction was identified by SDS-polyacrylamide electrophoresis and Western blotting analysis to confirm its purity and antigenicity. Only the fraction of high purity was dialyzed in phosphate buffer (pH 7.4) to prepare DA703-polypeptide D1. [Reference- FIG. 23]

Example 13

Expression and Purification of Polypeptides D2, D3 and D4 from E. Coli Transformants of Recombinant Expression Plasmids pDA356, pDA168 and pda99

E. coli DH5α strains transformed with the expression plasmids pDa356, pDA168 and pDA99, respectively, were cultured at 37 ° C in an LB medium (1% bactotriptone, 0.5% enzyme extract, 1% NaCl) containing 50 μg / ml of ampicillin, followed by absorbance (A600). When 0.5) became 0.5, IPTG (isopropy1-thio-β-D-galactoside) was added to 1 mM and incubated for 16 hours. The culture medium was taken at each time, and the cell lysate was subjected to SDS polyacrylamide gel electrophoresis and Western blotting. As a result, the fusion protein of the envelope region derived from pDA356 was 65 kilodalton, and the nonstructural antigen NS3 derived from pDA168. The fusion protein of 69 kilodaltons, non-structural antigen NS3 derived from pDA168, the fusion protein of 69 kilodaltons, the fusion protein of non-structural antigen NS4 derived from pDA99 was confirmed in the 57 kilodalton position, respectively.

Each transformation selected for large purification of DA356, the envelope portion of non-A hepatitis B virus, DA168 of the nonstructural protein NS3 region, and D2, D3 and D4 polypeptides of DA99 of the NS4 region Sieves were incubated for 20 hours at 37 ° C. in a fermentor using 2 liter culture medium, and then IPTG (isopropylthio-β-D-galactoside) was added to 3 mM at 600 nm absorbance at 600 nm during incubation. After incubation, the cells were recovered by centrifugation of the culture solution, washed twice with phosphate buffer (pH 7.4), and then pulverized by the ultrasonic grinding (400 watts, 3 times each 3 minutes) and centrifuged (30 at 10,000 xg). Min) only soluble protein sites of the supernatant were obtained.

Since the expression protein for each antigen is a fusion protein with maltose binding protein, the fusion protein is first adsorbed with maltose binding protein affinity amylose resin (New England Biolabs, USA) and then adsorbed as maltose of 10 mM. Eluted. The primary purified fusion protein is DEAE Sepharose Anion Exchange Chromatography for the purpose of enhancing its purity. After further purification, the activated factor Xa (Boehringer Mannheim, Germany) has a high substrate specificity for the binding site between two proteins of the fusion protein. In order to separate the maltose binding protein and the viral polypeptide from the cleaved fusion protein, the final product of the novel gene (cDNA) polypeptides D2, D3 and D4 was purified purely by gel filtration chromatography. 21, 22 and 23].

Example 14

Measurement of Non-A Non-B Hepatitis Virus Antibodies by Enzyme Immunoassay Using Purified Polypeptides D1, D3, and D4

In the present invention, the antigen polypeptides D1, D3, and D4, which have a strong response to serum of non-A hepatitis B patients, were purified and analyzed from serologic samples of 32 patients with liver disease using enzymatic immunoassay. The presence or absence of the non-A hepatitis B virus antibody was measured.

Higher accuracy was achieved with all D1, D3 and D4 antigens than with only the D1 antigen [see FIG. 24]. The experiments of this enzyme immunoassay were as follows. Each purified antigen polypeptide was dispensed at 100-150 μl per well of microplates (Nunc. Immunoplate, 2x8 strips) in 0.1 N NaHCO ₃ buffer at a concentration of 100 ng-1 μg / ml and then 16- 16 in a 4 ° C. or 28 ° C. chamber. The response was 24 hours. After the reaction, the filtrate in the well was removed by suction, and each well was washed three times with 200 μl of a washing solution containing 0.05% Tween 20 to minimize non-specific reactions. The antibody present in the sample was measured as follows using the microplate prepared as above. 100 μl of the blood sample diluted 1:10 to 1: 100 with 0.01 M PBS (pH 7.2) was added to the corresponding well of the microplate and reacted at 37 ° C. for 1 hour to form an antigen-antibody complex. After the reaction, the unbound sample filtrate was removed by suction, and each well was washed three times with 200 μl of a washing solution containing 0.05% of Tween 20.

100 μl of goat anti human immunoglobulin-peroxidase conjugate was added to each well and reacted at 37 ° C. for 1 hour to combine with the antigen-antibody complex to form a large polymer of antigen-antibody-secondary antibody-peroxidase. After the reaction, each well was washed three times with 200 μl of a washing solution containing 0..05% of Tween 20, followed by 35% peroxidation of a substrate effervescent tablet containing 12.8 mg of OPD (Ortho Phenylene Diamine · 2HCl). 100 μl of the dissolved substrate solution was added to 5 ml of substrate urine buffer containing 0.02% of hydrogen, followed by enzyme-substrate reaction at room temperature for 30 minutes.

After the reaction, 1N-H ₂ SO ₄ solution was added to 100μl per well to stop the reaction, and the absorbance was measured by using an absorbance analyzer.

Example 15

Vaccine Production Using Purified Envelope Protein

100 ml of suspension solution (PBS pH 7.2 Tween 80 0.01% Gelatin 0.02%) was added to the precipitate obtained by centrifuging PBS (pH 7.2) buffer containing purified envelope antigen at 80,000xg for 6 hours. The concentration of the antigen added is 10 mg to 50 mg per ml to prepare the vaccine stock solution.

Again dilution solution (PBS ph 7.2, Thimerosal 0.01%, EDTA 0.004%) was passed through a 0.02μm gelman filter to 10μg per ml of final dilution so that 1ml per vial was added. After dispensing and capping, the vaccine was prepared.

Claims (9)

A non-A non-B hepatitis virus complementary gene (cDNA) nucleotide sequence comprising the nucleotide sequence shown in FIG. The non-A and non-B component according to claim 1, comprising a base sequence derived from the non-A and non-B hepatitis virus complementary genes (cDNA). Hepatitis virus complementary gene (cDNA) sequence. A base for detecting non-A and non-B hepatitis DNAs by hybridizing the non-A and non-B hepatitis virus nucleotide sequences according to claim 1 or 2. A method for detecting non-A, non-B hepatitis DNA, characterized in that it is used as a sequence probe. A recombinant plasmid containing the nucleotide sequence according to claim 1. E. coli transformants transformed with the recombinant plasmid according to claim 4. A protein or polypeptide produced by the transformed strain of claim 1. A protein or polypeptide encoded by the nucleotide sequence of claim 1. Non-A and non-A present in serum samples containing or suspected of containing non-A and non-B hepatitis virus antibodies using the protein or polypeptide of claim 6 as an antigen. ) How to detect hepatitis B virus antibody. The method of claim 8, wherein the antigen to be used is in a suitable solid phase, and the antibody bound to the antigen immobilized on the solid phase is detected using an anti-human immunoglobulin antibody labeled with an enzyme or a radioisotope or an antigen used in the solid phase. How to.