CN1465700A - Hepatitis C virus envelope protein E2 gene capable of full-length expression in Escherichia coli and its encoded protein and application - Google Patents

Abstract

The present invention discloses a hepatitis C virus capsule protein E2 gene capable of implementing total-length expression in colibacillus and its coding protein. Said hepatitis C virus capsule protein E2 with total length expression has antigenicity of E2 and its immunogenicity, can be used for immunizing animal and can produce antibody for resisting E2, so that it can create the condition for researching the antigenicity and immunogenicity of complete HCV E2, the purified and complete total-length E2 protein and prepared E2 antibody can be used for diagnosis, prevention and therapy or hepatitis C.

Description

Hepatitis C virus envelope protein E2 gene capable of full-length expression in Escherichia coli and its encoded protein and application

technical field

The invention relates to the field of genetic engineering, in particular to a hepatitis C virus envelope protein E2 gene capable of full-length expression in Escherichia coli, its encoded protein and its application in the preparation of drugs for diagnosis, prevention and treatment of hepatitis C .

Background technique

Hepatitis C virus (HCV, Hepatitis C Virus) is the pathogenic agent of hepatitis C that seriously endangers human health at present, and transmits hepatitis C through blood. According to statistics, 1-3% of the world's population has been infected by the virus. 85% of them will develop chronic hepatitis, and 20% of them will eventually lead to cirrhosis and liver cancer. The development of vaccines to prevent hepatitis C, the development of effective therapeutic drugs, and the development of effective diagnostic techniques are the primary tasks for controlling the spread of hepatitis C.

Existing studies have shown that HCV is a positive-strand RNA virus with a genome of about 10,000 nucleotides in length, containing only a single reading frame, and encoding a polyprotein of 3,000 amino acids. By comparison with other viruses of the same genus, two glycosylated envelope proteins, E1 and E2, are thought to be located on the surface of the virion. The immune response elicited by the envelope proteins of certain types of viruses can produce neutralizing antibodies and effectively clear the virus.

Due to the low titer of HCV in the blood of patients, there is still a lack of effective in vitro propagation methods, and the virus cannot be directly studied, so it can only be studied through in vitro translation systems and various gene recombinant expression systems, such as Escherichia coli, insect viruses, Systems such as mammalian cells and vaccinia virus express HCV proteins.

HCV envelope proteins (E1, E2) are considered to contain major immune determinants, especially E2, so the study of envelope protein E2 is undoubtedly of great significance in the research of HCV molecular virology and vaccine development.

HCV E2 encodes amino acids 384-746, and contains two hypervariable regions HVR1 (coding 386-411) and HVR2 (coding 470-480) (Hijikata M et al. Biochem.Biophys.Res.Commun.1991 175:220-228; Weiner AJ et al. Virology 1991 180:842-848). The carboxy-terminal region of E2 is strongly hydrophobic (coding 662-746), and contains the transmembrane domain (TMD, transmembrane domain) coding 718-746, which contains the retention signal of E2 in the endoplasmic reticulum system (Duret S et al. J. Biol. Chem. 1998 273:32088-32095; Cocquerel L et al. J. Virol. 199872:2183-2193). E2 is expressed in yeast expression system (Mustlli AC et al. Res.Microbiol.1999150: 179-187), in insect virus expression system (Hüssy R et al. Virus Research 1996 45: 45-57), in mammalian cell expression system (Rosa D et al. Proc.Natl.Acad.Sci.U.S.A.199693:1759-1763), in the recombinant vaccinia expression system (Choo QL et al. Proc.Natl.Acad.Sci.U.S.A.1994 91:1294-1298) etc. have all realized expression .

If the carboxy-terminal hydrophobic region is removed or a secretion signal is added, secreted expression can be achieved in the above-mentioned eukaryotic expression system. Although its expression product has glycosylation processing and may be closer to the natural virus structure, it is still difficult to obtain a large amount of HCV E2 protein due to factors such as low expression level, difficult purification, and high cost. In the E. coli expression system, the propagation is fast and the cost is low. However, due to possible reasons such as the strong hydrophobicity of the carboxy-terminus of the extracellular domain, the full-length E2 of HCV cannot be expressed, and the full-length and complete E2 expression protein cannot be obtained. Usually, the hydrophobic region of the carboxy-terminus (662-746 amino acids) must be removed, such as expressing only the E2 protein whose carboxy-terminal hydrophobic region has been removed from the 384-661 amino acid (such as Hüssy R et al. J.Hepatology 1997 26: 1179-1186, Mita E et al. Biochem. Biophy. Res. Commun. 1992 183:925-930). There is no report on the expression of the strong hydrophobic region at the carboxy-terminus of the ectodomain of envelope protein E2 alone, nor the successful expression of the full-length envelope protein E2.

Contents of the invention

The core of the present invention is to break through the limitation that the carboxy-terminal part of the ectodomain of HCV envelope protein E2 and the complete HCV envelope protein E2 have not been able to obtain full-length expression in Escherichia coli. The amino-terminus of the terminal part, that is, the central region of HCV, introduces a frameshift mutation encoding 4 amino acid residues, which does not change the basic structure and sequence of the carboxy-terminal part of the extracellular domain in the E2 gene and the complete E2 gene, and is expressed in E. coli , that is, the expression of the carboxy-terminal part of the extracellular domain of HCV E2 or the full length of the HCV E2 protein can be obtained.

The present invention provides a HCV envelope protein E2 gene capable of full-length expression in Escherichia coli, its nucleotide sequence is shown in <210>1 in the sequence list, and it is in the ectodomain carboxylase in the 1b subtype HCV envelope protein E2 gene The N-terminus of the terminal part, that is, the central region of the envelope protein E2, introduces a frameshift mutation encoding 4 amino acid residues at the coding position 568 to 571, which is to change the original amino acid sequence PCNI to RVTS code mutation. The carboxy-terminal part of the E2 ectodomain containing the frameshift mutation (coding 567-730) and the nearly complete E2m (coding 385-730) gene [the expression clone pQE8/E2(385-730)m of this gene has been transformed In Escherichia coli TG-1, namely TG-1/pQE8E2m, was preserved by the General Microorganism Center (CGMCC) of Beijing China Microbiological Culture Collection Management Committee, March 4, 2002, preservation number CGMCC No.0725], in the large intestine In the bacillus expression system, it has broken through the limitation that the carboxy-terminal part of the E2 extracellular domain and the E2 gene cannot be expressed in full length in E. coli, and can efficiently obtain the carboxy-terminal part of the full-length E2 extracellular domain (position 567-730) or near Expression of the complete E2 protein (position 385-730). The E2 protein can be conveniently enriched and purified from the carboxy-terminal part of the E2 extracellular domain of HCV (position 567-730) or nearly complete E2 by using the Ni ²⁺ -NTA affinity medium fused with the 6xHis sequence on the expression vector. Protein (position 385-730). The full-length expressed E2 protein (see <210>2 in the sequence listing for its amino acid sequence) has good E2 antigenicity and immunogenicity, can react with the E2 antibody present in the serum of HCV patients, and can be applied to E2 antibody enzyme-linked immunosorbent assay.

The invention also provides the application of the coding protein of the HCV envelope protein E2 gene capable of full-length expression in Escherichia coli in the preparation, diagnosis, prevention and treatment of hepatitis C medicaments.

The full-length expressed E2 protein immunized animals (mice or rabbits) can produce anti-E2 antibodies, and the prepared multiple antiserum can be compared with E2 expressed in a variety of different systems (such as recombinant vaccinia virus, mammalian cells, E. coli, etc.) Protein reaction, which can be used to detect the expression of E2 in various systems, such as western blot analysis, etc.

This invention provides a condition that can study the carboxy-terminal part of E2 ectodomain alone or completely study the properties of HCV E2 protein and the molecular virology of HCV E2, as well as carry out the function and properties of complete HCV E2 antigen and antibody and their application Research.

The gene of the nearly complete envelope protein E2 of hepatitis C virus or the carboxy-terminal part of the extracellular domain of the E2 gene and its encoded protein that can obtain the full-length expression in the Escherichia coli system provided by the present invention, the carboxy-terminal of the extracellular domain The amino terminal of the hydrophobic region, that is, the central region of the complete E2 gene, introduces a frameshift mutation encoding 4 amino acid sequences, and the specific methods include:

1. Construction of an expression clone containing the carboxy-terminal part (567-700) of the ectodomain of hepatitis C virus envelope protein E2 [pQE8/E2(567-700)];

2. A clone [pQE8/E2(567-700)m] containing a frameshift mutation encoding 4 amino acid residues at the N-terminus of the carboxy-terminal part of the E2 ectodomain (567-700);

3. Construction of an expression clone containing a frameshift mutation at the carboxy-terminal part (567-730) of the ectodomain of E2 [pQE8/E2(567-730)m];

4. Construction of complete expression clone of HCV envelope protein E2 gene (coding 385-730) containing frameshift mutation [pQE8/E2(385-730)m] and E2 gene (coding 385-730) without frameshift mutation Construction of the expression clone of [pQE8/e2(385-730)];

5. Full-length expression of HCV near-complete E2 (385-730)m containing HCV complete E2 with frameshift mutation in Escherichia coli and identification of the properties of the expression product.

Description of drawings

The present invention is further illustrated by the following figures and examples, but does not limit the scope of the present invention.

The accompanying drawings of the present invention are as follows:

Figure 1 is a schematic diagram of the construction of the expression clone pQE8/E2 (567-700) containing the carboxy-terminal part of the HCV E2 extracellular domain (567-700).

Explanation of various symbols:

Envelope protein E2, the number marked below is the sequence number of the amino acid encoded at this position of E2; A-restriction enzyme ApaI; H-HindIII; B BamHI; S-SmaI; Lig-link; Annotated as the name of the original plasmid before recombination;

Fig. 2 is a schematic diagram of the frameshift mutation in the carboxy-terminal part (567-700) of the extracellular domain of E2 in the expression clone pQE8/E2(567-700)m encoding the amino acid sequence from position 568 to position 571.

The sequence number of the amino acid encoded by the position E2 above: P-proline; C-cysteine; N-asparagine; I-isoleucine; R-arginine; V-valine Acid; T-threonine; S-serine; underlined under the codon represents the 568-571th amino acid residue that has undergone frameshift mutation;

Figure 3 is a schematic diagram of the construction of the clone pQE8/E2(567-730)m containing the carboxy-terminal part of the HCV E2 extracellular domain (position 567-730) with a frameshift mutation.

Sp-restriction enzyme SphI; Sa-SalI; H-HindIII; lig-linkage.

Envelope protein E2, the number marked below is the sequence number of the amino acid encoded at the position of E2; the left side is marked with the name of the plasmid after recombination, and the right side is marked with the name of the original plasmid before recombination;

Figure 4 is a schematic diagram of the construction of the HCV near-complete E2(385730)m expression clone pQE8/E2(385-730)m containing a frameshift mutation.

被膜蛋白E2，标在下方的数字为E2该位置编码氨基酸的序号；P1→，正向引物P1；←P2，反向引物P2；P3→，正向引物P3；←P4，反向引物P4；H-HindIII；B-BamHI；A-ApaI；N-NheI；lig-连接。

Envelope protein E2, the number marked below is the sequence number of the amino acid encoded at the position of E2; P1→, forward primer P1; ←P2, reverse primer P2; P3→, forward primer P3; ←P4, reverse primer P4; H-HindIII; B-BamHI; A-ApaI; N-NheI; lig-linkage.

The left side is marked with the name of the plasmid after recombination, and the right side is marked with the name of the original plasmid before recombination;

Figure 5 is the expression product of expression clones pQE8/E2(567-700), pQE8/E2(567-700)m, pQE8/E2(567-730)m containing the carboxy-terminal part of E2 ectodomain and the expression of complete E2 Clones pQE8/E2(385-730) and expression products of pQE8/E2(385-730)m were identified by Coomassie brilliant blue staining after electrophoresis.

A-pQE8/E2(567-700); B-pQE8/E2(567-700)m;

C-pQE8/E2(567-730)m; D-pQE8/E2(385-730);

E-pQE8/E2(385-730)m.

The estimated molecular weight indicated on the figure: A-17.8KD; B-17.8KD;

C-23.2KD; D-41.6KD; E-41.6KD;

Fig. 6 is the western blot analysis of the full-length expression product E2(384-730)m of the intact envelope protein E2, and the photo of ECL development.

A-HCV-infected serum S94; B-E2 multiple antiserum RE2116;

Figure 7 is the Coomassie Brilliant Blue staining identification after Ni column purification and electrophoresis of HCV E2 full-length expression product E2 (384-730) m.

A-sample before purification; B-sample after purification;

Figure 8 is the Western blot analysis of the specific binding of the polyantiserum prepared from E2(384-730)m to E2 derived from different recombinant expression systems.

A - E2 expressed by recombinant vaccinia virus vv/E2;

B - E2 expressed by E. coli TG-1/pQE8/E2(385-730)m.

Detailed ways

Example 1 Construction of expression clones containing HCV E2 extracellular domain carboxy-terminal part (567-700)

The cDNA clone pUC19/E1E2 or pTAg/E1E2 containing subtype 1b HCV HC-C2 strain (Wang Y, GenBank Accession No. D13094) was used as the original plasmid (Wang Y et al. JMed Virol 1993 40:254-360). The restriction endonucleases and ligases used in gene recombination are all products of Roche Company or Chinese TaKaRa Company. Plasmid preparation was carried out according to the reagents and methods provided by QIAGEN, Germany. Plasmid recombination and bacterial transformation were carried out according to the routine methods provided by Sambrook J et al. "Molecular Cloning". The recipient bacteria used was Escherichia coli TG-1.

Because the 565th position in the E2 gene originally contained the ApaI restriction recognition site, and the 700th position was the HindIII recognition site on the external carrier, it was first cut with ApaI enzyme and then flattened, then cut with HindIII enzyme, and divided into Extract the fragment containing E2 (567-700), insert it into the transitional plasmid pUC19 after SmaI and HindIII double enzymatic digestion, and construct pUC19/E2 (567-700), then cut back the E2 (567-700) fragment with BamHI and HindIII, insert The same enzymatic hydrolysis of the expression vector, the amino-terminus of the cloning site contains the BamHI and HindIII sites on the pQE8 (QIAGEN company) marked by 6xHis (6xHis), and it is constructed to contain the carboxy-terminal part of the E2 extracellular domain (567-700 ) expression clone pQE8/E2(567-700). (see picture 1).

The expression plasmids have passed the sequence determination (completed by Shanghai Boya Company).

Example 2 The clone pQE8/E2(567-700)m containing a frameshift mutation at the amino-terminus of the carboxy-terminal portion (567-700) of the HCV E2 ectodomain capable of full-length expression in Escherichia coli.

The clone pQE8/E2 (567-700) containing the carboxy-terminal part (567-700) of the HCV E2 ectodomain constructed in Example 1 cannot obtain the expression of the expected full-length E2 (567-700) in Escherichia coli (see Embodiment 5 A) in Fig. 5 . In the selection, pQE8/E2(567-700)m can obtain the expression of the full-length E2(567-700)m in Escherichia coli (see Example 5, B in Figure 5). After sequence determination, it was shown that in pQE8/E2(567-700)m, a C was missing at the first position of the codon encoding the 567th amino acid residue at the amino terminal of E2, and a C was missing at the first position of the codon encoding the 571st amino acid residue. There is an extra G at the 3rd position, but the amino acid sequence after 571 has not been changed, but only a frameshift mutation has occurred in the first 4 amino acid sequences. The original PCNI was replaced by RVTS (see Figure 2). See <210>1 and <210>2 in the sequence listing for the E2 gene sequence containing the frameshift mutation and its corresponding amino acid sequence.

Example 3 Construction of expression clone pQE8/E2(567-730)m of HCV E2 ectodomain carboxy-terminal part (position 567-730) containing frameshift mutation

In order to extend the carboxy-terminus of the carboxy-terminal part of the expressed E2 extracellular domain to the nearly complete 730th position, the expression plasmid pEH containing the HCV E2 carboxy-terminus to the 730th position (Li Yingchun et al., Chinese Science Series C, 1998, 28( 3), 204-210), first use SalI enzymatic digestion to make up, and then use SphI enzymatic digestion to cut out the fragment containing E2 and insert it into the pQE8/E2(567-700)m plasmid described in Example 2. After the enzymolysis, the pQE8/E2(567-730)m expression clone expressing the carboxy-terminal part (567-730)m of the HCV E2 extracellular domain was constructed by filling the expression vector with SphI enzymolysis to remove the fragment. See Figure 3.

Example 4 Construction of nearly complete HCV E2(385-730)m expression clone pQE8/E2(385-730)m containing frameshift mutation

In order to obtain the expression clone of nearly complete HCV E2 (385-730), the plasmid pEH (Li Yingchun et al., Chinese Science Series C, 1998, 28 (3), 204-210) containing complete HCV E2 was used as a template, and introduced The BamHI recognition site forward primer P1 5'-GGGGATCCAACACCTACGTGACGGGG-3' and the reverse primer P2 5'-CTTTTAAGCTTAGGTATGGTGGTG-3' introduced into the HindIII recognition site were used to amplify the HCV E2 (385-730) fragment by PCR technology, and then After digestion with BamHI and HindIII, it was inserted into the expression vector pQE8 after double digestion with BamHI and HindIII, and a pQE8/E2(385-730) expression clone expressing HCV E2(385-730) was constructed (see Figure 4A) .

In addition, using the expression clone pQE8/E2(567-730)m described in Example 3 as a template, use the forward primer P3 5'-GTCGGGCCCCCGTGTAACATCG-3' that introduces the ApaI recognition site and the reverse primer that introduces the NheI recognition site P4 5′-CAAGCTAGCTTGGATTCTCACC-3′, the ApaI-NheI fragment containing the frameshift mutation was amplified by PCR technology, and then inserted into the fragment after the same digestion with ApaI and NheI, and the original ApaI was separated -The pQE8/E2(385-730) expression plasmid behind the NheI fragment was transformed into an expression clone pQE8/E2(385-730)m( See Figure 4B).

Example 5 Expression clone pQE8/E2(567-700) containing the carboxy-terminal part of the extracellular domain of E2 and expression clones pQE8/E2(567-700)m and pQE8/E2(567 -730)m and the expression clone pQE8/E2(385-730) containing nearly complete E2 and the expression clone pQE8/E2(385-730)m containing nearly complete E2 with a frameshift mutation in the extracellular domain and expression products color identification.

TG-1 containing each recombinant plasmid was induced and cultured with a final concentration of 1mM IPTG, and the cells were collected and extracted with PBS buffer (pH8.0) containing 8M urea/20mM mercaptoethanol. After standard Laemmli (Laemmli K et al., Nature 1970; 223:680) SDS-PAGE electrophoresis, the extract was stained with Coomassie Brilliant Blue G-250. The results are shown in Figure 5.

Expression protein of recombinant clone A, E2(567-700); B, E2(567-700)m; C, E2(567-730)m and D, E2(385-730); E, E2(385-730 The molecular weights of )m deduced from the encoded amino acids are 17.8kDa, 17.8kDa, 23.2kDa and 41.6kDa, 41.6kDa, respectively. However, neither the clone pQE8/E2 (567-700) containing the carboxy-terminal part of the ectodomain nor the expression clone pQE8/E2 (385-730) of the complete E2 could obtain the expected full-length expression product in E. coli or could only obtain less than the full-length expression product. The products of long expression are shown in A and D in Figure 5. Only recombinant E2 ectodomain carboxy-terminal expression clones pQE8/E2(567-700)m, pQE8/E2(567-730)m and ectodomain containing frameshift mutations The recombinant expression clone pQE8/E2(385-730)m of the nearly complete E2 code mutation can express the full-length protein in Escherichia coli, see B, C, and E in Figure 5, and have and predict molecular weights of 17.8 kDa, 23.2 kDa and 41.6 kDa corresponded protein expression.

Example 6 The immunoblotting analysis of the antigenicity of HCV E2(385-730)m expressed in full length

The full-length expressed E2 (385-730)m was electrophoresed and then transferred to nitrocellulose membrane (product of Wattman Company). The primary antibody used in Western blot analysis was serum S94 from patients with hepatitis C (Wang Yu, provided by Peking University School of Medicine) ) or polyantiserum RE2116 (Liu Jing et al., Biotechnol Appl Biochem 2001; 34:109-119) prepared from E2 (450-565) protein expressed in Escherichia coli, the secondary antibody was linked with horseradish peroxidase Combined protein A (product of Sigma Company, 1:1000) or horseradish peroxidase-linked conjugate of pig anti-rabbit Ig (product of Dako Company, 1:1000), according to the ECL reagent and method provided by Amersham Company, generate fluorescence And tableting, the results are shown in Figure 6. The results showed that the HCV patient serum containing E2 antibody (Figure 6A) or the multiple antiserum prepared by expressing E2 (Figure 6B) could specifically bind to it, and at the predicted full-length E2 envelope protein molecular weight of 41.6kDa A specific band was produced, which indicated that although E2(385-730)m expressed in E. coli had not been glycosylated, it still had good antigenicity and could be recognized by the E2 antibody in the serum of patients with hepatitis C and expressed by E2 The E2 antibody in the prepared polyantiserum recognizes, and the expressed full-length E2(385-730)m has good antigenicity.

Example 7 Purification of full-length expressed HCV E2(385-730)m using Ni ²⁺ -NTA metal chelate affinity chromatography

The TG-1/pQE8/E2 (385-730)m cultured cells induced by IPTG were collected by centrifugation, suspended in PBS buffer (pH8.0) containing 8M urea/20mM mercaptoethanol for extraction, and the supernatant was collected by centrifugation. Under denaturing conditions, use Ni ²⁺ -NTA agarose gel metal chelate affinity medium (QIAGEN company product) to bind, wash with PBS buffer (pH6.3) containing 8M urea/20mM mercaptoethanol, and then wash with 8M urea /20mM mercaptoethanol in PBS buffer (pH4.3) for elution and collection, after each sample was loaded for electrophoresis, and stained, after electrophoresis with a known amount of bovine serum albumin (BioRad company), scan with optical density, compare and calculate protein quantity. The purification results are shown in Figure 7, where A is the sample before purification. After one-step purification under denaturing conditions, the full-length expressed E2(385-730)m protein accounts for more than 30% of the total expressed protein, and the yield rate is more than 1 mg of purified E2 protein per liter of culture (Fig. 7B ).

Example 8 The purified HCV E2(385-730)m protein was used for enzyme-linked immunoassay (ELISA) of E2 antibody specific binding in HCV patient serum

Utilizing the good antigen performance of the expressed E2(385-730)m and the specific binding characteristics of the E2 antibody in the serum, the purified E2(385-730)m was used to detect the E2 antibody in the clinical serum. Coat each well with 0.15μg E2(385-730)m, incubate at 37°C for 1 hour, overnight at 4°C, use PBS/1% bovine serum albumin BSA (w/v)/2% inactivated newborn bovine serum Sealed, and then carried out according to the HCVEIA 4.0 kit and method provided by UBI company. The serum was diluted 1:20, incubated at 37°C for 1 hour, the secondary antibody was goat anti-human Ig antibody-horseradish peroxidase-linked conjugate provided by UBI, diluted 1:150, reacted at 37°C for 30 minutes, o-phenylenediamine ( OPD) for color development, and the absorbance value was measured at 492nm.

100 healthy hematopoiesis sera (total antibody negative), 158 HCV total antibody positive sera, 20 hepatitis B patient sera and 20 non-A-E hepatitis sera were selected for determination. The results are shown in Table 1. As can be seen from Table 1, the E2 antibody in human serum coated with E2 (385-730) m has good specificity, and 20 hepatitis B patient sera and 20 non-A-E hepatitis patient serum are all Negative. The positive rate of E2 antibody in the total antibody-positive serum of HCV patients was 56%, and 3 of the healthy blood assistants were positive for E2 antibody, which was confirmed to be E2 antibody after Western blot analysis. The results show that the E2 antibody in human serum can be effectively detected by ELISA method by coating with expressed E2(385-730)m.

Table 1 Utilize E2(385-730)m to detect HCV E2 antibody in human serum

                                                                                                              

Positive copies Positive rate ^{Positive copies} positive rate % 0 0% Hepatitis C patients (158) 158 100% 89 56%

Example 9 The polyantiserum prepared from purified HCV E2(385-730)m can specifically recognize E2 envelope proteins from different recombinant expression sources

Select 1-1.5 kg of female New Zealand big-eared rabbits (purchased from Shanghai Experimental Animal Center), and mix the purified E2(385-730)m protein with an equal volume of Freund's complete adjuvant (all adjuvants are products of GibcoBRL Company) Afterwards, inject subcutaneously at the back of the neck, 300 μg for each immunization, and boost once 4 weeks later. The protein used for boosting is mixed with an equal volume of incomplete adjuvant. Finally, the antiserum antibody titer prepared by blood collection reached 3.2×10 ⁴ . Multiple antisera were applied to Western blot analysis, and the results are shown in Figure 8. The E2 antibody in the multiple antiserum can specifically bind to E2 expressed from different sources of recombinant expression, such as HCV envelope protein E2 expressed from recombinant vaccinia virus (Figure 8A) and Escherichia coli (Figure 8B) without saccharification processing. A is the cell lysate of HeLa cells infected with recombinant vaccinia virus containing HCV E2 (384-730) [see Li Yingchun et al. Chinese Science Series C, 1998 28 (3), 204-210)], the recombinant vaccinia virus can be seen in the figure The glycosylated E2 expressed in the system has a molecular weight of about 55kDa. B is the expression product of Escherichia coli TG-1 containing HCV E2(385-730)m. In the figure, a specific band with a molecular weight of 41.6kDa of E2(385-730)m that has not been glycosylated in Escherichia coli can be seen.

SEQUENCE LISTING<110>Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences<120>Hepatitis C virus envelope protein E2 gene and its encoded protein that can be fully expressed in Escherichia coli and its application<160>2 <170>PatentIn version 3.1<210>1<211>1038<212>DNA<213>Hepatitis C Virus (Hepatitis C Virus)<400>1acc tac gtg acg ggg ggg gcg gct gca cgc ggg gcc tcc ggg atc acg 48agc ctc ttt tca cgt ggt ccg tct cag aaa atc cag ctt gtg aac acc      96aat ggc agc cgg cac atc aac ggg act gcc ctg aac tgc aat gac tcc      144ttc aac act ggg ttc ctc gcc gcg ctg ttc tac gcg cac agg ttc aac      192tcg tcc gga tgc cca gag cgc atg gcc agc tgc cgc tcc att gac aag      240ttc gac cag gga cgg ggt ccc atc act tat tat cag ggt gac agc ccg      288gac cag agg cct tat tgc tgg cac tac cca cct cga ccg tgt ggt ata      336gtg ccc gcg tcg gag gtg tgt ggt cca gtg tat tgt ttc acc cca agc      384ccc gtt gtg gtg ggg acg acc gat cgc ctc ggc gtc cct aca tat aac      432tgg ggg gaa aat gag aca gac gtg ctg ctc ctt aac aac acg cgg ccg      480ccg caa ggc aac tgg ttc ggc tgt acg tgg atg aat acc acc ggg ttc      528acc aag acg ttc ggg ggc ccc cgt gta aca tcg ggg ggg gcc ggc aac      576aac acc ttg act tgc ccc acg gac tgc ttc cgg aag cac ccc gag gcc      624act tac aca aaa tgt ggt tcg ggg cct tgg ttg aca cct agg tgc tta      672gtt gac tat cca tac agg ctc tgg cat tac ccc tgc act gtt aac ttc      720acc atc ttt aag gtt agg atg tat gtg ggg ggc gtg gag cac agg ctc      768gac gcc gca tgc aac tgg act cga gga gaa cgt tgc gcc ttg gag gac      816agg gat aga tca gag ctc agc ccg ctg ctg ctg tct aca aca gag tgg      864cag ata ctg ccc tgt tcc ttc acc acc cta ccg gcc ctg tct act ggt      912ttg atc cat ctc cac cgg aac atc gtg gac gtg caa tac CTA TAC GGT 960ATA AGG TCA GCA GCA GTT GTC TCC TTT GCC AAA TGG GAG TAG TAG TAT GTC CTG 1008TT CTT CTT CTC GCA GCA 103 <210> 346 <212> PRT <213> Hepatitis C virus ( Hepatitis C Virus) <400> 2thr Tyr Val THR GLY GLY GLY Ala Ala ARG GLY ALA Serle Thr1 5 10 15SER Leu PHE Serg Gln Lys Ile Gln Leu Val ASN Thrs Thr

20 25 30Asn Gly Ser Arg His Ile Asn Gly Thr Ala Leu Asn Cys Asn Asp Ser

35 40 45Phe Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Ala His Arg Phe Asn

50 55 60r Serg Met Ala Serg SES ARG Serite ASP LYS65 70 75 80phe ARG GLN GLE ThR Tyr Tyr Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

85 90 95Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile

100 105 110Val Pro Ala Ser Glu Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser

115 120 125Pro Val Val Gly Thr Thr Asp Arg Leu Gly Val Pro Thr Tyr Asn

130 135 140TRP GLU ASN GLU Thr ASP Val Leu Leu ASN ThR ARG Pro145 150 155 160 160 160 160 160 PHLN TRP PHE Gs THR THR THR THR THR GLY PHR GLY PHE PHR GLY PHE PHR GLY PHE PHR GLY PHE PHR GLY PHE PHR GLY PHE PHR GLY PHE PHR GLY PHE PHE

165 170 175Thr Lys Thr Phe Gly Gly Pro Arg Val Thr Ser Gly Gly Ala Gly Asn

180 185 190Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala

195 200 205Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu

210 215 220VAL ASP TYR Pro Tyr ARG Leu TRP HIS TYR Pro Cys THR Val Asn PHE2225 230 235 240thr Ile PHE LYS Val Gly GLY GLU HIS ARG Leu

245 250 255Asp Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Ala Leu Glu Asp

260 265 270Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp

275 280 285Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly

290 295 300Leu Ile His Leu His ARG Asn Ile Val ASP Val Gln Tyr Leu Tyr Gly305 315 320ile ARG Ser Ala Val Val Val Val Sera Ile LYS TRU TYR Valr Val Leu

325 330 335 Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg

340 345

Claims (3)

1. A hepatitis C virus envelope protein E2 gene capable of full-length expression in Escherichia coli, the nucleotide sequence of which is <210>1 in the sequence listing. 2. A protein encoded by the E2 gene according to claim 1, whose amino acid sequence is <210>2 in the sequence listing. 3. The application of hepatitis C envelope protein E2 as claimed in claim 2 in the medicine of preparation, diagnosis, prevention and treatment of hepatitis C.

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