Brief Reports Open Access
Copyright ©The Author(s) 2001. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 15, 2001; 7(1): 98-101
Published online Feb 15, 2001. doi: 10.3748/wjg.v7.i1.98
Immunogenicity of HGV NS5 protein expressed from Sf9 insect cells
Hao Ren, Fen Lu Zhu, Shi Ying Zhu, Yan Bin Song, Zhong Tian Qi, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
Hao Ren, graduated from Shandong Medical University in 1996, got master degree in 1998 from Second Military Medical University, and now is a Ph.D. student, majoring molecular virology.
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No.39825116, 39970394.
Correspondence to: Prof. Zhong Tian Qi, Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
Telephone: 0086-21-25070267 Fax: 0086-21-25070265
Received: July 26, 2000
Revised: September 22, 2000
Accepted: September 29, 2000
Published online: February 15, 2001

Key Words: hepatitis agents, GB/immunology, recombinant proteins, electrophoresis, polyacrylamide gel, flaviviridae infections, Blotting, western, insect vectors, polymerase chain reaction


Although reliable assays for the detection of hepatitis C virus and E virus became available, still 10%-20% hepatitis are not caused by hepatitisA-E virus[1-3]. In 1996, two research groups isolated this agent independently and almost simultaneously and named hepatitis G virus and GB virus C, respectively[4-7]. The nucleotide and amino acid homologies between GBV-C and hepatitis G virus (HGV) were 85% and 95%[5-7]. Therefore, GBV-C and HGV were considered as two different isolates of the same virus, referred to as HGV in this paper. HGV is a single-strand, positive sense RNA virus with approximately 9.4 kb in length, and classified as a member of Flaviviridae. HGV is mainly transmitted through transfusion and could be responsible for chronic liver infection. HGV RNA has been detected in the serum of intravenous drug users (IVDUs), volunteer and commercial blood donors, and patients with cryptogenic hepatitis[8-10]. Until now, RT-PCR is the most commonly used method for the diagnosis of HGV infection. It is necessary to develop a more convenient antibody detection assay. The baculovirus expression system is of a strong polyhedrin promoter[11], and can carry out many types of postranslation modification for a variety of proteins. Most of the expressed proteins were usually shown to be antigenic, immunological, and functionally similar to their authentic counterparts[12-16]. In this study, we used the baculovirus expression system to express HGV NS5 protein in Sf9 cells, and studied its immunogenecity.


HGV positive sera were collected from HGV RNA positive hemodialyzed patients. The plasmid pFastBac HTa, E.coli DH10 Bac cell, Spodoptera frugiperda (Sf9) cell and recombinant plasmid HGV Iwh6 were prepared previously in this laboratory[17]. The pPROEX HTa, Lipofectin and Grace’s medium were purchased from GIBCO/BRL; and expandTM Long Template PCR System was purchased from Boehringer Mannherm Company. PCR primers were designed according to HGV-Iwh6 and synthesized by Sangon Biotechnology Company. Two restriction enzyme sites Bam H I and Kpn I were added to the 5’ end of sense and antisense primers separately. The primer sequences are sense: 5’-GCGGATCCCTATCGGCTGCTGTAGCTAAG-3’; antisense: 5’-GCGGTACCTTATTGAGCGGCCCTCTTAGC-3’.

Amplification and sequence analysis of HGV NS5 fragment

HGV NS5 fragment was amplified using HGV-Iwh6 clone as the template (PCR condition: predenature 94 °C 2 min, followed by 94 °C 30 s, 60 °C 1 min, 68 °C 2 min, 35 cycles, and extension 10 min before the ending of the reaction). The amplified fragments and pPROEX HTa were digested with Bam H I and Kpn I. Fragment and vector were recovered respectively and ligated by T4 DNA ligase to obtain recombinant plasmid pHTNS5. Sequence analysis was carried out using ABI PRISM 377 DNA sequencer (PE Company) with M13/pUC primer.

Cloning into transposing vector pFastBac HTa

pHTNS5 and transposing vector pFastBac HTa were digested with Bam H I and Kpn I, and were ligated by T4 DNA ligase. The ligation mixture was transformed into DH5α competent cell, the positive colonies were chosen on selecting agar plate (ampicillin 100 μg/mL) and identified with endonuclease digestion to obtain the recombinant plasmid pFHTNS5.

Transposon between pFHTNS5 and bacmid

Plasmid pFHTNS5 was transformed into DH10Bac competent cells containing bacmid with a mini-att Tn7 site and helper plasmid. Following hot-shock at 42 °C for 45 s, the transformation mixture was placed in a shaking incubator at 37 °C for 4 h. Recombinant bacmid was selected on selecting plate agar containing kanamycin 50 μg/mL, gentamicin 7 μg/mL, tetracyline 10 μg/mL, X-gal 200 μg/mL, and IPTG 40 μg/mL after 24 h-48 h incubation at 37 °C.

Transfection of Sf9 cells

Recombinant bacmid was extracted according to the procedure of Bac-to-Bac system. For transfection, Sf9 insect cells were grown to 60%-70% confluence. The recombinant bacmid DNA 2 μg was transfected into insect cells Sf9 with Lipofectin. After 5 d-6 d incubation at 27 °C until the morphology of the cells had obvious changes, Sf9 cells and viral supernatant were harvested respectively.

Expression of recombinant protein in insect cells and SDS-PAGE, Western-blot analysis

Twenty μL viral supernatant harvested from the transfected cells was used to infect fresh insect cells. After 5 d-6 d incubation at 27 °C, the cells were harvested for protein expression analysis. The cells were washed twice with PBS and analyzed by SDS-PAGE according to the standard procedure. Western-blot was performed using HGV RNA positive sera (1:40 dilution).

Amplification of HGV NS5 fragment and sequence analysis

PCR product was analyzed by agrose gel electrophoresis and the length was the same as expected (Figure 1). Sequence analysis showed that the HGV NS5 fragment was cloned into the vector with correct orientation (data not shown).

Figure 1
Figure 1 Analysis of recombinant plasmid by restriction endonuclease digestion. 1. λ DNA/Eco R I + Hind III; 2. PCR product; 3. pHTNS5/Bam H I + Kpn I; 4. pFHTNS5/Bam H I + Kpn I.
Construction of recombinant transposing plasmid pFHTNS5

Figure 2 shows the construction of recombinant transposing plasmid pFHTNS5. Figure 1 shows the analysis of recombinant plasmid on agarose gel by restriction endonuclease digestion which verified that target fragment was correctly cloned into the transposing vector. The results demonstrated a successful construction of recombinant transposing plasmid pFHTNS5.

Figure 2
Figure 2 Construction of recombinant plasmid pFHTNS5.
Screening of recombinant bacmid

After transforming competent cell DH10Bac with transposing plasmid pFHTNS, the recombinant bacmid was screened by colour selection. White clones (lacZ-) were selected as positive recombinant bacmid in a background of blue colonies (lacZ+). The recombinant bacmid was extracted according to the procedures described in the manual of Bac-to-Bac system.

Morphology of transfected or infected Sf9 cells

The morphology of Sf9 cells changed gradually after transfection or infection. The cells became big and round obviously at 4 d-5 d after transfection or infection. Cytopathic effects (CPE) were seen whereas no pathological effects were observed in normal cells (Figure 3).

Figure 3
Figure 3 Morphology of uninfected, transfected and infected Sf9 cells (100 ×). A: Uninfected cells; B: Transfected and infected cells
SDS-PAGE and Western blot analysis of the recombinant protein

Transfected or infected Sf9 cells were harvested and analyzed on 12.5% polyacrylamide gels. Figure 4 shows the result of expressed target HGV NS5 protein with a molecular weight of Mr 41500. Scanning results indicated that the recombinant protein amounted to 11.7% of the total proteins. Western blot results implied that the recombinant protein could react with HGV RNA positive sera (Figure 5).

Figure 4
Figure 4 SDS-PAGE analysis of expressed HGV NS5 protein. 1. Uninfected sf9 cells; 2. sf9 cells infected with recombinant viruses; 3. Protein relative molecular mass standards. Arrow indicates the position of recombinant protein.
Figure 5
Figure 5 Western-blot analysis of recombinant protein HGV NS5. 1. Uninfected sf9 cells; 2. sf9 cells infected with recombinant viruses; 3. Protein relative molecular mass standards.

Although easy and reliable assays for the clinical diagnosis of HBV and HCV infection have been established[18-26], there still existed 10%-20% parenterally and community acquired hepatitis cases of unknown cause[4,5,7]. Transmission and molecular biology of these viruses have been studied thoroughly[27-34]. Clinical studies suggest that some of these may be of viral origin. HGV is a potential aetiological agent for viral hepatitis. As a member of Flaviviridae, HGV is a single-stranded RNA virus with a genome of 9400 bp in length which includes 5’ non-coding region, structural gene region C, E1, E2, non-structural gene region NS2, NS3, NS4, NS5a, NS5b and 3’ non-coding region. The genome contains a single open reading frame (ORF) which encodes a 2900 amino acid polyprotein precursor. Many researches have been carried out since the discovery of HGV, the studies of its antigencity is one of them[17,35-39].HGV NS5B protein functions as RNA-dependent RNA polymerase]. In addition, Pilot-Matias et al[40] also found that C26, C27, C28 (2047-2376 aa) of HGV NS5 gene had potential antigen epitopes. Wang et al[41] reported that two linear epitopes (P22, P6) might exist in HGV NS5 gene. The obtained HGV NS5 recombinant protein will provide important materials for studying its structure and function.

The Bac-to-Bac system was established by Luckow[11] in 1993, and a variety of proteins have been expressed with the control of a strong polyhedin promoter since then. It is based on site-specific transposition (transposon Tn7) of an expression cassette into baculovirus shuttle vector (bacmid) propagated in Escherichia coli. After selection of blue-white clonies, recombinant bacmid DNA was extracted for the transfection of Sf9 cells. Insect cells can identify and run many modifications of post-translation and make the expressed protein close to natural protein.

In this study, HGV NS5 gene fragment amplified by PCR was confirmed by restriction enzyme and sequence analysis, and cloned into baculovirus transposing vector pFastBacHTa. Recombinant bacmid was obtained with site-specific transposition, Sf9 cell was transfected with recombinant bacmid or infected with viral supernatant. On the polyacrylamine gel, an expected protein band was seen at Mr 41500. Western blot found that HGV NS5 recombinant protein could react strongly with HGV RNA positive sera, which implied that recombinant HGV NS5 protein could be used as antigen to detect HGV infection.


Edited by Ma JY

1.  Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989;244:362-364.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 2495]  [Cited by in F6Publishing: 2324]  [Article Influence: 66.4]  [Reference Citation Analysis (0)]
2.  Dawson GJ, Chau KH, Cabal CM, Yarbough PO, Reyes GR, Mushahwar IK. Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides. J Virol Methods. 1992;38:175-186.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 184]  [Cited by in F6Publishing: 191]  [Article Influence: 6.0]  [Reference Citation Analysis (0)]
3.  Schlauder GG, Mushahwar IK. Detection of hepatitis C and E virus by the polymerase chain reaction. J Virol Methods. 1994;47:243-253.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 12]  [Cited by in F6Publishing: 12]  [Article Influence: 0.4]  [Reference Citation Analysis (0)]
4.  Simons JN, Pilot-Matias TJ, Leary TP, Dawson GJ, Desai SM, Schlauder GG, Muerhoff AS, Erker JC, Buijk SL, Chalmers ML. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci USA. 1995;92:3401-3405.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 378]  [Cited by in F6Publishing: 348]  [Article Influence: 12.0]  [Reference Citation Analysis (0)]
5.  Leary TP, Muerhoff AS, Simons JN, Pilot-Matias TJ, Erker JC, Chalmers ML, Schlauder GG, Dawson GJ, Desai SM, Mushahwar IK. Sequence and genomic organization of GBV-C: a novel member of the flaviviridae associated with human non-A-E hepatitis. J Med Virol. 1996;48:60-67.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in F6Publishing: 10]  [Reference Citation Analysis (0)]
6.  Linnen J, Wages J Jr, Zhang-Keck ZY, Fry KE, Krawczynski KZ, Alter H, Koonin E, Gallagher M, Alter M, Hadziyannis S. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science. 1996;271:505-508.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 957]  [Cited by in F6Publishing: 882]  [Article Influence: 31.5]  [Reference Citation Analysis (0)]
7.  Simons JN, Leary TP, Dawson GJ, Pilot-Matias TJ, Muerhoff AS, Schlauder GG, Desai SM, Mushahwar IK. Isolation of novel virus-like sequences associated with human hepatitis. Nat Med. 1995;1:564-569.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 799]  [Cited by in F6Publishing: 751]  [Article Influence: 25.9]  [Reference Citation Analysis (0)]
8.  Zhuang H. Study of hepatitis G virus infection in our country. Zhonghua Ganzangbing Zazhi. 1998;6:3-4.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  Chang JH, Wei L, Du SC, Wang H, Sun Y, Tao QM. Hepatitis G virus infection in patients with chronic non-A-E hepatitis. China Natl J New Gastroenterol. 1997;3:143-146.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  Wang XT, Zhuang H, Song HB, Li HM, Zhang HY, Yu Y. Partial sequencing of 5' noncoding region of 7 HGV strains isolated from different areas of China. World J Gastroenterol. 1999;5:432-434.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Luckow VA, Lee SC, Barry GF, Olins PO. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol. 1993;67:4566-4579.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  Deng XZ, Diao ZY, He L, Qiao RL, Zhang LY. HBeAg gene expression with baculovirus vector in silk worm cells. World J Gastroenterol. 1999;5:167-171.  [PubMed]  [DOI]  [Cited in This Article: ]
13.  Bae JE, Toth TE. Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants. Vet Microbiol. 2000;75:199-204.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 7]  [Cited by in F6Publishing: 8]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
14.  Nermut MV, Hockley DJ, Bron P, Thomas D, Zhang WH, Jones IM. Further evidence for hexagonal organization of HIV gag protein in prebudding assemblies and immature virus-like particles. J Struct Biol. 1998;123:143-149.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 55]  [Cited by in F6Publishing: 55]  [Article Influence: 2.1]  [Reference Citation Analysis (0)]
15.  Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T. Expression and self-assembly of empty virus-like particles of hepatitis E virus. J Virol. 1997;71:7207-7213.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Conner ME, Zarley CD, Hu B, Parsons S, Drabinski D, Greiner S, Smith R, Jiang B, Corsaro B, Barniak V. Virus-like particles as a rotavirus subunit vaccine. J Infect Dis. 1996;174 Suppl 1:S88-S92.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 97]  [Cited by in F6Publishing: 97]  [Article Influence: 3.5]  [Reference Citation Analysis (0)]
17.  Zhu FL, Qi ZT, Ren H, Song YB, Shao L. Splicing and cloning of the full-length genomic cDNA of GBV virus C/hepatitis G virus. Dier Junyi DaxueXuebao. 1998;19:301-306.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Song CH, Wu MY, Wang XL, Dong Q, Tang RH, Fan XL. Correlation between HDV infection and HBV serum markers. China Natl J New Gastroenterol. 1996;2:230-231.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Zhou P, Cai Q, Chen YC, Zhang MS, Guan J, Li XJ. Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C. China Natl J New Gastroenterol. 1997;3:108-110.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Assy N, Minuk G. A comparison between previous and present histologic assessments of chronic hepatitis C viral infections in humans. World J Gastroenterol. 1999;5:107-110.  [PubMed]  [DOI]  [Cited in This Article: ]
21.  Yu SJ. A comparative study on the proliferating activity between HBV-related and HCV-related small HCC. China Natl J New Gastroenterol. 1997;3:236-237.  [PubMed]  [DOI]  [Cited in This Article: ]
22.  Zhang LF, Peng WW, Yao JL, Tang YH. Immunohistochemical detection of HCV infection in patients with hepatocellular carcinoma and other liver diseases. World J Gastroenterol. 1998;4:64-65.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Zheng Z, Yang SW, Xiao W, Sun P, Li XJ, Hu YQ. Evaluation of the HBV in HBsAg by HBV DNA measured with PCR. World J Gastroenterol. 1998;4:77.  [PubMed]  [DOI]  [Cited in This Article: ]
24.  Tang RX, Gao FG, Zeng LY, Wang YW, Wang YL. Detection of HBV DNA and its existence status in liver tissues and peripheral blood lymphocytes from chronic hepatitis B patients. World J Gastroenterol. 1999;5:359-361.  [PubMed]  [DOI]  [Cited in This Article: ]
25.  Chen K, Han BG, Ma XK, Zhang HQ, Meng L, Wang GH, Xia F, Song XG, Ling SG. Establishment and preliminery use of hepatitis B virus preS1/2 antigen assay. World J Gastroenterol. 1999;5:550-552.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Gao JE, Tao QM, Guo JP, Ji HP, Lang ZW, Ji Y, Feng BF. Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins. China Natl J New Gastroenterol. 1997;3:114-116.  [PubMed]  [DOI]  [Cited in This Article: ]
27.  Zhu FL, Lu HY, Li Z, Qi ZT. Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli. World J Gastroenterol. 1998;4:165-168.  [PubMed]  [DOI]  [Cited in This Article: ]
28.  Sun DG, Liu CY, Meng ZD, Sun YD, Wang SC, Yang YQ, Liang ZL, Zhuang H. A prospective study of vertical transmission of hepatitis C virus. China Natl J New Gastroenterol. 1997;3:111-113.  [PubMed]  [DOI]  [Cited in This Article: ]
29.  Zhang SZ, Liang JJ, Qi ZT, Hu YP. Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells. World J Gastroenterol. 1999;5:125-127.  [PubMed]  [DOI]  [Cited in This Article: ]
30.  Wei L, Wang Y, Chen HS, Tao QM. Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing. China Natl J New Gastroenterol. 1997;3:12-15.  [PubMed]  [DOI]  [Cited in This Article: ]
31.  Zhang SL, Han XB, Yue YF. Relationship between HBV viremia level of pregnant women and intrauterine infection: neated PCR for detection of HBV DNA. World J Gastroenterol. 1998;4:61-63.  [PubMed]  [DOI]  [Cited in This Article: ]
32.  Jiang RL, Lu QS, Luo KX. Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3. World J Gastroenterol. 2000;6:220-222.  [PubMed]  [DOI]  [Cited in This Article: ]
33.  Dai YM, Shou ZP, Ni CR, Wang NJ, Zhang SP. Localization of HCV RNA and capsid protein in human hepatocellular carcinoma. World J Gastroenterol. 2000;6:136-137.  [PubMed]  [DOI]  [Cited in This Article: ]
34.  Zhao LS, Qin S, Zhou TY, Tang H, Liu L, Lei BJ. DNA-based vaccination induces humoral and cellular immune responses against hepatitis B virus surface antigen in mice without activation of C-myc. World J Gastroenterol. 2000;6:239-243.  [PubMed]  [DOI]  [Cited in This Article: ]
35.  Wang XT, Zhuang H, Song HB, Li HM, Zhang HY, Yu Y. Partial sequencing of 5' noncoding region of 7 HGV strains isolated from different areas of China. World J Gastroenterol. 1999;5:432-434.  [PubMed]  [DOI]  [Cited in This Article: ]
36.  Zhu FL, Qi ZT, Zhu SY, Ren H, Shao L. A Preliminary Study on Macaca Mulatta Infected by Intrahepatic Injection of GHV-C/HGV RNA Transcripts produced in vitro. Bingdu Xuebao. 2000;16:176-178.  [PubMed]  [DOI]  [Cited in This Article: ]
37.  Zhu FL, Ren H, Zhu SY, Song YB, Qi ZT. High-level Expression of GBV-C/HGV NS3 protein in Sf9 Insect cells Using Baca-to-2Bac Vectors. Bingdu Xuebao. 2000;16:273-275.  [PubMed]  [DOI]  [Cited in This Article: ]
38.  Zhu FL, Ren H, Song YB, Qi ZT. Expression of GBV-C/HGV NS3 protein in Escherichia coli. Zhonghua Weishengwuxue He Mianyixue Zazhi. 1999;19:475-478.  [PubMed]  [DOI]  [Cited in This Article: ]
39.  Zhu SY, Pan W, Qi ZT. Expression of E2 glycoprotein gene of hepatitis G virus in insect cells. Zhongguo Bingduxue. 1999;14:135-139.  [PubMed]  [DOI]  [Cited in This Article: ]
40.  Pilot-Matias TJ, Muerhoff AS, Simons JN, Leary TP, Buijk SL, Chalmers ML, Erker JC, Dawson GJ, Desai SM, Mushahwar IK. Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C. J Med Virol. 1996;48:329-338.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in F6Publishing: 1]  [Reference Citation Analysis (0)]
41.  Wang XT, Zhuang H, Li HM, Zhang HY, Qi ZB, Wang YC, Pan HP, Chen ZY. Evaluation of peptide antigens for detection of antibodies to GBV-C. Zhonghua Weishengwuxue He Mianyixue Zazhi. 1997;17:157-159.  [PubMed]  [DOI]  [Cited in This Article: ]