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Biotechnology and Applied Biochemistry (2001) 34, (109–119) (Printed in Great Britain)

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Expression, purification, immunological characterization and application of Escherichia coli-derived hepatitis C virus E2 proteins

Jing Liu*, Lixin Zhu*, Xinxin Zhang*†, Min Lu†, Yuying Kong*, Yuan Wang*¹ and Guangdi Li*

*Institute of Biochemistry and Cell Biology, Shanghai Institutes for Life Sciences, Chinese Academy of Sciences, Shanghai, 200031, People's Republic of China, and †Department of Infectious Diseases, Ruijin Hospital, Second Medical University of Shanghai, Shanghai, 200025, People's Republic of China

Key words: hexahistidine-tagged protein, humoral immune response, patients' sera, vaccine.

Abbreviations used: aa, amino acids; BHK, baby-hamster kidney; CHO, Chinese-hamster ovary; ELISA, enzyme-linked immunoadsorbent assay (EIA is used for the commercial kit); HCV, hepatitis C virus; HRP, horseradish peroxidase; IPTG, isopropyl b-D-thiogalactoside; LB, Luria–Bertani; b-ME, b-mercaptoethanol; MVA, modified vaccinia virus Ankara; NTA, nitrilotriacetate; PNGase F, peptide N-glycosidase F.

¹To whom correspondence should be addressed (e-mail wangyuan@server.shcnc.ac.cn).

The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells. Anti-E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees. Therefore E2 has become a major target for the development of anti-HCV vaccines. Two E2 fragments [amino acids (aa) 450–565 and aa 385–565] derived from a subtype 1b HCV genome were expressed as N-terminally hexahistidine-tagged proteins in Escherichia coli and purified to over 85% purity. Both proteins were specifically recognized by homologous hepatitis-C-patient's serum on Western blotting, suggesting that these E. coli-derived E2 proteins displayed E2-specific antigenicity. E2-116 (aa 450–565) elicited strong antibody responses in BALB/c mice and rabbits. Rabbit antiserum raised against renatured E2-116 (R_E2–116R) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting. E2-181 (aa 385–565) reacted with 40% of anti-HCV⁺ patients' sera in ELISA. R_E2-116R and E2-181 were successfully used in preliminary modified vaccinia virus Ankara- and DNA-based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice. The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.

Received 29 May 2001/3 August 2001; accepted 3 August 2001

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