AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHO...AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.展开更多
基金Supported by National High Technology Research and Development Program of China (863 Program), No. 2001AA215171
文摘AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.
