Expression of Hepatitis C Virus E2 Ectodomain in E.coli and Its Application in the Detection of Anti-E2 Antibodies in Human Sera  

作　　者：JingLIU Xin-XinZHANG Shen-YingZHANG MinLU Yu-YingKONG YuanWANG Guang-DiLI 

机构地区：[1]StateKeyLaboratoryofMolecularBiology,InstituteofBiochemistryandCellBiology,ShanghaiInstitutesforBiologicalSciences,theChineseAcademyofSciences,Shanghai200031,China [2]DepartmentofInfectiousDiseases,RuijinHospital,SecondMedicalUniversityofShanghai,Shanghai200025,China

出　　处：《Acta Biochimica et Biophysica Sinica》2004年第1期57-63,共7页生物化学与生物物理学报（英文版）

基　　金：This work was supported by a grant from the National High Technology Research and Development Program of China (863 Program) (No. 2001AA215171)

摘　　要：The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previously reported C-terminally truncated E2 fragment (aa 385-565). Association between anti-E2 antibodies in patient sera and HCV RNA status was also demonstrated using this E. coli-derived protein. E2N730(m) might serve as an inexpensive alternative to mammalian cell-expressed E2 proteins in clinical and research applications.The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previously reported C-terminally truncated E2 fragment (aa 385-565). Association between anti-E2 antibodies in patient sera and HCV RNA status was also demonstrated using this E. coli-derived protein. E2N730(m) might serve as an inexpensive alternative to mammalian cell-expressed E2 proteins in clinical and research applications.

关 键 词：hepatitis C virus envelope protein E2 expression and purification Escherichia coll 

分 类 号：R512.6[医药卫生—内科学]