Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera.
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 C730) with a four-amino-acid mutation (aa 568 C571 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 glycoproteins 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 C565). 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.
Liu
Jing
J
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. wangyuan@server.shcnc.ac.cn
Zhang
Xin-Xin
XX
Zhang
Shen-Ying
SY
Lu
Min
M
Kong
Yu-Ying
YY
Wang
Yuan
Y
Li
Guang-Di
GD
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D018937:Hepatitis C Antibodies / Q000097:blood* / Q000276:immunology*; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D018937:Hepatitis C Antibodies; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus