Expression and characterization of hepatitis C Virus E2 glycoprotein fused to hepatitis B virus preS1(21-47) fragment in CHO cells.
To stably express hepatitis C virus (HCV) E2 glycoprotein in CHO cells and facilitate the detection and purification of the expression products, the gene fragment encoding N-terminal 277 amino acids of this protein was fused to the fragment encoding hepatitis B virus (HBV) preS1(21-47) region and inserted into a secretion vector pSecTagB. CHO cells transfected with the recombinant plasmid carrying fusion gene were selected under growth pressure of Zeocin. Secreted fusion products and its cell-associated counterpart were detected by Western blot using E2 specific or preS1 specific antibodies. Glycans carried by the expression products were analyzed with glycan-type specific glycosidases. Most of the cell-associated E2 were found to be high-mannose-type glycosylated, while the secreted E2 proteins were found to be mostly complex-type glycosylated, suggesting further modification in Golgi apparatus upon secretion. Primary studies showed that the fusion antigen could be specifically bind to and elute from anti-preS1 antibody coupled Sepharose resin, suggesting that large-scale preparation of the fusion antigen is feasible with an immunoaffinity resin. This work will contribute to the further study of immunological properties of HCV E2 glycoprotein and also to the study of recombinant HBV/HCV vaccine.
Wang
Chun-Lin
CL
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200231, China. wangyuan@server.shcnc.ac.cn
Zhu
Li-Xin
LX
Liu
Jing
J
Zhang
Zu-Chuan
ZC
Wang
Yuan
Y
Li
Guang-Di
GD
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D006514:Hepatitis B Surface Antigens; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen; C091015:glycoprotein E2, Hepatitis C virus