title: [Eukaryotic expression and application of HCV Hebei strain E2 extracellular core region].
abstract: Objective To express core region of HCV1b (Hebei strain) E2 protein (E2c) by eukaryotic system, and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature, the E2c gene was modified from the HCV1b gene and synthesized via overlapping PCR. Thereafter, the E2c gene including tissue-type plasminogen activator (tPA) signal peptide was cloned into the pCI-neo eukaryotic expression vector, and the product was named pCI-tpa-1bE2c. After HEK293T cells were transfected with pCI-tpa-1bE2c, the supernatant was collected, condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin (GNA)-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2c protein was successfully expressed in HEK293T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1bE2c protein can be effectively expressed in HEK293T cells and applied clinically.
authors.affiliation: Chinese PLA Centre of Diagnosis and Treatment for Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
authors.forename: Chuantao
authors.initials: C
authors.lastname: Ye
authors.affiliation: Chinese PLA Centre of Diagnosis and Treatment for Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
authors.forename: Peiyu
authors.initials: P
authors.lastname: Bian
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Daihui
authors.initials: D
authors.lastname: Weng
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Chinese PLA Centre of Diagnosis and Treatment for Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China. *Corresponding authors, E-mail: jiazsh@fmmu.edu.cn.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Lei
authors.affiliation: Chinese PLA Centre of Diagnosis and Treatment for Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China. *Corresponding authors, E-mail: yflei@fmmu.edu.cn.
authors.forename: Zhansheng
authors.initials: Z
authors.lastname: Jia
mesh_terms: D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006526:Hepatitis C / Q000097:blood / Q000276:immunology* / Q000821:virology; D018937:Hepatitis C Antibodies / Q000097:blood / Q000276:immunology; D054884:Host-Pathogen Interactions / Q000276:immunology; D006801:Humans; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D018937:Hepatitis C Antibodies; D014759:Viral Envelope Proteins