三种丙型肝炎包膜感染性假病毒颗粒的制备及初步应用研究  

Development of Infectious Pseudo-particle Harboring Three Subtypes Hepatitis C Virus Glycoproteins and Their Application in Neutralization Assays

作　　者：张柯[1] 谭文杰[1] 邓瑶[1] 李津 吴小兵[1] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]中国天坛生物制品总公司,北京100024

出　　处：《病毒学报》2008年第4期287-294,共8页Chinese Journal of Virology

基　　金：国家863课题2007AA02Z455;自然科学基金30571673;30671961

摘　　要：本研究构建携带HCV代表株H77（1a）、中国HeBei株（1b）以及JFH-1株（2a）丙型肝炎病毒完整的E1-E2包膜糖蛋白基因的表达质粒，间接免疫荧光法及Western blot验证了其在细胞膜（293细胞）上的正确表达。三种包膜质粒分别与慢病毒包装质粒pHR’CMV△8．2及携带EGFP报告基因的自灭活（Self—Inactivating，SIN）转移质粒pCS-CG共转染293FT细胞，产生三种不同基因型的丙型肝炎包膜感染性假型（pseudotyped）病毒颗粒，免疫荧光与Western blot分析验证了E1／E2包膜糖蛋白在假病毒颗粒上的表达与掺人，利用p24 ELISA法及感染性实验对HCV假病毒进行滴定。从上清中获得的HCV假病毒可以在体外感染肝癌细胞系Huh7及Huh7-CD81（且后者上的感染效率约为前者上的2～3倍）；利用针对HCVE2蛋白的具有广谱交叉中和活性的单克隆抗体AP33建立了基于上述假病毒颗粒的丙肝病毒体外中和抗体滴定方法，并应用于丙型肝炎患者体内的中和抗体水平的研究。本研究成功包装了包括中国流行株在内的3种不同基因型的HCV包膜感染性假病毒颗粒并建立了基于假病毒颗粒的HCV体外中和抗体检测方法，为研究HCV感染早期特性及特异抗病毒药物的体外筛选提供了有效模型，并可用于HCV感染者体内及HCV基因工程疫苗免疫后中和抗体水平的分析。In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 （1a）, Hebei （1b） and JFH1 （2a） strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay（IFA） and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV El-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid （pHR＇CMV△R8.2）and a self-inactivated（SIN） transfer plasmid （pCS-CG） containing a reporter EGFP gene to produce infectious HCV pseudo-particles（pp）. Flow cytometry assays showed that the HCVpp could infect Huh7 and HuhT-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25ng/mL p24 or 10^4-10^5 TU （transducing unit）/ ml. An in vitro HCV neutralizing assays based on HCVpp （1a,1b,2a） were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp （1a, 1b,2a subtype） were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection （host tropisms, receptor binding, membrane fusion, et al. ） or screening anti-HCV drugs（such as inhibitor to virus entry）. This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.

关 键 词：丙型肝炎病毒 中和抗体 假病毒 

分 类 号：R373.1[医药卫生—病原生物学]