6种丙型肝炎假病毒颗粒的制备及其中和抗体初步筛检  

Generation of six genotypes of infectious HCV pseudo-particles and detection of neutralizing antibodies in HCV patients

作　　者：杨晓飞[1] 潘蕾[1] 王宇[1] 马力[1] 张颖[1] 周云[1] 郝春秋[1] 马志远[1] 贾战生[1] 

机构地区：[1]第四军医大学唐都医院感染科,西安710038

出　　处：《中华肝脏病杂志》2013年第12期903-906,共4页Chinese Journal of Hepatology

摘　　要：目的制备6种不同基因型HCV代表株完整包膜糖蛋白E1E2的假病毒颗粒（HCVPP），建立检测HCV感染者中和抗体的方法。方法将6种包膜质粒分别与慢病毒包装质粒pHR’CMVA8．2及携带绿色荧光蛋白报告基因的自灭转移质粒pCS-CG共转染293T细胞，产生6种不同基因型的HCVpp；收集上清液中HCVpp并与患者血清孵育，加入到huh-7细胞中，流式细胞术检测中和抗体对HCVpp感染huh-7的抑制作用，以此抗体滴定方法完成中和抗体体外检测。结果制备的6种HCVpp均可在体外感染Huh-7细胞；5例HCV携带者（病毒RNA定量为阴性、抗HCV抗体为阳性的患者）的血清对2种HCVpp（1b、2a型）的中和滴度≥1：40；2例已治愈的丙型肝炎患者对1种HCVpp（1b型）的中和滴度≥1：40，其中1例的中和滴度为1：200；184例慢性丙型肝炎患者的健康配偶对4种HCVpp（3a、4、5、6型）的中和滴度≥l：40。结论成功制备6种不同基因型的HCVpp，建立了基于HCVpp的中和抗体检测方法，为研究抗病毒药物的体外筛选提供了有效模型。Objective To generate hepatitis C virus pseudo-particles （HCVpp） containing the complete E1-E2 envelope glycoprotein, in order to establish a HCVpp database covering the six major genotypes ofHCV （Ib, 2a, 3b, 4, 5, and 6） and to develop a simple and effective method for detection of neutralizing antibodies in HCV patients. Methods HCVpp were generated for the six genotypes by co- transfecting 293T cells with a plasmid expressing the respective El-E2 （p HR, CMVA 8.2 construct） and a MLV-GFP plasmid. Titration of each HCVpp was carried out by p24 ELISA. Infectivity of each HCVpp was assessed by mixing the harvested supernatant of producer cells with sera from HCV patients, adding the mixture to Huh-7 cells, and detecting the subsequent titers of neutralizing antibodies against HCVpp. Results All six types of HCVpp were able to infect Huh-7 cells in vitro. For healthy HCV carriers, only two genotypes of HCVpp （ 1 b and 2a） produced neutralizing antibody titers 〉 1：40. For cured HCV patients, only the lb genotype produced neulralizing antibody titers 〉 1：40. One patient showed titer of 1：200 for genotype 4. A healthy spouse of a chronic hepatitis C patient showed liters 〉 1：40 for four genotypes of HCVpp （3a, 4, 5, 6）. Conclusion We generated six different genotypes of HCVpp successfully, established the in vitro neutralizing antibody detection method, and provided an effective model for screening antiviral drugs.

关 键 词：肝炎病毒  丙型 抗体  中和 假病毒颗粒 

分 类 号：R512.63[医药卫生—内科学]