CAJ | 학술논문

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' CMV△8.2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H CVpp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≥1:40. 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.
목적 제비6충불동기인형HCV대표주완정포막당단백E1E2적가병독과립(HCVPP),건립검측HCV감염자중화항체적방법. 방법 장6충포막질립분별여만병독포장질립pHR' CMV△8.2급휴대록색형광단백보고기인적자멸전이질립pCS-CG공전염293T세포,산생6충불동기인형적HCVpp;수집상청액중HCVpp병여환자혈청부육,가입도huh-7세포중,류식세포술검측중화항체대HCVpp감염huh-7적억제작용,이차항체적정방법완성중화항체체외검측.결과 제비적6충H CVpp균가재체외감염Huh-7세포;5례HCV휴대자(병독RNA정량위음성、항HCV항체위양성적환자)적혈청대2충HCVpp(1b、2a형)적중화적도≥1∶40;2례이치유적병형간염환자대1충HCVpp(1b형)적중화적도≥1:40,기중1례적중화적도위1:200;184례만성병형간염환자적건강배우대4충HCVpp (3a、4、5、6형)적중화적도≥1:40. 결론 성공제비6충불동기인형적HCVpp,건립료기우HCVpp적중화항체검측방법,위연구항병독약물적체외사선제공료유효모형.
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 (lb,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 cotransfecting 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 supematant 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 (1b and 2a) produced neutralizing antibody titers ＞ 1:40.For cured HCV patients,only the lb genotype produced neutralizing antibody titers ＞ 1:40.One patient showed titer of 1:200 for genotype 4.A healthy spouse of a chronic hepatitis C patient showed titers＞ 1:40 for four genotypes ofHCVpp (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.