痘苗病毒辅助的重组丙型肝炎病毒生产体系中生产细胞的选择

Selection of cell lines for production of recombinant hepatitis C virus using the vaccinia virus expression system

目的:探讨痘苗病毒辅助的重组丙型肝炎病毒制备体系中生产细胞的选择.方法:将已构建好的质粒pT7HCV及pVHCV分别共转染BHK21,HepG2,SMMC7721细胞株,4h后用痘苗病毒感染细胞,96h后用RT-PCR检测HCV的RNA表达,用荧光定量PCR方法检测培养上清中RNA的拷贝数,并用Western blot检测结构蛋白E2以及非结构蛋白NS5的表达.结果:RT-PCR检测到各细胞株培养上清中均有HCV的RNA的存在,定量PCR显示RNA的拷贝数在BHK21,HepG2,SMMC7721细胞中的产量分别为(2.97±0.28)×107,(1.99±0.17)×105,(1.19±0.17)×105(copies/mL).Westernblot检测到在这3种生产细胞中均有HCV非结构蛋白NS5的表达.结论:BHK21,HepG2,SMMC7721细胞株均可生产出重组丙型肝炎病毒颗粒,且以BHK21为生产细胞株时产量最高.

AIM：To compare the yield of recombinant hepatitis C virus （HCV） in different cell lines.METHODS：BHK-21,HepG2 and SMMC7721 cells were co-transfected with recombinant plasmid pT7HCV and pVHCV,respectively,followed by infection with vTF7-3 vaccinia viruses.Ninety-six hours later,the presence of HCV RNA in culture supernatants was detected by reverse transcription-polymerase chain reaction （RT-PCR）.The titers of HCV were measured by fluorescent quantitative PCR （FQ-PCR）.The expression of HCV antigens was detected by West-ern blot RESULTS： HCV RNA was detectable in all culture supernatants. The titers of HCV in BHK-21, HepG2 and SMMC7721 cells were （2.97 ± 0.28） × 10^7, （1.99 ± 0.17） × 10^5, （1.19 ±0.17） × 10^5 （copies/mL）, respectively. The expression of HCV E2, NS3 and NS5 antigens could be detected in all the three cell lines.CONCLUSION： Recombinant HCV can be produced in BHK-21, HepG2 or SMMC7721 cells. The yield of recombinant HCV is highest in BHK-21 cells.