乙型肝炎病毒C基因重组逆转录病毒的构建和在真核细胞的表达

Expression of HBcAg in eukaryotic cells by retroviral vector mediated gene transfer

目的 构建含HBVC基因的重组逆转录病毒以用于慢性乙型肝炎的免疫治疗。方法 用PCR法扩增HBV的C基因片段 ,定向插入逆转录病毒质粒载体pLXSN中 ,重组质粒用脂质体转染包装细胞PT67,G418筛选抗性细胞 ,用抗性细胞培养上清液中的重组病毒感染NIH3T3、EL4和鼠骨髓来源的树突状细胞 (DC) ,用PCR检测目的基因的插入 ,用间接免疫荧光和流式细胞仪检测目的基因的表达 ,基因修饰的DC免疫C57BL 6鼠观察其诱导细胞免疫的能力。结果 含HBVC基因重组质粒载体经PCR、酶切和序列分析鉴定为阳性 ,转染PT67后经G418筛选获得抗性细胞 ,抗性细胞培养上清液中含有重组逆转录病毒 ,病毒效价达 3× 10 5CFU ml,感染NIH3T3和EL4细胞后PCR鉴定含目的基因 ,间接免疫荧光、流式细胞仪鉴定有HBcAg表达 ,基因修饰后的DC免疫C57BL 6鼠能诱导特异性CTL应答。

Objective To construct recombinant retroviral vector expressing HBcAg in eukaryotic cells. Methods The HBV core gene fragment was amplified by using PCR from pADR which contains complement nucleotide sequence of HBV subtype adr and cloned into retroviral expression plasmid pLXSN, then transfected into packing cell (PT67) with lipofec AMINE. After 2 3 weeks selection with G418, large colonies were isolated and transferred to individual plates. Virus containing medium was collected and used to infect NIH3T3, EL4 and mouse bone marrow derived dendritic cells(DC). DNA was extracted from infected cells and tested by PCR. Indirect immunofluorescence and FACS were used to detect the expression of HBcAg. Cell mediated immunity of immunized C 57 BL/6 mice with transduced DC was analyzed. Results The insertion of HBV core gene fragment in the recombinant retroviral plasmid was confirmed by PCR as well as enzyme digestion with EcoR Ⅰ and BamH Ⅰ. The viral titer reached 3×10 5 CFU/ml. The result of PCR showed that the HBV core gene had been integrated into the genome of infected NIH3T3 cells. Indirect immunofluorescence and FACS analysis showed that HBcAg had been expressed in these cells. HBcAg specific CTL responses could be generated in mice immunized with retrovirus transduced DC. Conclusion HBV core gene had been integrated into eukaryotic cells with retroviral vector and target gene had been expressed efficiently. These results may have some impact on gene therapy of chronic hepatitis B .