靶向肝细胞重组逆转录病毒载体的构建

目的：包装5种携带增强型绿色荧光蛋白（EGFP）基因的重组逆转录病毒，筛选肝细胞靶向性好的病毒载体。方法：在前期构建的融合表达质粒基础上，构建4种新的表达质粒，采用脂质体法将这5种质粒分别与质粒pL-EGFP共转染已稳定表达Gag-Pol蛋白的NIH3T3包装细胞系中，48h后收获病毒上清，分别感染肝源性HepG2215细胞及非肝源性293T细胞，48 h后提取细胞总RNA，荧光定量PCR比较重组病毒的靶向性。结果：酶切鉴定结果表明4种新的表达质粒构建成功，包装的病毒滴度约为10⁵cfu/ml，以pcDNA3.1(-)-env^m-preS1-S2质粒包装的病毒有较好的肝细胞靶向性。 结论：包装的肝细胞靶向性好的重组逆转录病毒载体，为包装携带HDV核酶的病毒载体以及HDV核酶抑制乙型肝炎病毒复制表达的研究奠定了基础。

Construction of recombinant hepatocytes-targeting retroviral vectors

Objective: To package five recombinant retroviral vectors containing the enhanced green fluorescent protein (EGFP) gene and screen for the best hepatocytes-targeting retroviral vector. Methods: Four additional expression plasmids were constructed based on the previously constructed fusion expression plasmid. Following a simultaneous transfection of five plasmids and plasmid pL-EGFP into NIH3T3 packaging cells respectively, which can stably express gag-pol protein, recombinant retroviral vectors were achieved 48h post-transfection. The virus supernatant was collected and used to infect HepG2215 and 293T cells. The total RNA of 48 h post-infected cells was extracted, and the tropism of the recombinant vectors were compared by real-time PCR. Results: Four additional expression plasmids were successfully constructed by enzyme digest identification. The viruses titer was about 10⁵ cfu/ml, and the retrovirus stocks packaging with the plasmid pcDNA3.1(-)-env^m-preS1-S2 had a better hepatocellular tropism. Conclusion: The recombinant retroviral vector which has a better hepatocellular tropism would provide great help for packaging the virus vector carrying the HDV ribozyme and studying the inhibition on HBV replication and expression induced by the HDV ribozyme.