靶向肝细胞重组逆转录病毒载体的构建  

Construction of recombinant hepatocytes-targeting retroviral vectors

作　　者：戚鹏[1] 韩金祥[1] 鲁艳芹[1] 王传玺[1] 

机构地区：[1]山东省医学科学院医药生物技术研究中心,山东济南250062

出　　处：《山东大学学报（医学版）》2007年第3期223-228,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助项目(30371328);山东省自然科学基金资助项目(Q99015)

摘　　要：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,48 h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(-)-envm-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。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 simultaneons transfection of five plasmids and plasmid pL-EGFP into NIH3T3 packaging ceils respectively, which can stably express gag-pol protein, recombinant retroviral vectors were achieved 48 h post-transfection. The vires 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.

关 键 词：肝细胞 逆转录病毒科 逆转录病毒科蛋白质类 DNA突变分析 聚合酶链反应 

分 类 号：Q784[生物学—分子生物学]