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.
出处
《山东大学学报（医学版）》
CAS
北大核心
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)
