摘要
目的构建表达HCV核心区的重组慢病毒,以探讨HCV感染后该病毒核心蛋白对肝脏干细胞转分化的影响。方法利用PCR体系延伸互为模板的引物,通过T载体克隆法获得HCV core-Ant基因克隆,酶切后连入pLenti6/V5-D-TOPO质粒,得到pLenti6/V5-D-HCV core-Ant,连同pLP/VSVG、pLP1和pLP2四质粒共转染293ET细胞,获得了表达HCV core-Ant重组慢病毒。收集病毒上清,大量扩增,用免疫组化法检测目的基因在Hela细胞的表达。用类似的方法构建了表达EGFP的重组慢病毒并测定其滴度。结果克隆出HCV core-Ant基因,经酶切及测序证实结果正确;使用表达HCV core-Ant重组慢病毒感染Hela细胞,免疫组化实验证实胞浆有HCV core表达。使用表达EGFP的重组慢病毒感染3T3细胞见到绿色荧光,说明构建慢病毒载体有效。结论通过分子克隆体外重组技术成功制备了表达HCV core的重组慢病毒,为进一步研究丙肝病毒致癌机制奠定了基础。
Objective To construct a recombinant lentiviral vector for HCV core-Ant and then study its effect on the transdifferentiation of hepatic stem cells. Methods The HCV core-Ant was obtained by PCR of two primers template with each other and T-vector cloning method, and then subcloned to pLenti6/V5-D-TOPO. The restriction endonuclease and T4 DNA ligase were used to construct the vector, pLenti6/V5-D-HCV core-Ant and the ViraPowerTM PackagingMix （containing three packaging plasmids pLP1, pLP2 and pLP/VSVG） were cotransfected into 293ET cells to produce replication in competent lentivirus after transfection. The viral supernatant on 293T ceils was collected. The expression of the lentiviral vector containing HCV core-Ant in Hela cells was measured by immunohistochemistry. Then we constructed the lentiviral vector containing green flurosccent protein by the similar method, and the titers were determined. Results HCV core-Ant was identified and analyzed by restriction enzyme digestion and sequencing, respectively. The expression of the recombinant lentiviral vector plasmid containing HCV core-Ant in Hela cells was confirmed by immunohistochemistry. The 3T3 ceils transfected the lentiviral vector containing green flurosecent protein were found to show strong expression of GFP, which confirmed that the four-plasmid system of the lentiviral vector was successfully constructed. Conclusion The recombinant lentiviral vector expressing HCV core-Ant was successfully constructed by molecular cloning and recombination techniques in vitro, which will be beneficial to guiding further study on gene therapy of cancer.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2009年第4期415-418,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No .30571648)~~
