表达HCVcoreAnt的重组慢病毒的构建

1、表达HCV coreAnt的重组慢病毒的构建         11-01-07 13:13:00     作者：马列婷，李砚，王亚文    编辑：studa20【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 coreAnt基因克隆，酶切后连入pLenti6/V5DTOPO质粒，得到pLenti6/

2、V5DHCV coreAnt，连同pLP/VSVG、pLP1和pLP2四质粒共转染293ET细胞,获得了表达HCV coreAnt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 coreAnt基因，经酶切及测序证实结果正确;使用表达HCV coreAnt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

3、究丙肝病毒致癌机制奠定了基础。 【关键词】  丙型肝炎病毒核心蛋白;重组慢病毒;分子克隆ABSTRACT: Objective To construct a recombinant lentiviral vector for HCV coreAnt and then study its effect on the transdifferentiation of hepatic stem cells. Methods The HCV coreAnt was obtained by PCR of two primers template with each other and Tvect

4、or cloning method, and then subcloned to pLenti6/V5DTOPO. The restriction endonuclease and T4 DNA ligase were used to construct the vector. pLenti6/V5DHCV coreAnt and the ViraPowerTM PackagingMix (containing three packaging plasmids pLP1, pLP2 and pLP/VSVG) were cotransfected into 293ET cells to pro

5、duce replication in competent lentivirus after transfection. The viral supernatant on 293T cells was collected. The expression of the lentiviral vector containing HCV coreAnt in Hela cells was measured by immunohistochemistry. Then we constructed the lentiviral vector containing green flurosecent pr

6、otein by the similar method, and the titers were determined. Results HCV coreAnt was identified and analyzed by restriction enzyme digestion and sequencing, respectively. The expression of the recombinant lentiviral vector plasmid containing HCV coreAnt in Hela cells was confirmed by immunohistochem

7、istry. The 3T3 cells transfected the lentiviral vector containing green flurosecent protein were found to show strong expression of GFP, which confirmed that the fourplasmid system of the lentiviral vector was successfully constructed. Conclusion The recombinant lentiviral vector expressing HCV core

8、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.KEY WORDS: HCV core protein; recombinant lentiviral vector; molecular cloning丙型肝炎病毒(hepatitis C virus, HCV)感染大部分转变为持续感染，继而发展为肝硬化和肝细

9、胞癌(hepatocellular carcinoma, HCC)。许多研究表明HCV的核心蛋白具有致癌潜能，且与HCC的发生发展密切相关。本试验通过构建可以表达HCV coreAnt融合蛋白的真核表达载体，试图为进一步研究丙肝病毒产物核心蛋白对肝脏干细胞的转分化作用奠定基础。1 材料与方法1.1 材料 克隆载体pGEMTEasy质粒(50ng/L)，购自Promega公司。限制性内切酶BamH、Himd、Xho、Taq DNA聚合酶、T4 DNA连接酶购自华美生物工程公司。一抗为小鼠抗HCV核心蛋白单克隆抗体，购自北京奥博森公司，HRP羊抗小鼠抗体为北京中杉公司提供。pTE28质粒、pGEM

10、 TAnt载体、pEGMHCV core cDNA克隆质粒、病毒穿梭质粒pLenti6/V5DTOPO、pLP/VSVG、pLP1、pLP2、大肠杆菌DH5及293细胞系，由西安华广生物工程公司提供。1.2 方法1.2.1 构建pGEM THCV coreAnt载体 采用已经构建的HCV core cDNA克隆质粒为模板，使用PCR技术删除终止子，通过T载体克隆法构建pGEMTHCV core cDNA克隆质粒。上游引物：5GGGATCC ATG AGC ACG AAT CCTAA3，下游引物：5GAAGCTT TCA CAC TTG GTA GGC CGA AG3。PCR反应体系：10

11、15;Taq DNA聚合酶Buffer 10L，dNTP(2.5mmol/L)及上、下游引物各1L，Taq DNA聚合酶(2.5u/L)1L，消毒去离子水将总体积加至100L。PCR条件为：94变性60s，42退火60s，72延伸90s，30个循环后，再72延伸5min。经筛选阳性克隆、酶切鉴定及测序后，通过常规分子生物学亚克隆方法，利用已有的pGEM TAnt载体，连接Ant和HCV core cDNA在同一开放阅读框架(open reading fragment，ORF)，构建pGEM THCV coreAnt载体。1.2.2 pTE28 HCV coreAnt载体构建 碱裂解法大量制备重

12、组质粒pGEM THCV coreAnt，用限制性内切酶Xho彻底消化后再用BamH消化，切取HCV coreAnt用T4DNA连接酶连入带有相同末端的已线性化pTE28表达载体，建立表达HCV core和果蝇穿膜肽Ant的融合蛋白表达克隆。用连接反应产物转化制备好的感受态细菌E.coli DH5菌株，随机挑选单个菌落，接种于含有氨苄西林的LB培养基内，37增殖培养后，碱裂解法小量提取质粒。用限制性内切酶EcoR和BamH联合酶切，筛选出重组质粒pTE28 HCV coreAnt。1.2.3 pLenti6/V5DHCV coreAnt的构建 Xho和BamH联合酶切慢病毒穿梭质粒pLenti

13、6/V5DTOPO和pTE28 HCV coreAnt，低熔点凝胶回收HCV coreAnt和线性化的pLenti6/V5DTOPO，T4DNA连接酶连接，转化DH5ct感受态细菌，挑选转化的菌落，提取质粒，Xho和BamH双酶切筛选并鉴定阳性克隆。1.2.4 重组慢病毒的包装 采用磷酸钙沉淀法。pLenti6/V5DHCVcoreAnt同包装质粒pLP1、pLP2和包膜质粒pLP/VSVG四质粒共转染293ET细胞,获得了表达HCV coreAnt重组慢病毒。转染10d后，质粒在细胞内同源重组构建出活重组慢病毒，毒斑出现。反复冻融含病毒栓子的培养基，提取病毒，收集病毒上清。1.2.5 重组蛋白的免疫细胞化学检测 使用表达HCV coreAnt重组慢病毒感染Hela细胞，将细胞接种于铺有盖玻片的6孔板，培养2448h后，取出盖玻片，冷丙酮固定20min。30mL/L H2O2消除细胞内源性过氧化物酶活性。一抗为小鼠抗HCV核心蛋白单克隆抗体，二抗为