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目的构建丙型肝炎病毒(HCV)全基因重组质粒,并建立其转染细胞系。方法双酶切质粒JFH1,回收HCVDNA,分别与真核表达载体pIRESneo和逆转录病毒载体pLNCX连接,构建重组质粒pIRESneo-HCV和pLNCX-HCV。将重组质粒pIRESneo-HCV转染BHK细胞,pLNCX-HCV转染pA317细胞,通过PCR、间接ELISA和免疫荧光试验鉴定转染细胞。结果重组质粒PCR和酶切鉴定证明构建正确。转染细胞上清经PCR检测均可见目的基因条带;ELISA结果表明,转染细胞冻融上清均可与抗HCV小鼠血清发生强的结合反应;转染细胞均可与抗HCV小鼠血清反应,产生荧光,但荧光不多且斑点不亮。结论已成功构建HCV全基因重组质粒,并建立了可表达HCV蛋白的转染细胞系,为转基因动物模型的建立奠定了基础。
Objective To construct a recombinant plasmid of hepatitis C virus (HCV) and establish its transfected cell line. Methods The recombinant plasmid pIRESneo-HCV and pLNCX-HCV were constructed by double enzyme digestion of plasmid JFH1 and recovery of HCVDNA. The recombinant plasmids pIRESneo and pLNCX were respectively ligated with the eukaryotic expression vector pIRESneo and the retroviral vector pLNCX-HCV. The recombinant plasmid pIRESneo-HCV was transfected into BHK cells and pLNCX-HCV was transfected into pA317 cells. The transfected cells were identified by PCR, indirect ELISA and immunofluorescence assay. Results The recombinant plasmid PCR and restriction enzyme digestion proved that the construction was correct. The supernatant of the transfected cells could be detected by PCR. The results of ELISA showed that the frozen-thawed supernatants of the transfected cells could strongly bind with the anti-HCV mouse serum. The transfected cells could be incubated with anti-HCV mouse Serum reaction, produce fluorescence, but not much fluorescence and the spot is not bright. Conclusion The HCV genome-wide plasmids were successfully constructed and transfected cell lines expressing HCV protein were established, which laid the foundation for the establishment of transgenic animal model.