表达hCD4和hCCR5基因的慢病毒载体的构建

发布时间：2018-01-24 16:37

本文关键词： 慢病毒载体 h CD/h CCR 质粒基因表达　出处：《华夏医学》2016年05期 　论文类型：期刊论文

【摘要】：目的:构建一种高效转染h CD4和h CCR5基因的慢病毒载体。方法:应用Gateway技术构建可表达h CD4和h CCR5的质粒载体PLA.Ex Bi.P/puro-CMV-CD4-IRES-CCR5,并进行酶切和测序鉴定,进行慢病毒包装,用q RT-PCR方法检测293T细胞中h CD4/CCR5的m RNA表达。将本慢病毒载体转染于小鼠白血病细胞L615,应用q RT-PCR检测细胞内h CD4/CCR5 m RNA的表达及对HIV-1的易感性。结果:成功构建PLA.Ex Bi.P/puro-CMV-CD4-IRES-CCR5质粒表达载体,包装成的慢病毒载体在293T细胞中具有h CD4/CCR5 m RNA水平的高表达(P0.05);转染于L615细胞亦表现出h CD4/CCR5 m RNA水平的高表达(P0.05),并具备了HIV-1入胞感染的特点。结论:成功建立h CD4和h CCR5基因双启动的慢病毒载体,可使目的细胞高效表达h CD4及h CCR5分子,对HIV-1宿主细胞的制备、HIV/AIDS的发病机制和抗病毒研究具有积极的意义。
[Abstract]:Objective: to construct a lentivirus vector with highly efficient transfection of h CD4 and h CCR5 genes. Methods: the expression of h CD4 and h CD4 was constructed by using Gateway technique. Plasmid vector PLA.Ex Bi.P/puro-CMV-CD4-IRES-CCR5 of CCR5. The lentivirus was packaged by enzyme digestion and sequencing. The expression of h CD4/CCR5 m RNA in 293T cells was detected by Q RT-PCR method. The lentivirus vector was transfected into murine leukemia cell line L615. Q RT-PCR was used to detect the expression of h CD4/CCR5 m RNA and its susceptibility to HIV-1. Results: PLA.Ex was successfully constructed. Bi.P/puro-CMV-CD4-IRES-CCR5 plasmid expression vector. The lentivirus vector was highly expressed in 293T cells at the level of h CD4/CCR5 m RNA. The overexpression of h CD4/CCR5 m RNA was also observed in L615 cells. Conclusion: the lentivirus vector of h CD4 and h CCR5 gene was successfully established. It can effectively express h CD4 and h CCR5 molecules in target cells, which has positive significance for the preparation of HIV-1 host cells and the study on the pathogenesis and antiviral effect of HIV / AIDS.
【作者单位】：桂林医学院生物技术学院;中山大学生命科学院;广州中医药大学热带医学研究所;桂林医学院广西肝脏损伤与修复分子医学重点实验室;
【基金】：国家自然科学基金资助项目(81660755;81573861)
【分类号】：Q78
【正文快照】： 2作者简介:李雅婧(1993-),女,山西临汾人,桂林医学院2014级病理学与病理生理学专业硕士研究生,主要从事病原生物学研究。慢病毒是一类逆转录病毒,包括人免疫缺陷病毒(HIV)、猿免疫缺陷病毒(simian immunodeficiencyvirus,SIV)和猫免疫缺陷病毒(feline immunode ficiencyvirus,

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