小窝蛋白-1重组慢病毒载体的构建及鉴定

Construction and identification of recombinant Caveolin-1 lentivirus vector

目的构建小窝蛋白-1(Cav-1)重组慢病毒载体,并在293T细胞和小鼠中验证Cav-1过表达。方法将Cav-1基因克隆至慢病毒载体GV287,然后利用酶切、PCR扩增鉴定、阳性克隆鉴定及测序验证构建Cav-1重组慢病毒。将Cav-1重组慢病毒转染至293T细胞,通过荧光检测慢病毒转染效果,采用Western blot法检测Cav-1蛋白表达情况,同时转染C57BL6小鼠,免疫组化法检测小鼠肺组织中Cav-1的表达情况。结果经酶切、PCR扩增鉴定以及阳性克隆测序,提示Cav-1重组慢病毒构建正确。荧光检测显示转染Cav-1重组慢病毒后的293T细胞可见强荧光,Western blot法检测结果显示目的基因可表达,小鼠肺组织中Cav-1呈强阳性表达,且实时定量PCR检测Cav-1重组慢病毒滴度为1.3×10^12copies/ml,达到可利用标准。结论采用本研究方法可成功构建Cav-1重组慢病毒载体。

Objective To construct and identify a recombinant lentivirus vector containing high expression of caveolin-1(Cav-1)gene.Methods Cav-1 gene was cloned into lentivirus vector GV287, and then identified by restriction enzyme digestion and PCR. Positive clones were identified and sequenced to construct a recombinant lentivirus with high expression of Cav-1. After Cav-1 recombinant lentivirus was transfected into293 T cells, the effect of lentivirus transfection was detected by fluorescence, the expression of Cav-1 protein was detected by Western blot. Meanwhile,C57 BL6 mice were transfected with Cav-1 recombinant lentivirus, and the expression of Cav-1 in lung tissue of the mice was detected by immunohistochemistry. Results Restriction enzyme digestion, PCR amplification and sequencing of positive clones suggested that Cav-1 recombinant lentivirus was constructed correctly. Fluorescence assay and Western blot both indicated the expression of the target gene in 293 T cells transfected with Cav-1 recombinant lentivirus. Immunihistochemistry showed that Cav-1 was strongly expressed in lung tissue of the mice. The titer of recombinant lentivirus was 1.3×10^12 copies/ml, which was up to the available standard by RT-PCR. Conclusion The recombinant lentivirus vector of Cav-1 has been successfully constructed by this method.