小鼠FAM3C基因重组腺病毒载体的构建及其在肾系膜细胞中的表达

Construction of recombinant adenovirus Containing FAM3C gene and its effection in mouse glomerlar mesangial cells

目的:利用Ad Easy TMsystem构建携带小鼠FAM3C基因的重组腺病毒,转染至人胚肾细胞系HEK293细胞,检测外源FAM3C在细胞中的表达。方法:取小鼠肾脏提取RNA,创建c DNA文库,用PCR的方法扩增FAM3C基因,将其克隆到p EASY-1载体中,T3连接酶连接。经Bgl II单酶切后,接入p Ad Tra Ck-CMV穿梭载体,T4连接酶连接,构建重组腺病毒的穿梭质粒p Ad Tra Ck-CMV-FAM3C。将经Pme I线性化的p Ad Track-CMFAM3C电穿孔共转化入BJ5183重组细菌,获取重组腺病毒质粒Ad-FAM3C,再将经Pac I线性化的Ad-FAM3C重组病毒骨架质粒转染人胚胎肾细胞系HEK293细胞,包装并扩增病毒。用Ad-FAM3C感染小鼠肾系膜细胞,Western blot法检测FAM3C在小鼠肾系膜细胞中的表达。结果:DNA序列分析和琼脂糖凝胶电泳结果表明,已构建表达FAM3C基因的重组腺病毒,该腺病毒在体外能有效感染小鼠肾系膜细胞,且高表达FAM3C蛋白。结论:成功地构建了携带小鼠FAM3C基因的重组腺病毒,为进一步阐明FAM3C的功能及作用机制奠定实验基础。

[Objective] The study aims to construct a recombinant adenovirus vector containing FAM3 C gene, evaluate the transfection efficiency and identify its function in mouse glomerular mesangial cells(MMcs). [Methods] FAM3 C gene was amplified by PCR from mouse kidney c DNA library, and inserted into p EASY-1 vector using T3 ligase. Single scissored by Bgl II, FAM3 C gene was inserted into p Ad Track-CMV with T4 ligase. Linearized by Pme I and electronically transfected into BJ5183, the Ad-FAM3 C was then packaged by transfecting into HEK293 cells and identified by infecting mouse glomerular mesangial cells(MMCs). [Results] DNA sequencing and agarose gel electrophoresis pictures proved FAM3 C was inserted into the vectors. On the picture of western blot the expression of Ad-FAM3 C was enhanced in MMCs. Pathological sections indicated Ad-FAM3 C increase inflammatory response. [Conclusion]Ad-FAM3 C was successfully constructed.The study laid a foundation for further research of the function of FAM3 C in MMCs.