野生型和突变型小鼠动力蛋白激活蛋白1真核表达载体的构建及其在小鼠足细胞中的表达

Construction of mouse wide-type and mutant dynactin-1 vectors and their expression in mouse podocytes

目的构建野生型和突变型小鼠动力蛋白激活蛋白1(dynactin-1)真核表达载体,并观察其在小鼠足细胞中表达。方法以总RNA反转录合成的cDNA为模板,通过PCR扩增得到小鼠dynactin-1的全长cDNA,将该片段克隆到真核表达载体pcDNA3.1(+)-FLAG和pEGFP-N1;利用部位特异性突变插入方法构建突变型表达载体,经酶切和测序鉴定;各载体转染小鼠足细胞MPC5后,用蛋白质印迹分析法和免疫荧光显微镜法检测dynactin-1蛋白表达。结果 PCR扩增得到大小为3.8kb的小鼠dynactin-1片段,连接到载体后,酶切鉴定电泳结果分别显示pcDNA3.1(+)-FLAG(5.4kb)、pEGFP-N1(4.7kb)以及小鼠dynactin-1(3.8kb)片段,测序分析结果显示克隆的野生型和突变型小鼠dynactin-1序列与数据库序列相同;蛋白质印迹分析法检测到重组dynactin-1的蛋白条带;免疫荧光显微镜观察到dynactin-1主要表达在小鼠足细胞的细胞质。结论成功构建了野生型和突变型小鼠dynactin-1真核表达载体,并在足细胞中表达,为进一步开展细胞生物学研究奠定了实验基础。

Objective To construct mouse wide-type and mutant dynactin-1 expression vectors and investigate their expression in mouse podoeytes. Methods Mouse eDNA was synthesized from mouse total RNA and was used as a template for PCR amplification to obtain full length dynactin-1 eDNA. The DNA fragment was then cloned into pcDNA3.1（＋ ）-FLAG and pEGFP-N1 vector to produce wide-type dynactin-1 vector. The mutant dynactin-1 was obtained by site-direct mutagenesis kit. All the constructs were verified by restriction enzyme digestion, sequenced, and then transfected into mouse podocyte clone 5 （MPC5）. Western blotting analysis and immunofluorescence microscopy were employed to determine dynactin-1 protein expression. Results The amplified mouse dynactin-1 eDNA fragment was analyzed by agarose gel electrophoresis and a single discrete band of the correct size （3.8 kb） was observed. The vectors containing mouse dynactin-1 were subjected to restriction enzyme digestion and two vector fragments （pcDNA3.1[＋]-FLAG（5.4 kb） and pEGFP-NI[4.7 kb] individually） and the 3.8 kb insert fragment were observed by electrophoresis. The result of sequencing showed that the sequence of cloned dynaetin-1 was identical to that reported in Genbank, Dynactin-1 protein band with the correct relative molecular weight was detected by Western blotting analysis, and immunofluorescenee microscopy showed dynactin-1 protein expression in the cytoplasm of the mouse podocytes. Conclusion We have successfully constructed wide-type and mutant dynactin-1 vectors and expressed them in mouse podocytes, which provides a foundation for future research on dynactin-1.