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| 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 |
| 王春花1△,李林2△,傅鹏1,李金花1,3,原爱红1*,孙向华4,蒋晓峰1,余晨1* |
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(1. 同济大学附属同济医院肾内科,上海 200065;
2. 第二军医大学长征医院肾内科,上海 200003;
3. 江西省九江市中医医院肾六科,九江 332000;
4. 同济大学附属同济医院中心实验室,上海 200065
△共同第一作者
*通信作者) |
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| 摘要: |
| 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3.8 kb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(5.4 kb)、pEGFP-N1(4.7 kb)以及小鼠dynactin-1(3.8 kb)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。 |
| 关键词: 动力蛋白激活蛋白1 真核表达载体 足细胞 |
| DOI：10.3724/SP.J.1008.2012.00780 |
| 投稿时间：2012-05-29修订日期：2012-06-25 |
| 基金项目:上海市自然科学基金 (09ZR1434800). |
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| Construction of mouse wide-type and mutant dynactin-1 vectors and their expression in mouse podocytes |
| WANG Chun-hua1△, LI Lin2△, FU Peng1, LI Jin-hua1,3, YUAN Ai-hong1*, SUN Xiang-hua4, JIANG Xiao-feng1, YU Chen1* |
(1. Department of Nephrology, Tongji Hospital, Tongji University, Shanghai 200065, China;
2. Department of Nephrology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China;
3. The 6th Division of Nephrology, Jiujiang TCM Hospital, Jiujiang 332000, Jiangxi, China;
4. Department of Clinical Laboratory, Tongji Hospital, Tongji University, Shanghai 200065, China
△Co-first authors.
*Corresponding authors.) |
| Abstract: |
| ObjectiveTo construct mouse wide-type and mutant dynactin-1 expression vectors and investigate their expression in mouse podocytes. MethodsMouse cDNA was synthesized from mouse total RNA and was used as a template for PCR amplification to obtain full length dynactin-1 cDNA. 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. ResultsThe amplified mouse dynactin-1 cDNA 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-N1\[4.7 kb\] individually) and the 3.8 kb insert fragment were observed by electrophoresis. The result of sequencing showed that the sequence of cloned dynactin-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 immunofluorescence microscopy showed dynactin-1 protein expression in the cytoplasm of the mouse podocytes. ConclusionWe 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. |
| Key words: ConclusionWe 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. |
