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- 2016
人CAP1蛋白真核表达及细胞定位与功能
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Abstract:
摘要：目的 构建CAP1蛋白真核表达质粒并使之在细胞内得以表达，确定CAP1蛋白在细胞中的定位及其对细胞迁移的影响。方法 以HeLa细胞cDNA为模板，经PCR获取CAP1编码区cDNA，将该编码区cDNA序列插入pCMV-Myc质粒中，构建带Myc标签的真核表达重组质粒。重组质粒转染至293细胞中，Western blot方法检测其在真核细胞内的表达；重组质粒转染HeLa细胞，免疫荧光法检测其细胞内的定位，划痕实验观察其对细胞迁移的影响。结果 成功构建了CAP1真核表达重组质粒，并在真核细胞内成功表达，确定CAP1定位于细胞质中，划痕实验证明CAP1过表达的细胞迁移能力明显下降。结论 在真核细胞中成功表达了CAP1重组质粒且CAP1定位于细胞质，其过表达对细胞迁移有抑制作用，为研究CAP1的生理功能奠定实验基础。
ABSTRACT: Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1) and to explore its intracellular location and functions. Methods By using Hela cDNA as the template, the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid. The recombinant plasmid was transfected into 293 cells using lipofectamine 2000. The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence, respectively. Scratch-repair experiment was used to detect the cancer cells’ migration ability. Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells. The recombinant plasmid was successfully expressed in eukaryote cells. CAP1 was located in the cytoplasm. The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration. Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells. CAP1 protein was located in the cytoplasm. The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1
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