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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。
Objective To construct the eukaryotic expression plasmid of CAP1 protein and express it in the cell to determine the location of CAP1 protein in cells and its effect on cell migration. Methods cDNA encoding CAP1 was obtained by PCR using HeLa cell cDNA as a template. The cDNA sequence of the coding region was inserted into pCMV-Myc plasmid to construct a recombinant plasmid with the Myc tag. The recombinant plasmids were transfected into 293 cells. Western blot was used to detect the expression in eukaryotic cells. HeLa cells were transfected with the recombinant plasmids. The intracellular localization of the recombinant plasmids was detected by immunofluorescence assay. Results The recombinant plasmid of CAP1 was successfully constructed and successfully expressed in eukaryotic cells. CAP1 was localized in the cytoplasm. Scratch experiments showed that CAP1 overexpression significantly decreased the ability of cell migration. Conclusions CAP1 recombinant plasmid was successfully expressed in eukaryotic cells and CAP1 was located in the cytoplasm. Overexpression of CAP1 inhibited cell migration and laid the experimental foundation for the study of the physiological function of CAP1.