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目的:构建hCAP真核表达载体并检测融合蛋白在细胞内的表达及定位。方法:提取工具细胞CV-1的mRNA,反转录为cDNA。PCR扩增hCAP基因的cDNA全长,并将其克隆至pEGFP-C1表达载体中。进一步将构建的重组载体进行酶切和测序鉴定,并转染到工具细胞COS-7中,提取细胞蛋白进行Western blot。最后利用激光共聚焦显微镜观察pEGFP-hCAP在NIH3T3成纤维细胞内的定位。结果:hCAP基因cDNA全长克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段为3 879 bp,测序证实成功。Western blot检测GFP-hCAP融合蛋白表达,相对分子质量(Mr)约为169 000。pEG-FP-hCAP在NIH3T3细胞中主要定位于细胞周边。结论:成功构建了真核表达载体pEGFP-hCAP,融合蛋白在NIH3T3细胞中主要定位于细胞周边。
Objective: To construct eukaryotic expression vector of hCAP and to detect the expression and localization of the fusion protein in the cell. Methods: The mRNA of CV-1 was extracted from the tool cells and reverse transcribed into cDNA. The full-length cDNA of hCAP gene was amplified by PCR and cloned into pEGFP-C1 expression vector. The constructed recombinant vector was further digested with restriction endonucleases and sequenced. The recombinant vector was transfected into COS-7, which is a tool for cell culture. Finally, the localization of pEGFP-hCAP in NIH3T3 fibroblasts was observed by laser confocal microscopy. Results: The full-length cDNA of hCAP gene was cloned into the eukaryotic expression vector pEGFP-C1. The fragment was 3 879 bp. The sequencing proved successful. The expression of GFP-hCAP fusion protein was detected by Western blot. The relative molecular mass (Mr) was about 169,000. pEG-FP-hCAP mainly located in the periphery of NIH3T3 cells. Conclusion: The eukaryotic expression vector pEGFP-hCAP was constructed successfully. The fusion protein mainly located in the periphery of NIH3T3 cells.