BC022687基因真核表达质粒的构建及在CHO细胞内的蛋白表达和定位

目的:BC022687可能是调节纤毛形成的蛋白。本研究构建BC022687基因真核表达载体并探讨融合蛋白在细胞内表达及定位。方法:以小鼠睾丸cDNA文库为模板,PCR扩增全长BC022687编码序列,测序后亚克隆至携带绿色荧光蛋白(GFP)基因的pEGFP-C1真核表达载体中。将构建的重组质粒转染到CHO细胞中,提取细胞蛋白进行Western印迹检测。利用共聚焦激光扫描显微镜观察BC022687/GFP融合蛋白在CHO细胞内定位。结果:翻译BC022687蛋白的cDNA序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段大小950 bp。Western印迹检测到相对分子质量约为64 000的融合蛋白表达。BC022687/GFP融合蛋白在细胞内定位以细胞质为主,并在中心体、纤毛形成的模板表达。结论:成功构建BC022687全长基因真核表达载体,为进一步研究该蛋白的功能奠定了基础。

Construction of a recombinant plasmid of BC022687 and identification of its expression and localization in CHO cells

Objective: To construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary(CHO) cells.Methods: The BC022687 coding sequence was amplified by polymerase chain reaction(PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein(GFP).After the target region was sequenced,the recombinant plasmid was transfected into CHO cells,and its expression in the CHO cells was determined by Western blot.The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy.Results: BC022687 was successfully cloned into the mammalian expression vector pEGFP-C1,with the restriction fragment length of 950 bp.The expression of the fusion protein was confirmed,with the relative molecular weight of 64 000.The GFP-tagged BC022687 protein was mainly 1ocalized in the cytoplasm,and also presented in the centrioles in the transfected CHO cells.Conclusion: The successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.