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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。
Purpose: BC022687 may be a protein that regulates cilia formation. This study constructed BC022687 gene eukaryotic expression vector and to explore the expression and localization of the fusion protein in the cell. Methods: The coding sequence of BC022687 was amplified by PCR from the mouse testis cDNA library and subcloned into pEGFP-C1 eukaryotic expression vector carrying green fluorescent protein (GFP) gene after sequencing. The constructed recombinant plasmids were transfected into CHO cells and cell proteins were extracted for Western blot analysis. The localization of BC022687 / GFP fusion protein in CHO cells was observed by confocal laser scanning microscopy. RESULTS: The cDNA sequence of BC022687 protein was cloned into the eukaryotic expression vector pEGFP-C1. The size of the fragment was 950 bp. Western blot detected a relative molecular mass of about 64 000 expression of the fusion protein. The BC022687 / GFP fusion protein is localized cytoplasm within the cell and expressed in a centrosome, cilia-forming template. Conclusion: The full-length BC022687 eukaryotic expression vector was successfully constructed, which laid the foundation for further study on the function of this protein.