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目的:构建小鼠Spag6L基因真核表达质粒,并研究其在细胞内的表达与定位。方法:以小鼠睾丸cDNA基因文库为模版,PCR扩增Spag6L基因全长序列,测序正确后亚克隆至pEGFP-N2和pcDNA3真核表达载体中并酶切鉴定,将构建成功的重组表达质粒转染至COS-7与CHO细胞中,Western blot法检测COS-7细胞中SPAG6L蛋白表达,激光共聚焦扫描显微镜观察SPAG6L蛋白在CHO细胞内的定位。结果:重组质粒pEGFPSpag6L和pcDNA3-Spag6L经双酶切后产生的1.5kb的目的插入片段,经测序证实与Spag6L基因一致。GFPSPAG6L融合蛋白分子质量为82kU,SPAG6L蛋白分子质量为56kU。SPAG6L蛋白在CHO细胞中主要定位于细胞质,以微管和囊泡表达为主。结论:构建的pEGFP-Spag6L和pcDNA3-Spag6L真核表达质粒成功,为进一步探究Spag6L基因与Spag6基因的关系以及Spag6L基因在精子发生中的功能与作用奠定了基础。
Objective: To construct the eukaryotic expression plasmid of mouse Spag6L gene and study its expression and localization in the cell. Methods: The full-length sequence of Spag6L gene was amplified by PCR using the mouse testis cDNA gene library as a template. The sequence was subcloned into pEGFP-N2 and pcDNA3 eukaryotic expression vector and identified by restriction enzyme digestion. The constructed recombinant plasmid The expression of SPAG6L protein in COS-7 cells was detected by Western blot and the localization of SPAG6L protein in CHO cells by laser scanning confocal microscope. Results: The 1.5kb insertion fragment of recombinant plasmid pEGFPSpag6L and pcDNA3-Spag6L was double digested and confirmed to be identical to the Spag6L gene by sequencing. The molecular weight of GFPSPAG6L fusion protein was 82kU, and the molecular weight of SPAG6L protein was 56kU. SPAG6L protein mainly localized in the cytoplasm of CHO cells, mainly in microtubules and vesicles. CONCLUSION: The eukaryotic expression plasmids pEGFP-Spag6L and pcDNA3-Spag6L are successfully constructed, which lays the foundation for further exploring the relationship between Spag6L gene and Spag6 gene and the function and function of Spag6L gene in spermatogenesis.