Spag6L基因真核表达质粒在细胞内的蛋白表达与定位及启动子转录调节活性研究

发布时间：2018-07-09 16:22

  本文选题：Spag6L基因 + 精子发生　； 参考：《武汉科技大学》2016年硕士论文

【摘要】：目的:构建小鼠Spag6L基因的真核表达质粒,并研究其在哺乳动物细胞内的表达与定位。构建上游启动子荧光素酶报告基因重组质粒,并检测其转录活性。方法:以小鼠的睾丸c DNA基因文库作为模版,PCR扩增Spag6L基因全长序列,测序正确后亚克隆至p EGFP-N2和pc DNA3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。构建上游启动子荧光素酶报告基因重组质粒Spag6L/PGL3,用双荧光素酶法检测其转录活性并与Spag6进行比较。结果:重组质粒Spag6L/pEGFP和Spag6L/pc DNA3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。GFP-Spag6L融合蛋白分子量为82k D,Spag6L蛋白分子质量为56k D。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。Spag6L上游启动子转录调节序列荧光素酶活性明显强于Spag6。结论:构建Spag6L/p EGFP、Spag6L/pc DNA3和Spag6L/PGL3重组质粒成功,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作用奠定了基础。
[Abstract]:Aim: to construct the eukaryotic expression plasmid of mouse Spag6L gene and study its expression and localization in mammalian cells. The recombinant plasmid of luciferase reporter gene of upstream promoter was constructed and its transcriptional activity was detected. Methods: the full-length sequence of Spag6L gene was amplified by PCR using mouse testis c DNA library as template, and subcloned into pEGFP-N2 and pcDNA3 eukaryotic expression vectors and identified by restriction endonuclease digestion. The recombinant expression plasmid was transfected into COS-7 and Cho cells to detect the expression of Spag6L protein by Western-blot. The localization of Spag6L protein in Cho cells was observed by confocal laser scanning microscopy. The upstream promoter luciferase reporter gene recombinant plasmid Spag6L / PGL3 was constructed and its transcriptional activity was detected by double luciferase method and compared with that of Spag6. Results: the recombinant plasmids Spag6L / pEGFP and Spag6L / pcDNA3 were inserted into the 1.5kb fragment. The molecular weight of the recombinant plasmid Spag6L / pEGFP and Spag6L / pcDNA3 was 56kD.Spag6L, which was consistent with that of Spag6L gene. The molecular weight of the fusion protein was 82kD / Spag6L, which was mainly located in the cytoplasm of Cho cells. The luciferase activity of transcriptional regulatory sequence of upstream promoter of microtubule and vesicle was significantly higher than that of Spag6L. Conclusion: the recombinant plasmids Spag6L / p EGFPN Spag6L / pcDNA3 and Spag6L / PGL3 were successfully constructed, which laid a foundation for further study of the relationship between Spag6L gene and Spag6 gene and the role of Spag6L gene in spermatogenesis.
【学位授予单位】：武汉科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R698.2

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