探讨ARM结构的缺失对SPAG6在真核细胞中定位的影响

发布时间：2018-03-11 20:34

本文选题：男性不育　切入点：SPAG6　出处：《武汉科技大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：构建SPAG6基因全长及六个不同长短ARM序列的真核表达载体pEGFP-N2-SPAG6-△ARM，探讨融合蛋白在细胞内表达及定位。观察全长及六个ARM结构缺失后对真核细胞CHO细胞中SPAG6蛋白定位的影响。方法：1.利用NCBI数据库，寻找小鼠SPAG6蛋白的保守功能区，分析全长蛋白序列，检索出SPAG6有七个ARM区域；2.以健康成年雄性小鼠的睾丸组织为来源，建立小鼠睾丸cDNA文库；3.以小鼠睾丸cDNA文库为模板，PCR扩增SPAG6全长及六个不同长短ARM结构的编码序列；4. TA克隆后测序；5.构建携带不同长短ARM序列的真核表达载体pEGFP-N2-SPAG6-△ARM，对阳性克隆进行酶切和测序鉴定，将构建的重组质粒转染到CHO细胞中；6.分别提取细胞蛋白进行Western blot检测，利用共聚焦激光扫描显微镜观察七个不同SPAG6/GFP融合蛋白在CHO细胞内的定位。结果：酶切和测序鉴定表明，，全长及六个缺失不同长短ARM结构的真核表达质粒构建成功，转染实验发现重组质粒均能够在CHO细胞中表达，但仅全长表达产物定位于微管，缺失任何一个ARM区域都可影响在细胞中的微管定位。结论：SPAG6在真核细胞内的正确定位依赖于全长。该研究为进一步研究SPAG6蛋白的结构与功能奠定基础。
[Abstract]:Aim: to construct the eukaryotic expression vector pEGFP-N2-SPAG6-ARMof SPAG6 gene and six ARM sequences of different length, to investigate the expression and localization of the fusion protein in cells, and to observe the effect of the full length and six ARM deletion on the localization of SPAG6 protein in eukaryotic CHO cells. Methods 1. By using NCBI database, the conserved functional region of mouse SPAG6 protein was found, and the full-length protein sequence was analyzed. The seven ARM regions of SPAG6 were found, which were derived from testicular tissue of healthy adult male mice. The mouse testis cDNA library was established. The mouse testis cDNA library was used as template to amplify the full length of SPAG6 and the coding sequence of six ARM structures of different length and length. The recombinant plasmid pEGFP-N2-SPAG6- was constructed and cloned by TA and sequenced by sequencing 5. The eukaryotic expression vector pEGFP-N2-SPAG6-. The positive clones were digested and sequenced. The recombinant plasmid was transfected into CHO cells. The proteins were extracted for Western blot detection, and the localization of seven different SPAG6/GFP fusion proteins in CHO cells was observed by confocal laser scanning microscope. Results: restriction endonuclease digestion and sequencing showed that the eukaryotic expression plasmids with full length and six different length ARM structures were successfully constructed. The transfection experiments showed that the recombinant plasmids could be expressed in CHO cells, but only the full-length expression products were located in microtubules. The absence of any ARM region can affect the localization of microtubules in cells. Conclusion the correct localization of SPAG6 6 in eukaryotic cells depends on its full length. This study lays a foundation for further study on the structure and function of SPAG6 protein.
【学位授予单位】：武汉科技大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

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