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目的本研究构建精子相关抗原6(SPAG6)基因全长及6个不同长短ARM序列的真核表达载体pEGFPN2-SPAG6-△ARM,并使其在CHO细胞中表达,观察全长及6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7个ARM区域。利用PCR技术扩增出6个缺失不同ARM结构的SPAG6 cDNA,测序后亚克隆至携带绿色荧光蛋白基因的pEGFP-N2真核表达载体中,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,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7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6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在哺乳动物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。
OBJECTIVE: To construct the eukaryotic expression vector pEGFPN2-SPAG6- △ ARM with full-length gene of sperm-associated antigen 6 (SPAG6) and six ARM sequences with different lengths and to express in CHO cells. The full-length and six ARM structures Effect of deletion on localization of SPAG6 protein in eukaryotic CHO cells. Methods The NCBI database was used to search for the conserved functional region of mouse SPAG6 protein. The full-length protein sequence was analyzed. Seven SPAR6 ARM regions were found. Six of the SPAG6 cDNAs with different ARM structures were amplified by PCR and subcloned into the pEGFP-N2 eukaryotic expression vector carrying the green fluorescent protein gene after sequencing. The positive clones were digested and sequenced. The constructed recombinant The plasmids were transfected into CHO cells, and the cell proteins were respectively extracted for Western blot. Confocal laser scanning microscopy was used to observe the localization of seven different SPAG6 / GFP fusion proteins in CHO cells. Results The results of enzyme digestion and sequencing showed that the full-length and 6 eukaryotic expression plasmids with different lengths of ARM were successfully constructed. The transfection experiments showed that the recombinant plasmids could be expressed in CHO cells. However, only the full- , The absence of any one ARM region can affect microtubule localization in the cell. Conclusion The correct localization of SPAG6 in mammalian cells depends on the full length. This study lays the foundation for further study on the structure and function of SPAG6 protein.