ARM结构的缺失对SPAG6在哺乳细胞中定位的影响  

Effect of ARM domain deletion on SPAG6 subcellular localization in mammalian cells

作　　者：汪智琼[1,2] 张玲[1] 石玉琴[1] 李红飞[1] 张志兵[1,3] 

机构地区：[1]武汉科技大学医学院公共卫生学院,武汉430065 [2]华中科技大学同济医学院附属同济医院 [3]Virginia Commonwealth University, Richmond, VA

出　　处：《中国男科学杂志》2015年第1期3-8,共6页Chinese Journal of Andrology

基　　金：国家自然科学基金(81172462)、(81300536)、(81302401);湖北省自然科学基金(2012FFB04904)、(2013CFB331)资助

摘　　要：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pEGFP-N2-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 examine the effect of armadillo （ARM）domains on sperm- associated antigen 6 （SPGA6） localization in CHO cells. Methods The sequences coding different ARM domains were amplified by polymerase chain reaction （PCR）, and were subcloned into the pEGFP-N2 vector. These plasmids were transfected into CHO cells. The expression of the GFP-tagged SPAG6 proteins was proved by Western blot analysis, and localization of GFP-tagged full length and truncated SPAG6 proteins was observed by using laser scanning confocal microscopy. Results Truncated SPAG6 proteins with series deletion of ARM domains were successfully constructed into the mammalian expressing vector carrying the green fluorescent protein gene, pEGFP-N2. The expression of the fusion protein was confirmed by Western blot analysis. The full length GFP-tagged SPAG6 protein was 1ocalized on the microtubule in the transfected CHO cells, but none of the truncated proteins was localized on the microtybule. Conclusion Microtubule localization of mouse SPAG6 protein is dependent on full length sequence. Construction of the plasmids expressing full length and truncated SPAG6 proteins provides an important tool for further study SPAG6 function.

关 键 词：精子相关抗原6基因 ARM结构域 不育 男性 

分 类 号：R698.2[医药卫生—泌尿科学] R394.33[医药卫生—外科学]