武汉工厂员工行为规范.ppt

万方数据
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;,
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;
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。

关键词: 男性不育;SPAG6;ARM 结构域;蛋白定位
I
万方数据
Abstract
Objective:To construct the eukaryotic plasmid containing the SPGA6 (full length and
different ARM domains sequences), express the protein in the mammalian cell,and
further examine the effect of armadillo (ARM) domains on Sperm- associated antigen 6
(SPGA6) localization in CHO cells.

Methods: 1. Looking for SPAG6 protein in mice conservative functional areas, analyze
the full-length protein sequences, retrieving the SPAG6 seven ARM domains; 2. cDNA
library was constructed by reverse transcription with testicular tissue from healthy,
matured male mice; 3. The SPAG6 coding sequences (C-terminal&full length) were
amplified by PCR with the cDNA template; The sequences coding different ARM
domains were amplified by polymerase chain reaction (PCR) ; 4. the sequences were
analized after the TA Clone; 5. The correct SPAG6 and different ARM domains
sequences were subcloned into the pEGFP-N2 plasmids were transfected
into CHO . 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 deleti