绿色荧光蛋白和大鼠睾丸AQP7融合蛋白表达载体构建及其表达  

Reconstruction and Expression of Green Fluorescent Protein and Aquaporin 7 Fusion Recombinant Vector

作　　者：杨柯[1] 李峰[1] 赵丹[1] 李大男[1] 刘庆双[1] 刘喜春[1] 张键[1] 赵雪俭[1] 杨宝学 

机构地区：[1]吉林大学基础医学院生殖病理生理研究室,吉林长春130021 [2]加利弗尼亚大学医学院

出　　处：《中华男科学杂志》2004年第11期819-823,共5页National Journal of Andrology

摘　　要：目的 :构建真核表达的pEGFP C1与大鼠睾丸AQP7的融合蛋白表达载体。　方法 :RT PCR的方法扩增Wistar大鼠睾丸AQP7cDNA编码区全长序列 ,测序鉴定 ,将AQP7cDNA融合于pEGFP C1基因的下游。以脂质体转染方法将pEGFP C1 AQP7融合蛋白转入中国仓鼠卵巢 (CHO)细胞 ,应用免疫细胞化学方法和Western印迹技术对转染细胞株进行鉴定。　结果 :①Wistar大鼠AQP7cDNA序列登录到GenBank ,登录号 :AY15 7737;②通过鉴定证明CHO细胞已经稳定表达了pEGFP C1 AQP7融合蛋白 ,其相对分子质量为 5 30 0 0 ;③绿色荧光蛋白作为标记物观察到AQP7在CHO细胞的定位。　结论 :稳定表达pEGFP C1 AQP7融合蛋白的CHO细胞株的建立 ,为AQP7在细胞内定位与功能研究奠定了基础。Objective: To study the membrane mobility of aquaporin 7 (AQP7) by cloning stably transfected CHO cells with expression of pEGFP-C1-AQP7, in which AQP7 cDNA was fused downstream and in frame to pEGFP-C1 gene. Methods: The full sequence of AQP7 was amplified by RT-PCR and then recombined in the downstream of the green fluorescent protein gene in the pEGFP-C1 vector . The recombinant vector pEGFP-C1-AQP7 was stably transfected into CHO cells. With fluorescent microscopy, immunocytochemical stain and Western blot, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization. Results: ① The sequence of AQP7 cDNA of the Wistar rat was logged into the GenBank(access number: AY157737). ②Identification demonstrated that pEGFP-C1-AQP7 fusion protein stably expressed in CHO cells. ③With fluorescence microscopy, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization. Conclusion: The CHO cell line with stable pEGFP-C1-AQP7 expression was set up successfully for advanced research.

关 键 词：水通道蛋白 融合蛋白 绿色荧光蛋白 睾丸 大鼠 

分 类 号：R346[医药卫生—基础医学]