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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 construct a fusion protein expression vector of eukaryotic expression pEGFP C1 and rat testis AQP7. Methods: The full - length coding sequence of AQP7 cDNA in testis of Wistar rats was amplified by RT - PCR and sequenced. The AQP7 cDNA was fused downstream of pEGFP C1 gene. The pEGFP C1 AQP7 fusion protein was transfected into Chinese hamster ovary (CHO) cells by lipofectamine. The transfected cell lines were identified by immunocytochemistry and Western blotting. RESULTS: ① The AQP7 cDNA sequence of Wistar rats was registered in GenBank with the accession number: AY15 7737; ② The pEGFP C1 AQP7 fusion protein was confirmed to have been stably expressed in CHO cells with a relative molecular mass of 5 30 0 0; ③ GFP as a marker The localization of AQP7 in CHO cells was observed. Conclusion: The establishment of CHO cell line stably expressing pEGFP C1 AQP7 fusion protein lays the foundation for the study of AQP7 localization and function in cells.