大鼠Ad-pDOV-mCMV-TRPC4-EGFP-3FLAG质粒的构建、基因结构分析及鉴定

Construction of Ad-CN1035-pDOV-mCMV-TRPC4-EGFP-3FLAG Plasmid in Rat and An Analysis of Its Gene Structure

目的:大鼠Ad-pDOV-mCMV-TRPC4-EGFP-3FLAG基因重组表达质粒的构建及其基因结构分析。方法:通过UCSC(http://genome.ucsc.Edu/)分析大鼠瞬时受体电位通道4(TRPC4)及其家族基因组结构,并通过NCBI确定了TRPC4的蛋白结构域,通过Bio GPS(http://biogps.org/#goto=welcome)分析其表达模式;PCR扩增大鼠TRPC4蛋白编码区域(CDS),并通过限制性内切酶与穿梭载体pDOV-mCMV-MCS-EGFP-3FLAG进行连接,其产物转化细菌感受态细胞;挑取阳性克隆进行菌落PCR鉴定,并对阳性克隆进行测序验证,测序正确的克隆即为目的质粒,命名为pDOV-mCMV-TRPC4-EGFP-3FLAG;将该质粒转染HEK293细胞进行腺病毒包装,并命名为Ad-pDOV-mCMV-TRPC4-EGFP-3FLAG,通过Western blot验证蛋白的表达。结果:生物学分析发现TRPC4蛋白保守的TRP-2、ANK及TRP结构域,通过Bio GPS分析发现TRPC4在杏仁核和海马中表达明显,在血管内皮细胞中也有表达;PCR克隆获得3 000 bp的TRPC4 CDS序列,CDS被亚克隆到表达载体pDOV-mCMVMCS-EGFP-3FLAG中,酶切鉴定及测序验证正确;进行腺病毒包装后的病毒可以在HEK293细胞中表达TRPC4-EGFP-3FLAG融合蛋白,Western blot证实其为含flag标签的蛋白且大小为128 KDa。结论:成功构建了大鼠TRPC4的腺病毒表达载体Ad-pDOV-mCMV-TRPC4-EGFP-3FLAG。

Objective：To construct TRPC4 expression vector and to analyze rat TRPC4 protein domain.Methods：TRPC4 domain of rats and its family genome structure was analyzed by using UCSC（http：//genome.ucsc Edu/）.The structural domain of it was determined with NCBI,and its expression pattern analyzed with Bio GPS.TRPC4 coding sequence（CDS） was amplified by PCR and sub-cloned into pDOV-mCMV-MCS-EGFP-3FLAG vector.Right clones were testified by PCR,and further confirmed by sequencing.To get Ad-CN1035-pDOV-mCMV-TRPC4-EGFP-3FLAG adenovirus.pDOV-mCMV-TRPC4-EGFP-3FLAG was transfected into 293 based on Ad Max system,and western blot was used to test the TRPC4-EGFP-3Flag fusion protein.Results：Conservative structural domains TRP-2,ANK,and TRP of TRPC4 protein were found through biological analysis.As well,Bio GPS analysis results showed TRPC4 expresses obviously in amygdala,and hippocampus,and in heart and endothelial cells too.TRPC4 full-length CDS was about3000 bp and sub-cloned into the adenovirus expression vector pDOV-mCMV-MCS-EGFP-3FLAG.The sequence was confirmed by enzyme digestion test.The TRPC4-EGFP-3FLAG fusion protein in packaged adenovirus expressed in HEK293 cells,and was identified by western blot.The size of the flag-tagged protein was about 128 Kda.Conclusion：Rat Ad-pDOV-mCMV-TRPC4-EGFP-3FLAG expression vector was constructed successfully.