含人Apelin-R重组腺病毒载体pDC316-apelinR-EGFP的构建

Construction of recombinant adenovirus vector pDC316-apelinR-EGFP encoding human Apelin-R

目的构建含有人Apelin受体(Apelin-R)基因的pDC316-apelinR-EGFP腺病毒穿梭载体,为糖尿病相关基因Apelin的体内研究提供新方法。方法 PCR方法分别扩增Apelin-R基因,测序正确后与载体pDC316-EGFP连接,构建重组腺病毒载体pDC316-apelinR-EGFP;脂质体法与腺病毒骨架载体共转染HEK293细胞进行重组腺病毒包装,稀释法测定腺病毒滴度。RT-PCR和Western blot方法检测Apelin-R的表达。结果限制性核酸内切酶鉴定及测序结果均证明Apelin-R基因成功扩增并克隆到重组腺病毒载体pDC316-apelinR-EGFP。在HEK293中成功包装出含Apelin-R的腺病毒Ad-apelinR-EGFP,病毒滴度为3.1×109TCID50/ml,感染Ad-apelinR-EGFP的HEK293E细胞中可检测到Apelin-R的mRNA和蛋白表达。结论成功构建了真核表达载体pDC316-apelinR-EGFP,得到了表达绿色荧光蛋白的Apelin-R腺病毒,该病毒有望用于糖尿病的体内研究实验。

Objective To construct recombinant adenovirus vector pDC316-apelinR-EGFP encoding human Apelin-R(Apelin receptor,Apelin-R)gene in order to provide a new method for in vivo studies of diabetes-related genes Apelin.Methods The Apelin-R gene regions were amplified by polymerase chain reaction and cloned into shuttle vector pDC316-EGFP vector to generate the plasmid pDC316-apelinR-EGFP respectively after the gene regions verified by DNA sequence analysis.Then pDC316-apelinR-EGFP was cotransfected into HEK293E cells with the adenovirus backbone plasmid by Lipofectamine 2000 to get packaged.The titer of recombinant adenovirus was detected by TCID50.The expression of Apelin-R was detected by RT-PCR and Western blot.Results Restriction endonuclease digestion analysis and DNA sequencing results showed that the adenovirus vector pDC316-apelinR-EGFP was constructed in the sequence as designed.Recombinant adenovirus Ad-apelinR-EGFP was obtained in HEK293E cells and virus titer was 3.1×109 TCID50/ml.After infection,the expression of Apelin-R mRNA and protein could be detected in HEK293E cells.Conclusions The recombinant adenovirus vector pDC316-apelinR-EGFP has been constructed successfully,which provides experimental basis for further studying the diabetes in vivo.