广西巴马小型猪ABCA1基因真核表达载体的构建与鉴定

Construction and Identification of the ABCA1 Gene Expression Vector of Guangxi Bama Miniature Pig

本研究目的是克隆广西巴马小型猪ABCA1基因CDs序列并构建真核表达载体。利用RT-PCR技术从广西巴马小型猪肝脏组织中扩增出ABCA1基因编码序列,直接插入至pEGFP-N1真核表达载体的KpnⅠ酶切位点,构建出重组质粒。之后将重组质粒pEGFP-N1-ABCA1转染PK15细胞,并于24 h、48 h后观察细胞荧光表达情况。收集转染48 h后的PK15细胞通过qRT-PCR检测ABCA1 mRNA相对表达量。结果表明:本实验成功构建了广西巴马小型猪ABCA1基因真核表达载体,重组质粒pEGFP-N1-ABCA1转染PK15细胞后,有绿色荧光蛋白表达,且与转染pEGFP-N1和空白组相比,pEGFP-N1-ABCA1转染组的ABCA1 mRNA表达量显著提高。本研究为下一步研究ABCA1基因的功能和调控及生产转ABCA1基因广西巴马小型猪奠定基础。

The objective of this study was to clone the ATP-Binding Cassette Transporter Al（ABCA1） gene sequence and construct the eukaryotic expression vector of Guangxi Bama miniature pig.The coding sequence（CDs）of ABCA1 gene was amplified from the liver tissue of Guangxi Bama miniature pig using RT-PCR technique and the ABCA1 was inserted into the Kpn I enzyme loci of pEGFP-Nl to construct the pEGFP-Nl- ABCA1 eukaryotic expression vector.The plasmids were transfected into PK15 cells using lipofectamine and investigated the green fluorescence under microscope at 24 h and 48 h.The relative transcription level of ABCA1 gene mRNA were determined by qRT-PCR at the 48 h transfected PK15 cells.The results showed that the pEGFP-Nl- ABCA1 vector was constructed successfully and the green fluorescence was observed in the transfected cells.Compared with pEGFP-Nl,NC,the relative level of ABCA1 gene mRNA increased significantly in PK15 cells which transfected with pEGFP-Nl-ABCA1.In conclusion,this study laid a foundation for further analyzing the function and regulation of ABCA1 and producing the ABCA1 pig in future.