牛sox2基因的克隆及重组反转录病毒载体的构建

Cloning of bovine sox2 gene and construction of its retrovirus vector

摘要:

为了构建包含牛sox2基因编码序列的重组反转录病毒载体, 获得有感染性的病毒颗粒, 本研究以胎牛原始生殖嵴为材料, 用RT-PCR方法克隆出牛sox2基因的开放阅读框序列, 将其亚克隆至pMD18-T载体并测序。结果表明获得的基因片段与发表的牛sox2基因序列(GenBank Accession No. NM-001105463)高度同源; 将测序正确的重组T载体用EcoR I和Bgl II酶切, 基因片段插入反转录病毒载体pMSCVneo的相同酶切位点, 成功构建了重组反转录病毒载体pMSCV-sox2。采用脂质体法用pMSCV-sox2转染包装细胞PT67, 同时用pMIG(含绿色荧光蛋白)作为阳性对照, 经流式细胞仪测定, 该载体转染效率达到68.3%; 转染后PT67细胞经G418筛选得到稳定的产毒细胞株, 其病毒滴度达 8.16×107 CFU/mL, 为下一步特定因子诱导牛体细胞转变为牛iPS细胞的研究奠定了基础。

Abstract:

In order to construct the recombinant retrovirus vector of bovine sox2 gene and obtain infectious retroviral particles, we successfully amplified the ORF (open reading frame) of bovine sox2 gene from the primodial genital ridges of bovine embryo by RT-PCR. The cDNA of ORF was subcloned to pMD18-T vectors and verified that its sequence was highly homologous to the GenBank counterpart (GenBank Accession No. NM-001105463) by sequencing. The correct fragment was digested by EcoR I/Bgl II from recombinant pMD18-T vector and inserted into the same restriction sites of retroviral vector pMSCVneo. We got recombinant retrovirus vector pMSCV-sox2 which was transfected into PT67 by lipofectamine 2000 with pMIG (including green fluorescence protein) as a control. Flow cytometry analysis showed that its transfected efficiency was 68.3%. Subsequently, we established the stable cell strain by G418 selection which could produce virus. Its viral titer was up to 8.16×107 CFU/mL. This greatly facilitates the further study of bovine induced pluripotent stem cells induced from bovine somatic cells by defined factors.