【摘要】 克隆猪ATF4基因CDS,构建pcDNA3.1(+)-ATF4真核超表达载体,为下一步在细胞水平和个体水平研究该基因功能奠定基础。提取RNA,运用RT-PCR和巢式PCR技术扩增猪ATF4全部编码序列,克隆转化到pMD18-T载体中,测序验证后,酶切连接到pcDNA3.1(+)载体中,构建成pcDNA3.1(+)-ATF4真核超表达载体,对其进行酶切和测序鉴定并在细胞水平上进行表达量的鉴定。实验结果表明,在转染了pcDNA3.1(+)-ATF4载体的细胞中,ATF4 mRNA表达水平明显增加,成功构建了pcDNA3.1(+)-ATF4真核超表达载体,为进一步研究该基因的功能奠定了基础。更多还原

【Abstract】 We construct an eukaryotic expression vector pcDNA3.1(+)-ATF4 by cloning porcine ATF4 gene,which provide us advantage for further study of gene function at the cellular and individual level.Extracted RNA and using RT-PCR and nested PCR method,we amplified coding sequence of porcine ATF4 and cloned it into the pMD18-T vector and then constructed pcDNA3.1 (+)-ATF4 vector which was identified by restriction enzyme analysis and DNA sequencing.The results show that we successfully construct an eukaryotic expression vector pcDNA3.1(+)-ATF4.Transiently transfected cells and effect of expression is obvious,which will contribute to further study on the ATF4 function and to the establishment of condition.更多还原