SPF 鸡胚 St3galⅠ基因的克隆及其在 MDCK 细胞中的表达

摘要

为了提高 MDCK 细胞表面禽流感病毒（AIV）受体的水平，以12日龄的 SPF 鸡胚为试材，通过反转录 PCR扩增唾液酸转移酶Ⅰ基因（St3galⅠ），并将回收的片段插入到 pMD18-T 载体中。酶切 pMD18-T-St3galⅠ和 pCI-neo质粒，将目的片段进行连接，构建含有 St3galⅠ基因的真核表达载体。利用脂质体介导法转染 MDCK 细胞，在 G418的筛选下，获得具有抗性的转基因细胞系。通过细胞克隆化培养和 PCR 检测，筛选可稳定表达 St3galⅠ基因的细胞株。结果表明，从鸡胚中克隆的 St3galⅠ基因成功的插入到 pCI-neo 质粒，从而实现真核表达载体 pCI-neo-St3galⅠ的构建。在 G418选择压力下，初步获得了稳转目的基因的 MDCK 细胞系。将抗性细胞混合克隆进行稀释，经选择后挑选出30个单克隆抗性细胞株。分别以转染细胞基因组 DNA 和总 RNA 为模板，经 PCR 检测显示，目的基因已整合入MDCK 细胞的基因组中并可稳定的进行表达。%In order to improve the abundance of avian influenza virus(AIV)receptor on the surface of MDCK cells,12-day-old SPF chicken embryos was as material and St3galⅠ gene was cloned by RT-PCR.Next,the target fragment was inserted to the pMD18-T vector.Subsequently,the pMD18-T-St3galⅠ and pCI-neo plasmids were di-gested and connected.Thus,the eukaryotic expression vector containing St3galⅠ gene was constructed.Afterwards, the pCI-neo-St3gal Ⅰ plasmid was purified and transfected into MDCK cell by liposome-mediated method.The transgenic cell lines were screened by G418.The cell strains expressing St3galⅠ gene stably were initially obtained by cell cloning culture and PCR assay.The results showed that St3galⅠ gene from chicken embryo was successfully inserted into pCI-neo plasmid,leading to the construction of the eukaryotic expression vector pCI-neo-St3galⅠ .In the G418 selection pressure,MDCK cells stably transfected target gene were initially obtained.The resistant cells were diluted,and 30 selected monoclonal resistant cell lines were selected.With genomic DNA and total RNA of transfected cells as a template,PCR analysis showed that the target gene had been integrated into the genome of MDCK cells and stably expressed.