大约克猪 SLA-1原核表达载体的构建及表达

摘要

In order to construct the prokaryotic expressing vector of SLA-1 derived form York-shire pig and express the interest of protein,a pair of primers was designed to amplify the extra-cellular domain of SLA-1 gene from Yorkshire pig (named SLA-1-DYKe)by PCR.Then the PCR product was cloned into pMD® 1 9-T Simple Vector and transformed into Escherichia coli TOP10. After cleaved by Nde Ⅰ and Xho Ⅰ,the positive clones were selected to be sequenced.Analy-zing by biological soft,the fragment from positive clone with correct sequence was inserted into pET-28a (+)and transformed into E .coli BL21 (DE3).After induction and expression,the in-terest of protein was detected by SDS-PAGE.The results showed that the extracellular domain of SLA-1-DYKe was successfully amplified with the fragment length of 837 bp.The interest of SLA-1 gene was successfully cloned into pMD® 1 9-T Simple Vector and the positive recombinant plasmids with correct sequences were obtained.The SLA-1-DYKe from positive recombinant plasmids was further inserted into pET-28a(+).After transformed into E .coli BL21(DE3)and induction,the SLA-1-DYKe was successfully expressed.The molecular weight of the protein was about 34 ku.It was concluded that the prokaryotic expressing vector of SLA-1 was constructed successfully from Yorkshire pigs and then the expressed protein was obtained,which would lay a base for studying on the structure and function of SLA-1 from Yorkshire pig in the future.%为构建大约克猪 SLA-1胞外区的原核表达载体及表达目的蛋白,试验设计1对引物,经 PCR 扩增获得大约克猪 SLA-1胞外区基因(命名为 SLA-1-DYKe),将此片段克隆至 pMD®19-T Simple Vector,转化大肠杆菌TOP10感受态细胞,经 Nde Ⅰ和 Xho Ⅰ双酶切筛选阳性克隆菌并测序,将目的基因插入到原核表达载体 pET-28a (+)中,转化至宿主菌 BL21(DE3)进行诱导表达,用 SDS-PAGE 检测目的蛋白的表达情况,大量诱导提取包涵体并检测。结果显示,PCR 成功扩增 SLA-1-DYKe 的胞外区,得到大小为837 bp 的目的基因,目的基因成功克隆至pMD®19-T Simple Vector,并获得序列正确的重组质粒。以得到的重组质粒成功构建了 SLA-1-DYKe/pET-28a (+)表达载体,目的蛋白大小约为34 ku。本研究成功构建了大约克猪 SLA-1原核表达载体,获得了表达蛋白,为今后研究大约克猪 SLA-1的空间结构和基因功能奠定了基础。