大约克猪SLA-1原核表达载体的构建及表达  

Construction and Expression of SLA-1 Prokaryotic Expressing Vector in Yorkshire Pig

作　　者：张秀娟[1] 杨杰[1] 孙永强[1] 高凤山[1] 

机构地区：[1]大连大学生命科学与技术学院,大连116622

出　　处：《中国畜牧兽医》2016年第12期3121-3126,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目:猪源病毒CTL多肽表位与SLA-Ⅰ结晶研究(31172304);2015年辽宁省大学生创新创业训练计划项目:育-大约克猪SLA-1原核表达载体的构建及表达研究(20150086);2014年大连大学大学生创新创业训练计划项目重点项目:育-大约克猪SLA-1原核表达载体的构建及表达研究(2014066)

摘　　要：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。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.

关 键 词：大约克猪 SLA-1 基因 原核表达载体 

分 类 号：Q786[生物学—分子生物学]