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当前位置: 首页 > 文章 > 大约克猪SLA-1原核表达载体的构建及表达 中国畜牧兽医 2016,43 (12) 3121-3126
Position: Home > Articles > Construction and Expression of SLA-1 Prokaryotic Expressing Vector in Yorkshire Pig China Animal Husbandry & Veterinary Medicine 2016,43 (12) 3121-3126

大约克猪SLA-1原核表达载体的构建及表达

作  者：
张秀娟;杨杰;孙永强;高凤山
单  位：
大连大学生命科学与技术学院
关键词：
大约克猪;SLA-1基因;原核表达载体
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NdeⅠ和XhoⅠ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837bp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34ku。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。
译  名：
Construction and Expression of SLA-1 Prokaryotic Expressing Vector in Yorkshire Pig
作  者：
ZHANG Xiu-juan;YANG Jie;SUN Yong-qiang;GAO Feng-shan;College of Life Science and Technology,Dalian University;
关键词：
Yorkshire pig;;SLA-1 gene;;prokaryotic expressing vector
摘  要：
In order to construct the prokaryotic expressing vector of SLA-1derived form Yorkshire pig and express the interest of protein,apair of primers was designed to amplify the extracellular domain of SLA-1gene from Yorkshire pig(named SLA-1-DYKe)by PCR.Then the PCR product was cloned into pMD~19-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.Analyzing 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 interest 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-1gene was successfully cloned into pMD~19-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-1was 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-1from Yorkshire pig in the future.

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