摘要
为构建托佩克猪SLA-1重链基因原核重组表达载体并研究其在pET-21a(+)中的表达,设计引物,PCR扩增托佩克猪SLA-1基因胞外区(命名为SLA-1-TPKe),并克隆至pMD19-T Simple Vector,经酶切鉴定后将SLA-1-TPKe基因与表达系统pET-21a(+)连接,转化到BL21(Rosseta)菌进行诱导表达,SDS-PAGE检测目的蛋白。提取SLA-1-TPKe包涵体,并经SDSPAGE检测。PCR结果显示,SLA-1-TPKe大小为851 bp,成功克隆到pMD19-T Simple Vector,酶切鉴定大小为831 bp,连接到pET-21a(+)并转化到BL21(Rosseta)菌,经诱导表达和SDS-PAGE检测目的蛋白的大小为31 kD。目的蛋白以包涵体形式存在,纯化后蛋白纯度达到90%以上。
In order to construct the recombinant prokaryotic expressed vector of the heavy chain of SLA-1 derived from Topigs pig and to study its expression in pET-21a（＋）, a pair of primers was designed to amplify the extracellular domain of SLA-1-TPK gene（named SLA-1-TPKe）followed by sub-cloning the gene into p MD19-T Simple Vector. After identification by cleavage with Nde I and Xho I, the SLA-1-TPKe was ligated to p ET-21a（＋）and transformed into BL21（Rosseta）to be induced to express followed by analysis of the expressing products by SDS-PAGE. Finally, the inclusion bodies of the SLA-I-TPKe was isolated and detected by SDS-PAGE. The PCR result was shown that the length of the SLA-1-TPKe was about 851 bp which was consistent with the calculated value. Then, the SLA-1-TPKe was successfully cloned into the pMD19-T Simple Vector and identified by cleavage with Nde I and Xho I and the inserted fragment was about 831 bp. In succession, the gene was successfully inserted into p ET-21a（＋）followed by transforming into Escherichia coli BL21（Rosseta）. After induction and expression, the SLA-1-TPKe was successfully expressed with the interest of protein about 31 kD. The protein was expressed mainly as inclusion body protein with the purity of 90%.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第3期135-139,共5页
Biotechnology Bulletin
基金
国家自然科学基金项目(31172304)
大连大学本科生创新项目(2013249)
关键词
托佩克猪
SLA-1
重链基因
表达
包涵体
Topigs pig
SLA-1
the heavy chain gene
expression
inclusion protein
