烟台黑猪SLA-I重链基因末端生物素修饰及表达  

Biotinylated the Carboxyl Terminal of Heavy Chain of SLA-I from Yantai Black Pig and Its Expression

作　　者：刘筏[1] 杨金刚[1] 翟晓鑫 董宋鹏 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：《生物技术通报》2014年第1期191-195,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31172304);辽宁省大学生创新项目(201311258009);大连大学本科生创新项目(2012029)

摘　　要：为构建烟台黑猪SLA-2重链基因偶合生物素化酶BirA底物肽(BirA substrate peptide,BSP)并研究其在pET-28a(+)中的表达,设计烟台黑猪SLA-2-BSP复合基因引物,PCR扩增烟台黑猪SLA-2-YTH-BSP复合基因,并克隆至pMD 19-T Simple Vectorp,经酶切鉴定后将SLA-2-YTH-BSP复合基因与表达系统pET-28a(+)链接,并转化到BL21(Rosseta)菌进行诱导表达,SDS-PAGE检测目的蛋白。表达蛋白经过Ni-NTA亲和纯化,并经SDS-PAGE检测蛋白纯化效果。PCR结果显示SLA-2-BSP大小为900 bp,并成功克隆到pMD 19-T Simple Vector,酶切鉴定大小为876 bp,该基因链接到pET-28a(+)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32.4 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。成功构建烟台黑猪SLA-I重链偶合生物素化酶BirA底物肽(BSP)的pET-28a(+)的重组表达系,为下一步的SLA-I类分子四聚体研究鉴定基础。To construct the heavy chain of SLA-2 conjugated with the BirA substrate peptide （ BSP ） and express the recombinant genes in pET-28a （ ＋ ） for Yantai Black Pig （ YTH ） , a pair of primers to express the recombinant SLA-2-YTH-BSP was designed and the recombinant SLA-2-YTH-BSP was amplified by PCR followed by sub-cloning the gene into pMDI9-T Simple Vector. After identification by cleavage with Nde I and Xho I, the SLA-2-YTH-BSP was ligated to pET-28a （ ＋ ） and the recombinant plasmids was transformed into BL21 （ Rosseta ） to be induced to express followed by analysis of the expressing products by SDS-PAGE. The expressed interest of protein was purified by Ni-NTA column and detected by SDS-PAGE. The PCR results showed that the length of nucleotides of SLA-2-YTH-BSP was about 900 bp which was consistent with the calculated value. Then, the SLA-2-YTH-BSP with 876 bp was successfully inserted into the pMD-19-T Simple Vector identified by cleavage with Nde I and Xho l, and then the genes were inserted into pET-28a （ ＋ ） and transformed into Escherichia coli BL21 （ Rosseta ） . After induction, SLA-2-YTH-BSP was successfully expressed with the interest of protein about 32.4 kD. After purification, the purity of the interest of the inclusion protein reached to 90%. It was concluded that the recombinant expressing system containing SLA-2 conjugated with BSP in pET-28a （ ＋ ） was successfully constructed and the research would pave the base to construct tetramer in SLA class I.

关 键 词：烟台黑猪 SLA-2 重链基因 生物素化酶BirA底物肽 纯化 

分 类 号：S828[农业科学—畜牧学]