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.
出处
《生物技术通报》
CAS
CSCD
北大核心
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)
