摘要
采用PCR技术从草分枝杆菌(Mycobacterium phlei)基因组中扩增出亮氨酰-t RNA合成酶基因leu RS,依次克隆入p MD19-T Simple克隆载体及p ET28a(+)表达载体,并在大肠埃希菌(Escherichia coli)BL21(DE3)中表达。表达产物用Ni^(2+)螯合的His Trap^(TM) HP亲和色谱纯化,测定纯化所得目的蛋白的酶活力。结果显示,经PCR扩增得到2.8 kb的DNA片段,重组质粒p ET28a(+)-leu RS经酶切鉴定和测序分析,表明构建正确。SDS-PAGE显示,有与目的蛋白大小相当(相对分子质量约1.10×10~5)的蛋白质表达,表达的重组蛋白占菌体可溶性蛋白的32.8%,纯化后的蛋白质纯度达93.8%,酶活力为13.2 u/ml。
The leucyl-tRNA synthetase gene from Mycobacterium phlei genomic DNA was cloned into the cloning vector pMD19-T Simple and then cloned into the expression vector pET28a （＋）. The expression of M. phlei Leu RS was carried out in Escherichia coli BL21 （DE3）. The expression product was purified by HisTrapTM HP affinity chromatography and the enzymatic activity was measured. It was found that a DNA fragment at the length of 2.8 kb was amplified. Both restriction analysis and sequencing analysis proved that the recombinant plasmid pET28a （＋）-leu RS was correctly constructed. The expressed product with a relative molecular weight of 1.10× 10^5 was observed by the SDSPAGE. The product contained about 32.8 % of total somatic soluble protein, with the purity of 93.8 % and the enzymatic activity of 13.2 u/ml.
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2016年第11期1374-1378,共5页
Chinese Journal of Pharmaceuticals
基金
辽宁省教育厅高等学校创新团队项目(LT2014023)
沈阳市科学技术计划项目(F15-199-1-27)
国家基础科学人才培养基金项目(J1103606)
