禽源巴氏杆菌链霉素耐药基因StrA的克隆及原核表达

Cloning and Expression of Avian Pasteurella Multocida Streptomycin Resistance in Pronucleus

根据巴氏杆菌链霉素耐药基因 Str A序列 ,设计合成 1对引物 ,以临床分离的禽源巴氏杆菌链霉素耐药菌株质粒DNA为模板 ,通过 PCR技术 ,扩增出禽源巴氏杆菌的 Str A基因片段。将长约 80 4 bp的 Str A目的基因克隆到 p GEM-T载体上 ,通过核苷酸序列测定 ,结果表明 ,克隆的 Str A基因长约 80 4 bp,与文献报道 (NC- 0 0 1774 )的同源性为99.8% ,只有 1个碱基不同 (6 9位 T→ C) ,但所编码的氨基酸没有改变。将 Str A基因按正确的阅读框架定向克隆到原核表达载体 p ET- 2 8c(+)中 ,重组质粒酶切鉴定正确后 ,将构建好的原核表达质粒 p ET- 2 8c(+) - Str A转化到受体菌BL- 2 1(DE3)中 ,经 IPTG诱导后 ,进行 SDS- PAGE电泳 ,经检测 ,Str A外源基因的表达产物占菌体总蛋白的 12 %。

We designed and synthesised a pair of primer according to pasteurella gene rank, the rank of 350～1 153 bp were amplified by pasteurella strain of streptomycin resistance .PCR products bonded with expression vecter of plasmid carrier,804 bp aim gene were cloned into pGEM-T vecter,constructed cloning vecter pGEM-TstrA。The sequences of StrB gene have 99.8% homology with that of the standard strain(NC-001774). Only one base occurrenced mutation,but coding amino acid did not alterated。The amplificated product was cloned into expression vector pET-28c(+)after both restriction digest.The recombinant plasmid that has right reading frame was transformed into competent cell of BL-21(DE3)and was induced by IPTG.The expressed fusion protein was visualized by SDS-PAGE and the efficency was estimated at 12%.