志贺毒素B(ShT-B)亚单位基因的克隆、表达与纯化

Cloning Expression and Purification of Shiga Toxin B Subunit Gene in E.coli

用KpnⅠ和HindⅢ双酶切pGEM-TB3 克隆质粒,得到为225bp成熟ShT-B基因片段,将其插入到经同样双酶切的pQE30表达载体中,构建了ShT-B的重组表达质粒pQE30-B2,转化到E.coliM15,经IPTG诱导后,重组质粒目的蛋白均得到表达表达蛋白约占菌体总蛋白的16%,主要为可溶性形式,约9. 2%。为重组抗原的制备提供了必要的物质基础。

Objective To express the ShT-B gene fragment in E.coli. Methods pGEM-ShTB 3 cloning vector and prokaryotic pQE-30 expression vector were respectively digested with the endonucleases KpnⅠand Hind Ⅲ, and then the small fragment of ShT-B and the large fragment of linearized pQE-30 were ligated under T4 DNA ligase, resulted in recombinant vector, named as pQE30-ShTB 2,The resulting expression vector pQE30-ShTB 2 was selected and identified by the colony-PCR and endonucleases digestion, finally expressed in E.coli M15 cell. Results The transgenetic strain E.coli M15 with pQE30-ShTB 2can expressed an ShT-B recombinant protein induced by IPTG. SDS-PAGE showed the size of expressed recombinant protein was similar with ShT-B molecular weight in 10KD, and the target fusion protein occupied 16% of total cell protein and the cell supernatant is occupied 9.2%