Vero毒素-1B亚基基因克隆及表达

Construction and expression of the plasmid vector of Vero toxin-1B subunit

目的:构建表达Vero毒素-1B亚基的重组质粒。方法:用PCR法扩增并用低熔点琼脂糖法回收Vero毒素-1B亚基基因,将其与质粒pGEX-4T-2重组,通过酶切图谱和序列分析法筛选出重组质粒。将含重组质粒的宿主菌诱导表达后,提取全菌总蛋白进行SDS-PAGE电泳分析。结果:获得了克隆有Vero毒素-1B亚基基因的重组质粒,经诱导后表达分子量为34 kDa的新蛋白。结论:重组质粒pVT1B的构建和表达成功说明克隆的Vero毒素-1B亚基基因具有完整的阅读框架,使简便获得Vero毒素-1B亚基成为可能,为以后研究新型的肠出血性大肠杆菌疫苗及治疗用药奠定了基础。

Objective：To construct and express the plasmid vector of Vero toxin - 1B subunit from E. coli O157： H7. Methods： The Vero toxin - 1B subunit gene was amplified from the genome of E. coli O157：H7 and was recombined with plasmid pGEX - 4T -2. The recombined plasmid with proper orientation was identified with enzyme analysis and gene sequencing. The host bacteria containing recombinant plasmid grew with IPTG induction, and the complete protein of the bacteria was extracted for SDS - PAGE. Results：The recombinant plasmid, pVT1B, expressed a new protein with a weight of 34 kDa. Conclusion：The success of construction and expression of recombinant plasmid pVT1B indicated the toxin -1 B subunit gene obtained by PCR has a complete reading frame. The above work has laid a necessary foundation for future research on a new vaccine of E. coli O157： H7.