结核杆菌Ag85B的基因克隆及在大肠杆菌中的表达

目的通过结核杆菌Ag85B基因的克隆和在大肠杆菌表达的研究，探讨Ag85B蛋白作为候选疫苗用于结核免疫预防和治疗的可行性。方法用PCR技术，扩增人结核杆菌Edman株Ag85B基因序列，并将扩增的DNA片段插入克隆质粒载体pUC18，用质粒酶谱和DNA序列分析鉴定重组克隆。克隆的Ag85B基因插入原核表达质粒pBV220，构建重组Ag85B表达质粒，用SDS-PAGE和Westernblot鉴定大肠杆菌表达的重组蛋白。结果成功克隆了结核杆菌Ag85B基因，构建了Ag85B的原核表达载体，获得了高效表达菌株，目的蛋白的表达量达菌体总蛋白的25％～30％。结论获得了高效表达人结核杆菌Ag85B成熟蛋白的工程菌株，为Ag85B保护性抗原位点分析和新型结核疫苗的研制奠定了基础。

CLONING AND EXPRESSION OF MYCOBACTERIUM TUBERCULOSIS Ag85B IN E. COLI

Objective:To analyze the protective epitopes of Antigen 85B(Ag85B)of M. tuberculosis,and investigate the potential use of Ag85B as vaccine for the treatment and prevention of tuberculosis, expression recombinant of mature Ag85B was constructed. Methods: Ag85B gene was amplified with PCR, inserted into pUC18 and then sequenced. The cloned Ag85B was seperated and inserted in the downstream of PL promotor of expression vector pBV220. The SDS-PAGE and Western Blot were used to identify the recombinant protein. Resuits:Both cloning and expression recombinants of Ag85B were obtained. DNA sequencing indicated that cloned Ag85B gene was similar to that reported previously. The mature Ag85B was expressed at high level(25 % ～30 %of total bacterial protein)in E. coli. Conclusion:The cloning and high level expression of Ag85B in E. coli provide basis for future study on the preventive and therapeutic vaccine of tuberculosis.