【摘要】 目的通过原核表达获得结核分枝杆菌Ag85A蛋白。方法用PCR从结核分枝杆菌H37Rv菌株中扩增出编码Ag85A的fbpA基因,克隆入原核表达载体pProEXHTb,产生重组质粒pPro85A后,转化至大肠杆菌感受态细胞BL21并诱导大量表达。用镍纯化系统纯化重组Ag85A蛋白,用不同分枝杆菌感染的小鼠血清通过ELISA确定其免疫反应性。利用PCR技术鉴定fbpA基因在不同分枝杆菌的分布。结果32 ku的Ag85A蛋白获得高效表达和纯化。表达Ag85A蛋白的fbpA基因在结核分枝杆菌H37Rv、H37Ra、BCG、草分枝杆菌、土地分枝杆菌、耻垢分枝杆菌和次要分枝杆菌中均有表达,但在牝牛分枝杆菌中未表达。结核病患者和结核分枝杆菌毒株H37Rv感染小鼠血清所产生的抗Ag85A抗体滴度最高。结论重组Ag85A蛋白已成功表达纯化,并保留了免疫反应性。更多还原

【Abstract】 Objective To obtain M.tuberculosis Ag85A protein by prokaryotic expression.Methods The fbpA gene encoding M.tuberculosis Ag85A protein was amplified by polymerase chain reaction(PCR) from M.tuberculosis H37RV strain.The PCR product was cloned into prokaryotic expression vector pProEXHTb to generate the recombinant plasmid pProfbpA,which was then transformed into the competence Escherichia coli BL21 cells.The recombinant Ag85A protein was successfully expressed by isopropyl thio-β-D-galactoside(IPTG) induction and purified by the Ni-purification system.The distribution of fbpA gene in different nonpathogenic mycobacterial strains was screened by PCR and ELISA was performed to determine the immunoreactivity of the recombinant Ag85A protein with serum from mice with mycobacterial infections.Results 32 ku Ag85A protein was successfully expressed and purified.It was confirmed by PCR and ELISA that fbpA gene presented in the genomes of M.tuberculosis H37Rv,H37Ra,BCG,M.smegmatis,M.terra,M.trivial and M.phlei,but being absent in the genomes of M.vaccae.The highest Ag85A antibody titer was found in serum of TB patients and mice infected by M.tuberculosis H37Rv.Conclusion The recombinant Ag85A protein was successfully expressed and purified.更多还原