结核分枝杆菌分泌蛋白Ag85B-ESAT6的融合表达及纯化

目的　融合表达结核分枝杆菌分泌蛋白Ag85B ESAT6 ,为结核病的预防提供有效亚单位疫苗。方法　设计含不同酶切位点的Ag85B和ESAT6引物 ,采用聚合酶链反应 (polymerasechainreaction ,PCR)的方法从结核分枝杆菌毒株H3 7Rv DNA中分别扩增出相应大小的DNA片段 ,将片段分别与PGEM T easy载体连接测序。将测序正确的Ag85B和ESAT6按照不同的酶切位点克隆入PPROEXHT表达载体 ,挑选出阳性克隆 ,将诱导的表达产物进行聚丙烯酰胺凝胶电泳 (SDS PAGE)分析 ,同时与含6个组氨酸 (histidine 6×his)的单克隆抗体 (monoclonalantibody ,mAb)进行固定化蛋白质免疫学测定(Western blot) ,将用含Ni 鳌合剂的Ni NTA亲合柱纯化的表达产物免疫小鼠 ,用结核分枝杆菌培养上清滤液蛋白 (culturefiltrateproteins ,CFP)作为抗原 ,酶联免疫吸附试验 (enzyme linkedimmunosorbentassay ,ELISA)测定免疫小鼠血清特异性抗体的滴度。结果　PCR获得的Ag85B和ESAT6序列与基因文库 (GenBank)报道的完全一致。两者在大肠杆菌DH5α株中融合表达的产物约为 370 0 0 ,与预计大小相吻合。Western blot结果显示 ,在相对分子量约 370 0 0处有表达产物与 6×hismAb特异性结合带。表达产物为包涵体 ,通过Ni NTA纯化系统 ,可得到纯化的目的蛋白。Ag8

Mycobacterium tuberculosis secreting protein Ag85B-ESAT6 fused expression and purification

OBJECTIVE: To investigate the fused expression of secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis, and to provide a promising preventive subunit vaccine against tuberculosis. METHODS: The gene encoding Ag85B and ESAT6 protein was amplified by PCR from genome of Mycobacterium tuberculosis H(37)Rv strain, and inserted into cloning vector P(GEM)-T-easy. After sequence analysis, and digestion by restriction endonuclease, Ag85B-ESAT6 was cloned into corresponding sites of the expression vector P(PRO) EXHT, and the recombinant plasmid was transformed into expressive strain E. coli DH5 alpha, induced with IPTG and fusion protein was purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with two fusion protein was detected by ELISA. RESULTS: The sequences of Ag85B and ESAT6 by PCR amplification were identical to those reported by GenBank. The recombinant plasmid fused expression protein of Ag85B-ESAT6 with relative molecular mass (Mr) of 37,000, which was confirmed by Western-blot analysis with specific monoclonal antibody against 6 x HismAb. The fused expression protein was insoluble. It could be purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with fusion Ag85B-ESAT6 was 1:1000 and that of mouse immunized with fusion protein ESAT6-Ag85B was 1:5000. CONCLUSIONS: Secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis was successfully fused expressed in E. Coli DH5 alpha. It may become a new type of vaccine against tuberculosis.