结核分枝杆菌潜伏感染相关蛋白ICL的表达及鉴定

目的　表达结核分枝杆菌潜伏感染相关蛋白ICL ,鉴定其生物学活性并制备其多克隆抗体。方法　采用PCR法从结核分枝杆菌H37Rv基因组DNA中扩增出icl基因片段 ,将其插入原核表达质粒pQE30 ,构建重组质粒 pQE30 icl。将pQE30 icl转化大肠杆菌 ,用IPTG诱导目的基因表达。Western blot免疫印迹法鉴定后纯化该表达产物 ,测定其酶活性并用此纯化蛋白免疫新西兰兔制备多克隆抗体。结果　序列分析发现PCR扩得的icl有两个核苷酸突变 ,但均为无义突变。在大肠杆菌中表达的目的产物经SDS PAGE分析约为 4 7kDa。免疫印迹分析证实目的蛋白与阳性结核病患者抗血清产生特异性反应。纯化的目的蛋白异柠檬酸裂解酶的酶活性为 16 .7u/mg。双向免疫扩散法测定目的蛋白免疫兔血清的抗体效价为 1 32。结论　研制出的结核分枝杆菌重组ICL具有异柠檬酸裂解酶活性和抗原性 ,重组ICL和抗ICL免疫血清的制备为抗结核分枝杆菌潜伏感染的研究奠定了必要的物质基础。

Expression and identification of Mycobacterium tuberculosis latent infection implicated protein ICL

To express and purify the Mycobacterium tuberculosis latent infection implicated protein (ICL),determine the bidogical activity and prepare the polyclonal antibody against ICL,the gene encoding ICL was amplified by PCR from genome of Mycobacterium tuberculosis H37Rv strain and inserted into prokaryotic expression vector pQE30 to obtain recombinant plasmid pQE30 icl. The recombinant vector pQE30 icl was transformed into E.coli after digestion by restriction endonuclease and sequence analysis, induced with IPTG. The product was analyzed by SDS PAGE and Western blotting. Then the expressed protein was purified by Ni NTA purification system. The enzyme activity of the purified protein was determined. The polyclonal antibody was prepared by immunization with the purified protein in New Zealand rabbits, and the specific antibody titer was determinded by double immunodiffusion.It was found that the sequence of icl obtained by PCR amplification had two nucleotides mutations,both were nonsense mutations.The expressed protein induced by IPTG in E.coli had relative molecular mass of 47 kDa. The Western blotting analysis showed the expressed protein could react with the serum from tuberculosis patients speicfically. The enzyme activity of the purified protein was 16.7u/mg. The specific antibody titer in the serum of rabbits immunized with the purified protein was 1∶32.Mycobacterium tuberculosis latent infection implicated protein ICL with enzyme activity was expressed successfully in E.coli. The polyclonal antibody of ICL was produced successfully in rabbit. Our study established some material foundations for researches against latent infection of Mycobacterium tuberculosis.