结核分枝杆菌MPT64蛋白的表达、纯化及初步应用

Expression of MPT64 protein of Mycobacterium tuberculosis in Escherichia coli and its purification application.

目的 获得重组MPT64蛋白 ,研究其免疫学特性 ,评价其在结核病血清学诊断中的价值。方法 应用基因工程技术表达、纯化MPT64蛋白 ,通过Westernblotting分析其抗原性。分别以结核分枝杆菌PPD或纯化的rMPT64蛋白为抗原 ,通过ELISA方法及结核病一步法检测血清中抗结核抗体。结果 重组质粒pET1 5b MPT64测序表达除 83位密码子由CCA突变为CCG外 ,其余密码子均与报道的相同 ,但其氨基酸序列无变化。它在大肠杆菌BL2 1 (DE3)细胞内以包涵体形式存在 ,表达量占菌体总蛋白的 2 0～ 30 % ,分子量约 2 6kDa ,Westernblotting分析它与抗结核分枝杆菌多克隆抗体具有良好的免疫反应性。纯化后的rMPT64样品经SDS PAGE和光密度扫描分析表明其纯度约为 80 %左右 ,每1 0 0ml培养菌可获得 1 0mg左右的重组蛋白。以 33例正常人血清的OD值 + 2S为正常界限值 ,PDD的特异性和敏感性分别为 94% ( 31 /33)、63.7% ( 2 1 /33)、66.7% ;rMPT64纯化蛋白分别为 94% ( 31 /33)、30 .3( 1 0 /33) ;一步法分别为 88% ( 2 9/33)、66.7% ( 2 2 /2 3)。结论 pET1 5b MPT64大肠杆菌工程菌能以包涵体形式高效表达重组MPT64蛋白 ,该蛋白具有较高的抗原特异性和免疫反应性。rMPT64纯化蛋白有可能成为结核病血清学诊断的组合抗原之一。

Objective To obtain the recombinant MPT64 protein,to study its immunological characteristics,and to evaluate its potential value for serodiagnosis of tuberculosis.Methods The gene coding MPT64 protein inserted into a expression vector pET 15b,and then transferred to E.coli BL21(DE3).The expressed product was purified by metal chelation chromatography,and analyzed its immunogenicity by western blotting.66 human serum was detected the antibodies against M.tuberculosis rMPT64 and PPD antigens by ELISA,and antibodies against M.tuberculosis antigens by tuberculosis one step test.Results Sequencing recombinant plasmid pET 15b MPT64 showed that there was a mutation (CCA→CCG) at codon 83,but its amino acid had no change.The recombinant MPT64 protein existed in inclusion bodies of E.coli ,and amounted to 20～30% of total bacterial proteins.Its molecular weight was about 26 kilodalton.It can produce good immunoreactivity with serum from goats vaccinated with M.tuberculosis by western blotting.The purity of final product was about 80%.10mg of rMPT64 protein per 100ml culture could be obtained.The positive cutoff values were OD 492 plus 2 standard deviation of 33 negative serum detected by ELISA.Of 33 serum from tuberculosis patients,the specificity and sensitivity of PPD as antigen of ELISA were 94% and 63.7%,That of rMPT64 were 94% and 30.3%,and that of tuberculosis one step test were 88% and 66.7%,respectively.Conclusion The recombinant MPT64 protein could be highly expressed in the form of inclusion bodies in E.coli. It had good antigenic specificity and immunoreactivity,and might be selected as one of serodignostic antigen of tuberculosis.