分泌蛋白MPT64鉴定结核分枝杆菌复合群的应用研究

Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64

目的构建重组MTB分泌蛋白MPT64，制备多克隆抗体，评价其在鉴定MTB复合群中的应用价值。方法通过PCR扩增mpt64基因，构建pET21a—mpt64重组质粒；纯化重组蛋白并免疫兔制备多克隆抗体；纯化和标记抗体，建立双抗体夹心酶联免疫吸附试验（ELISA）方法；检测分枝杆菌标准菌株和临床分离株培养液中MPT64蛋白，抽提样本基因组扩增mpt64基因。结果成功构建重组质粒，纯化重组蛋白并制备多克隆抗体，检测抗体浓度为0．85g／L，ELISA法检测敏感度为0．01mg／L，MTB复合群中H37Rv、牛分枝杆菌和非洲分枝杆菌为阳性，24株非MTB均为阴性；rapt64基因扩增的结果与ELISA法检测结果完全相符。ELISA法检测MTB临床分离株的敏感度为97．3％（110／113），57株非MTB均未检出MPT64蛋白，检测特异度为100．0％。基因扩增MTB中2株为阴性，非MTB均为阴性。结论采用ELISA法检测分泌蛋白MPT64来鉴定MTB复合群，是一种准确、快速和简便的方法，值得临床推广应用。

Objective To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64. Methods The gene mpt64 was amplified by polymerase chain reaction （PCR） from the genome of Mycobacterium tuberculosis H37 Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay （ELISA） was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR. Results A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0. 01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% （111/113） and 97. 3% （ 110/113 ） respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains. Conclusion Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.