结核分枝杆菌PPE36-p27蛋白的原核表达及其在结核诊断中的应用

Prokaryotic expression of Mycobacterium tuberculosis PPE36-p27 protein and its application in diagnosis of tuberculosis

目的原核表达结核分枝杆菌标准菌株PPE36-p27蛋白,并应用于结核特异性细胞免疫检测。方法以结核分枝杆菌H37RV基因组DNA为模板,采用PCR法扩增PPE36基因,纯化回收后插入至原核表达载体pEASY,将测序正确的重组质粒转入感受态E.coli BL21(DE3),诱导表达PPE36-p27蛋白,经SDS-PAGE鉴定及NI-NTA亲和层析柱纯化。将纯化后的PPE36-p27作为特异性刺激原,建立新型T细胞检测方法 ELISPOT-IL-12技术。结果重组质粒pEASY-PPE36经双酶切及测序鉴定,构建正确。PPE36-p27蛋白的相对分子质量约30 000,纯化后蛋白浓度为14.69μg/m L。重组蛋白PPE36-p27应用于ELISPOT-IL-12检测方法时,可刺激机体产生细胞因子IL-12,与包被的抗IL-12抗体产生特异性结合,可见明显斑点。结论 PPE36-p27蛋白在原核表达系统成功表达,以其为刺激原建立了ELISPOT-IL-12检测技术,可应用于结核的早期诊断。

Objective To express the PPE36-p27 protein of standard strain of Mycobacterium tuberculosis in prokaryotic cells and use the expressed product for determination of M. tuberculosis antigen-specific cellular immunity. Methods PPE36 gene was amplified by PCR using the genomic DNA of M. tuberculosis H37RV as a template and inserted into expression vector pEasy. The constructed recombinant plasmid PEasy-PPE36 was transformed to E. coli BL21 （DE3）, and the expressed PPE36-p27 protein was identified by SDS-PAGE, purified by Ni-NTA chromatography and used as a specific stimulator to develop a novel ELISPOT-IL-12 assay for T cells. Results Restriction analysis and sequencing proved that recombinant plasmid pEasy-PPE36 was constructed correctly. The expressed PPE36-p27 protein, with a relative molecular mass of about 30 000, reached a concentration of 14. 69/xg/mL after purification, and stimulated the production of IL-12 in ELISPOT-IL-12 assay. The produced IL-12 showed specific binding to the coated antibody against IL-12, which formed obvious spots. Conclusion PPE36-p27 protein was successfully expressed in prokaryotic cells, with which an ELISPOT-IL-12 assay was developed and used t＇or the early diagnosis of tuberculosis.