结核分枝杆菌mpt59和esxA融合基因编码蛋白的原核表达及免疫原性观察

Prokaryotic expression and immunogenicity analysis of mpt59 and esx A fusion gene from Mycobacterium tuberculosis

目的:获取结核分枝杆菌mpt59和esx A基因编码的原核表达融合蛋白,并将该蛋白初步用于结核患者的快速诊断。方法:PCR扩增含有柔性链接肽段的esx A核苷酸序列,并将其连接至原核表达载体p ET28a上,再将mpt59连接至p ET28a-esx A上。该融合蛋白在大肠杆菌BL21中原核表达后,用镍珠子进行亲和纯化。采用垂直电泳和免疫印迹分析重组蛋白,并用于结核患者的诊断。结果:成功构建了原核表达载体p ET28a-esx A-mpt59,转化大肠杆菌BL21后经诱导产生了高水平的表达产物。用该纯化蛋白进行ELISA检测,结果表明其诊断结核的灵敏度为97.8%,特异度为100%。结论:构建了含mpt59和esx A融合抗原基因的原核表达载体,并诱导表达了融合蛋白,为进一步研究结核分枝杆菌疫苗或诊断试剂奠定了基础。

Aim：To obtain bacterially expressed prokaryotic protein encoded by mpt 59 and esxA from Mycobacterium tuberculosis , and to apply this protein for rapid diagnosis of tuberculosis patients .Methods：The nucleotide sequence of es-xA gene containing flexible peptide linker was obtained from Mycobacterium tuberculosis using PCR and was linked to pET28a expression vector .Then mpt59 gene was cloned into the pET 28a-esxA.The fusion protein was expressed in E .coil BL21 and purified by Ni-NTA.The purified fusion protein was confirmed by SDS-PAGE and Western blot analysis , and was used for TB diagnosis .Results：The recombinant expression vector pET 28a-esxA-mpt59 was successfully constructed . The E.coli BL21 strains with recombinant vector showed high level expressions of fusion protein after IPTG induction .The ELISA assay with purified protein for detecting Mycobacterium tuberculosis antibody showed a sensitivity of 97.8% and a specificity of 100%.Conclusion：The expression of recombinant fusion protein of Mycobacterium tuberculosis mpt 59 and esxA lays a basis for further studies on researches such as vaccine or diagnostic reagent .