结核分枝杆菌Mtb81蛋白的表达及应用研究

目的克隆结核分枝杆菌Mtb81蛋白的编码基因Rv1837c,并在大肠杆菌中表达、纯化,获得重组蛋白Mtb81。方法以结核分枝杆菌H37Rv基因组为模板,PCR扩增Rv1837c基因序列,克隆入原核表达载体pET-28a,构建重组表达质粒,转化大肠杆菌后用IPTG诱导表达,纯化表达产物,获得重组Mtb81。ELISA检测重组Mtb81蛋白的敏感性和特异性。结果重组质粒pETRv1837c测序表明具有正确的编码序列,质粒构建成功,重组蛋白Mtb81在大肠杆菌中以包涵体和可溶性形式稳定表达。以Mtb81为抗原ELISA检测结核病患者血清抗体总敏感性为20.83%,特异性为95.83%。结论结核分枝杆菌Mtb81重组蛋白能在大肠杆菌工程菌种成功表达,为进一步的应用研究奠定基础。

Prokaryotic expression of Mtb81 protein of Mycobacterium tuberculosis application

Objective To obtain the recombinant Mtb81 protein of M. tuberculosis. Methods By the utilization of PrimerPremier 5.0 software and according to the DNA sequences of the Mtb81 protein, a pair of primers was designed for amplification of the target gene from H37Rv. Mtb81 was inserted into the expressional plasmids pET-28a. The recombinant plasmid was transformed into the competent cells of E. coli BL21（DE3） strains. The positive transformant was selected with IPTG inducement. Results The recombinant expression plasmid was constructed. The Mtb81 protein existed in the inclusion bodies of E. coli. ELISA results showed that the sensitivity and specificity of the antibody to the recombinant fusion protein Mtb81 were 20.83% and 95.83% respectively. Conclusion The recombinant protein has successfully been expressed and purified.