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目的:克隆编码结核分枝杆菌调节基因RelA并在大肠杆菌中表达,纯化后制备兔抗RelA的抗体。方法:利用PCR从结核分枝杆菌H37Rv株中扩增RelA基因,构建重组表达质粒pET-32a(+)-RelA;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行镍离子亲和层析纯化,通过SDS-PAGE和Westem blot鉴定目的蛋白的表达及反应原性;以表达的RelA蛋白免疫家兔,制备抗RelA的多克隆抗体并进行效价及特异性鉴定。结果:扩增了RelA基因,克隆于表达载体pET-32a(+)中,PCR筛选和酶切鉴定获得阳性克隆,测序证实正确。经诱导在大肠杆菌中表达出相对分子质量(Mr)为120 000的目的蛋白;纯化的RelA免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶6 400以上,且具有良好的特异性。结论:已成功构建RelA基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗RelA抗体,效价及特异性均良好,为进一步研究RelA蛋白在结核病中的致病机制奠定了基础。
OBJECTIVE: To clone the RelA gene encoding Mycobacterium tuberculosis and express it in Escherichia coli, and then purify it to prepare rabbit anti-RelA antibody. Methods: RelA gene was amplified from Mycobacterium tuberculosis strain H37Rv by PCR and the recombinant plasmid pET-32a (+) - RelA was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) The target protein was purified by nickel ion affinity chromatography under denaturing conditions. The expression and the reactivity of the target protein were identified by SDS-PAGE and Western blot. The rabbits were immunized with the expressed RelA protein to prepare anti-RelA polyclonal Antibodies were titrated and characterized. Results: The RelA gene was amplified and cloned into the expression vector pET-32a (+). The positive clones were obtained by PCR screening and restriction enzyme digestion. The sequencing was confirmed correctly. The target protein of Mr 120 000 was expressed in E.coli. The purified RelA immunized rabbits could effectively stimulate the production of specific antibodies, and the antiserum titer reached 1: 400 , And has good specificity. CONCLUSION: The prokaryotic expression vector of RelA gene has been successfully constructed and expressed highly in E. coli. Rabbit anti-RelA antibody was prepared with good titer and specificity, which laid the foundation for further study on the pathogenesis of RelA protein in tuberculosis basis.