结核分枝杆菌调节蛋白RelA的原核表达及多克隆抗体制备

Preparation of polyclonal antibody and prokaryotic expression of recombinant protein RelA from Mycobacterium tuberculosis

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。

AIM： To prepare polyclonal antibodies against RelA protein of Mycobacterium tuberculosis.METHODS： RelA gene segment was inserted into pET-32a（＋） and the recombinant protein RelA was expressed in E.coli under IPTG induction.The protein was purified and identifed by SDS-PAGE and Western blot.Polyclonal antibody to RelA was got by immunizing rabbits with the protein.Quality and quantity of the antibody was identified.RESULTS： RelA gene segment was successfully inserted into pET-32a（＋） and recombinant protein RelA was obtained.The polyclonal antibody to RelA had a good specificity,and the titer reached more than 1∶ 6 400.CONCLUSION： RelA recombinant protein and rabbit anti-RelA polyclonal antibody with high specificity were obtained,which provided good tools for further studying functional characterization of RelA.