重组钩端螺旋体多抗原位点同源合成基因的表达及其抗原性鉴定

Expression and antigenicity identification of several linked antigen epitopes of consensce genes of Leptospira

目的:构建重组钩端螺旋体(钩体)优势抗原表位原核表达系统,以期获得高纯度的钩体优势抗原表位重组融合蛋白,为进一步研究钩体快速检测方法奠定基础。方法:利用基因重组的方法构建表达质粒pET-rLP。经IPTG诱导后,获得高效表达带组氨酸标签的以包涵体形式存在的融合蛋白,包涵体蛋白经尿素变性溶解,采用镍离子亲和纯化,利用SDS-PAGE、Western blotting和间接ELISA法鉴定。结果:在相对分子质量为20 000处有1条特异性蛋白条带,为重组钩体优势抗原表位融合蛋白;ELISA结果显示其可与钩体阳性血清特异性结合,而与其他血清无交叉反应。结论:成功构建重组钩体优势抗原表位原核表达系统,并获得高纯度的重组钩体优势抗原表位融合蛋白。

Objective To construct the prokaryotic expression system of the recombinant advantage antigen epitopes of Leptospira,and to gain the high purity expression products of the recombinant advantage epitope fusion protein of Leptospira and to provide the basis for research on the rapid method to detect Leptospira.Methods The plasmid pET-rLP was constructed by gene recombinant method.The fusion protein with His-tag was efficiently expressed in the form of inclusion body after IPTG induction.The inclusion body was washed by urea,dissolved and purified by Ni2＋ affinity chromatography,and it was identified by SDS-PAGE,Western blotting and ELISA methods.Results There was a specific protein band,which identified the high expression of the recombinant advantage epitope fusion protein of Leptospira by SDS-PAGE analysis and Western blotting,at the molecular weight of 20 000.The ELISA results showed specific reactions with Leptospirosis positive sera,but no cross-reaction with the other positive sera sample（salmonella） using the expression protein.Conclusion The prokaryotic expression system of the recombinant advantage antigen epitopes of Leptospira is constructed successfully,and the recombinant advantage epitope of fusion protein of Leptospira with high purity is gained successfully.