幽门螺杆菌HP0762蛋白的原核表达纯化及多克隆抗体制备

Expression and Purification of the Recombinant HP0762 from HeliCobacter pylori and Preparation of its Polyclonal Antibody

目的:表达和纯化幽门螺杆菌HP0762蛋白,并制备该蛋白的多克隆抗体。方法:从幽门螺杆菌SS1中经PCR扩增得到了hp0762基因,将其克隆至含有6×His编码序列的原核表达载体pET-28a(+)中,再将重组质粒转化大肠杆菌BL21(DE3),在IPTG诱导下进行蛋白表达;用HiTrap Chelating HP亲和柱纯化重组蛋白,Western印迹进一步鉴定;以纯化后的蛋白为抗原免疫新西兰大耳白兔,制备该蛋白的多克隆抗体;用ELISA和Western印迹检测抗血清。结果:目的蛋白在大肠杆菌BL21(DE3)中获得了可溶性表达,纯化后纯度可达90%以上;制备了针对HP0762重组蛋白的抗血清,抗体ELISA效价为1:256000,Western印迹分析表明该抗体能特异性识别内源性HP0762。结论:完成了HP0762蛋白的原核高效表达与纯化,并制备了其高效价的多克隆抗体,为进一步对其进行疫苗研制与基因功能研究奠定了基础。

Objective：To obtain the recombinant HP0762 protein from Helicobacter pylori SS1 and prepare its antibody used for further studies.Methods：The sequence of hp0762 was obtained by PCR from H.pylori SS1 genome and was cloned into the prokaryotic expression vector pET-28a （＋） with 6×His coding sequence.The recombinant plasmid was transformed into host strain E.coli BL21（DE3） and the recombinant HP0762 was expressed by IPTG induction.The recombinant protein was purified through HiTrap Chelating HP affinity columns and then identified by Western blotting analysis.Purified protein was used as the antigen to immunize New Zealand rabbits for three times to raise polyclonal antibody.Antiserum was analyzed by ELISA and Western blot.Results：Reconstructed hp0762 gene was successfully expressed in E.coli BL21（DE3） in soluble form,when purified with Ni column,HP0762 protein covered more than 90% total proteins.The polyclonal antibody against the protein with high titer was obtained,ELISA assay titer of antiserum from vaccinated rabbits reached 1：256 000.The results of Western blotting showed HP0762 protein exhibited a better immunogenicity and its polyclonal antibody could specifically recognize endogenous HP0762 protein.Conclusion：The HP0762 protein was successfully purified and the antiserum with high titer was obtained,which will lay the foundation for further research of the vaccine and the gene function.