猪丹毒杆菌表面抗原SpaA的原核表达纯化及免疫原性验证

Prokaryotic expression,purification and immunogenicity verification of SpaA protein of Erysipelothrix rhuriopathiae surface antigen

本研究制备了一种具备优良免疫原性的猪丹毒杆菌表面抗原重组蛋白。采用基因调取的方式,构建质粒并亚克隆到表达载体pET28a上,进一步将改造质粒转化到大肠杆菌感受态BL21细胞中,构建了1株猪丹毒SpaA全基因重组表达菌株。之后通过IPTG诱导表达、镍离子层析柱分离纯化、梯度透析获得高纯度重组SpaA蛋白。经免疫攻毒试验验证,重组SpaA蛋白能够有效保护小鼠和猪免于猪丹毒杆菌强毒攻击,其中小鼠的重组SpaA蛋白最小免疫剂量为每只2.0μg,100μg的重组SpaA蛋白能够保护猪免于猪丹毒杆菌强毒株感染。以重组SpaA蛋白作为组分与猪多杀性巴氏杆菌病活疫苗共同免疫小鼠,结果显示该混合疫苗对于猪丹毒杆菌及猪多杀性巴氏杆菌均有良好的保护效果。本研究结果证实,重组SpaA蛋白是我国现行猪丹毒杆菌传统灭活疫苗升级换代的理想候选抗原,同时也具备良好的抗原相容性,其高纯度及低免疫剂量的特点也为其他组分提供了充足的剂量空间。

In this study,a recombinant protein with excellent immunogenicity was prepared.The target gene was transferred and subcloned into pET28a expression vector.The modified plasmid were further transformed into the Escherichia coli strain BL21(DE3),and a whole gene recombinant expression strain of Erysipelothrix rhuriopathiae SpaA was constructed.After that,the highly purified recombinant SpaA protein was obtained by IPTG induced expression,nickel ion chromatography column separation and purification,and gradient dialysis.The immune challenge test verified that the recombinant SpaA protein could effectively protect mice and pigs from the virulent attack of Erysipelothrix rhuriopathiae.The minimum immune dose of the recombinant SpaA protein was 2.Oμg/mouse,and 100μg recombinant SpaA protein could protect pigs from the infection of virulent strain of Erysipelothrix rhuriopathiae.Recombinant SpaA protein was used as a component to immunize mice with attenuated live swine pasteurella vaccine.The results showed that the mixed vaccine had qualified protective effects on swine erysipelas and swine pasteurella,which confirmed that the recombinant SpaA protein is an ideal candidate antigen for upgrading the traditional inactivated vaccine of Erysipella suis in China,and it also had desired antigen compatibility.Its high purity and low immune dose characteristics also provided sufficient dose space for other components.