2型猪链球菌层粘连蛋白结合蛋白的原核表达及免疫原性检测

Expression and immunogenicity identification on laminin binding protein of Streptococcus suis 2

目的表达2型猪链球菌(Streptococcus suis2,S.suis2)层粘连蛋白结合蛋白(Lmb)并检测其免疫原性。方法 PCR检测lmb基因在不同血清型S.suis中的分布。将S.suis2中国强毒株05ZYH33的lmb基因克隆至表达载体pET32a,转化大肠杆菌E.coliBL21,IPTG诱导表达,His亲和层析柱纯化重组蛋白。Western blot检测Lmb的免疫原性。结果 lmb基因存在于大多数S.suis血清型中。诱导表达并纯化后获得较高纯度的重组蛋白。重组Lmb能够和感染05ZYH33全菌的猪恢复期血清反应。结论 lmb基因在S.suis不同血清型中广泛分布,Lmb在细菌感染宿主过程中表达,可以作为疫苗开发的候选分子。

To detect the distribution of lmb gene in different serotypes of Streptococcus suis（S.suis）,bioinformatics analysis of the whole genome of Streptococcus suis 2（S.suis 2） was carried out to find the lmb gene.The results indicated that the ORF SSU05-0330 encoded the lmb gene of S.suis 2 virulent strain 05ZYH33.PCR with a pair of primers specific to lmb showed that lmb gene could be found in 30 S.suis serotypes.The lmb encoding ORF from the genomic DNA in the virulent strain 05ZYH33 was amplified by PCR using a pair of specific primers and subcloned into pET32a expression vector with double digestion of EcoRⅤ and XhoI.Subsequently,the recombinant plasmid was transformed to E.coli BL21（DE3） after the identification of restriction endonuclease digestion and DNA sequencing.After induction with IPTG,E.coli cells expressing HtpS were harvested by centrifugation and lysed by sonication.Following sonication,bacterial lysate was subjected to centrifugation for the removal of the insoluble pellets.The supernatant was filtered with a 0.22 μm pore-size filter and purified using a Ni-NTA column.SDS-PAGE demonstrated that E.coli BL21 containing the recombinant plasmid could express a distinct band with a molecular weight of 50 kDa,which was similar to the predicted band of recombinant Lmb protein.Western blot was carried out to detect the immunogenicity of Lmb,and sera of convalescent-phase swine collected from SPF-pigs survived from infection by S.suis 2 05ZYH33 were used as the first antibody.The result showed that recombinant Lmb could react with convalescent-phase sera from pigs infected by S.suis 2,indicating that HtpS was expressed and exposed in vivo and could be recognized by the immune system and elicit a host response during natural infection of S.suis 2.Taken together,Lmb is an in vivo expressed immunogenic protein,and potential to be a vaccine candidate of S.suis 2.