2型猪链球菌组氨酸三聚体蛋白家族05SSU1267的克隆、表达及免疫保护作用研究

Cloning and expression of Histidine triad protein gene 05SSU1267 and evaluation of its protective efficacy against Streptococcus suis serotype 2

目的对2型猪链球菌(Streptococcus suis serotype 2,S.suis 2)05ZYH33组氨酸三聚体蛋白家族(Histidine triadprotein,Htp)成员编码基因05SSU1267进行克隆、表达,并研究其免疫保护作用。方法根据05ZYH33基因组编码的05SSU1267基因序列设计引物,PCR扩增目的片段。将目的基因克隆入原核表达载体pET32a,并转化E.coli BL21(DE3)。通过IPTG诱导表达、His-Tag亲和层析柱纯化获得重组蛋白,并将纯化后的蛋白用于Western blot分析。以纯化的重组蛋白免疫新西兰兔制备兔多抗血清,并通过ELISA检测多抗血清的效价。重组蛋白免疫Balb/c小鼠,研究其免疫保护作用。结果 Western blot显示重组蛋白可以与兔抗05ZYH33全菌血清反应,显示该蛋白具有较好的免疫反应原性;ELISA结果显示重组蛋白免疫新西兰兔后获得高效价的多抗血清;动物保护实验表明该蛋白具有一定的免疫保护作用。结论在原核系统成功表达了05ZYH33 Htp蛋白05SSU1267,动物实验结果表明其能够在Balb/c小鼠中发挥一定的免疫保护作用,研究结果为Htp蛋白作为2型猪链球菌疫苗候选分子以及进一步了解该蛋白家族的生物学功能奠定基础。

To determine the potential protective efficacy of gene 05SSU1267 of Streptococcus suis serotype 2（S.suis 2） strain 05ZYH33 encoding a Histidine triad protein（Htp）,we cloned and expressed the gene in this study.A pair of specific primers was designed for 05SSU1267.The target gene was amplified using the genomic template of 05ZYH33 by PCR,and then was cloned into prokaryotic expression vector pET32a.The resulting plasmid was confirmed by direct DNA sequencing and transformed into E.coli BL21.Recombinant protein was obtained after induction with IPTG and purified using His-Resin,which was then used to immunize rabbits for prepare polyclonal antibody against 05SSU1267.ELISA analysis revealed that the titer of rabbit serum reached more than 1∶1 024 000;Western blot experiment showed that the recombinant protein can react with rabbit anti-05ZYH33 serum,indicating that 05SSU1267 could be expressed in the infection process of S.suis 2 and was immunogenic.Animal experiment demonstrated that vaccinated Balb/c mice with 05SSU1267 conferred a significant protection.These results indicate that 05SSU1267 was successfully expressed in prokaryotic system and the expressed protein can serve as a vaccine candidate,which build a fundament for further study on function of Histidine triad protein of S.suis 2.