论文部分内容阅读
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。
Objective To clone and express the gene encoding SSR1267 of the member of Streptococcus suis serotype 2 (S. suis 2) 05ZYH33 Histidine triadprotein (Htp) and study its immunoprotection. Methods According to the sequence of 05SSU1267 gene of 05ZYH33 genome, primers were designed and amplified by PCR. The target gene was cloned into the prokaryotic expression vector pET32a and transformed into E. coli BL21 (DE3). The recombinant protein was induced by IPTG and purified by His-Tag affinity chromatography. The purified protein was used for Western blot analysis. Rabbit polyclonal antiserum was prepared by immunizing New Zealand rabbits with the purified recombinant protein and the titer of multiple antisera was detected by ELISA. Balb / c mice were immunized with the recombinant protein to study its immunoprotection. Results Western blot showed that the recombinant protein could react with rabbit anti-05ZYH33 whole bacteria serum and showed good immunoreactivity. ELISA showed that the recombinant protein was immunized with high titer of polyclonal antiserum. Animal protection experiments showed that Protein has a certain degree of immune protection. Conclusions 05SSY1267, a 05ZYH33 Htp protein, was successfully expressed in prokaryotic system. The results of animal experiments show that it can exert some immunoprotective effect in Balb / c mice. The result of this study is that Htp protein can be used as a candidate vaccine for Streptococcus suis type 2 vaccine, The biological function of the protein family lays the foundation.