副猪嗜血杆菌外膜蛋白PlpD基因的原核表达及免疫保护性分析

PROKARYOTIC EXPRESSION AND IMMUNE PROTECTION OF OUTER MEMBRANE PROTEIN PLPD OF HAEMOPHILUS PARASUIS

为研究副猪嗜血杆菌PlpD基因的免疫保护性,根据Gen Bank上已公布的PlpD基因序列设计该基因的特异性引物,以本实验室保存的副猪嗜血杆菌5型菌株为模板,PCR扩增目的片段,构建重组质粒p ET28-PlpD,并将其转至表达菌BL21(DE3)中进行诱导表达。SDS-PAGE电泳检测显示,在温度为16℃时使用终浓度为1 mmol/L IPTG诱导12 h,目的蛋白可溶性表达量最高,Western blot结果表明该目的蛋白具有较好的反应原性。将获得的融合蛋白PlpD进行纯化后制备成亚单位疫苗,经腹腔注射免疫小鼠,3免后用高于副猪嗜血杆菌的致死剂量1.5×109CFU进行攻毒,结果显示PlpD基因对小鼠具有一定的免疫保护性。

In order to study the immune protection of PlpD of Haemophilus parasuis（Hps）, specific primers were designed according to the published information and the genomic sequence of serotype type 5 of Hps kept in our laboratory was used as template for PCR amplification. Subsequently, the recombinant expression plasmid was constructed and transformed into E. coli BL21（DE3） for expression of PlpD protein. After induction with 1mmol/L IPTG for 12 h at 16℃, the recombinant PlpD protein was expressed abundantly as detected in SDS-PAGE. Western blot analysis showed that the expressed protein had good immunoreactivity. The recombinant PlpD was purified for preparation of subunit vaccine that was used to intraperitoneally immunize mice three times. The immunized mice were then challenged with 1.5×109CFU of Hps and some degree of protection was observed.