副猪嗜血杆菌血清5型TbpA蛋白的表达及其免疫原性分析

Expression of tbpA gene from Haemophilus parasuis serotype 5 and identification of the recombinant protein

为原核表达副猪嗜血杆菌血清5型TbpA蛋白,本研究利用特异性引物扩增tbpA基因,并将其克隆到pMD18-T载体中进行序列测定。再将其亚克隆于pET-28a中构建重组表达质粒pET-tbpA。将其转化受体菌E.coli BL21(DE3),经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约106 ku,并以包涵体形式存在。表达的重组蛋白经纯化后,免疫6周龄昆明小鼠制备免疫血清,ELISA分析表明制备的血清抗体效价在1∶3 200以上,表明TbpA具有良好的免疫原性。

To express Haemophilus parasuis serotype 5 TbpA protein, the tbpA gene was amplified by PCR and cloned into vector pMD-18T for sequencing. The tbpA gene was subcloned into vector pET-28a. The resultant recombinant plasmid pET-tbpA was transformed into BL21 （DE3） and induced with IPTG. The results of SDS-PAGE and westem blot indicated that the expressed TbpA protein was about 106 ku and mainly existed in form of inclusion bodies. The anti-tbpA serum was prepared in Kunming mice immunized with purified TbpA with the titer of 1：3,200, which showed that the express product of TbpA had strong immunogenic reaction.