多杀性巴氏杆菌PurF蛋白的原核表达及多克隆抗体制备

Prokaryotic Expression and Preparation of Polyclonal Antibodies against Pasteurella multocida PurF Protein

为了获得多杀性巴氏杆菌PurF蛋白及其多克隆抗体,通过PCR扩增了多杀性巴氏杆菌C51-17株purF基因,将其克隆到表达载体pPROEX-HTa中,构建重组表达质粒pHT-PurF,转化至大肠埃希菌BL21(DE3)感受态细胞中,经IPTG诱导表达。SDS-PAGE检测表明,获得重组蛋白分子质量约56.5ku,主要以包涵体形式存在;Western blot检测表明,表达的蛋白可与多杀性巴氏杆菌制备的高免血清发生特异性反应。将制备的重组蛋白免疫Balb/c小鼠制备多克隆抗体,ELISA检测血清抗体效价,结果显示制备的多克隆抗体滴度达到1∶128 000,Western blot表明可与多杀性巴氏杆菌PurF蛋白发生反应。本研究制备了多杀性巴氏杆菌的PurF蛋白及其多克隆抗体,为进一步研究PurF蛋白在多杀性巴氏杆菌致病中的作用奠定了基础。

To acqmre the recombinant PurF protein of Pasteurella multocida and the polyclonal antibodies of PurF,the purF gene of C51-17 strain of P. multocida was amplified by PCR and cloned into a prokary otic expressive plasmid pPROEX-HTa. The recombinant plasmid pHT-purF was transformed into E. coli BL21（DE3） and induced by IPTG. SDS-PAGE showed that the recombinant protein was about 56.5 ku andin the form of inclusion body. Western blot analysis indicated that the rPurF protein was recognized by the positive serum of P. rnultocida. Balb/c mice were immunized with rPurF protein to prepare polyclonal antibodies. The ELISA titers of polyclonal antibodies could reach as high as 1 ： 128 000. The polyclonal antibodies were recognized by the native PurF protein from P. multocida. The polyclonal antibodies of PurF were the fundament to further study the role of PurF protein in the pathogenesis of P. multocida.