牛分枝杆菌PtpA基因的原核表达及其多克隆抗体的制备

Expression of PtpA gene of Mycobacterium bovis and preparation of polyclonal antibody

为深入研究牛分枝杆菌PtpA基因在细胞免疫过程中所发挥的具体调控作用。本研究以牛分枝杆菌基因组为模板扩增得到PtpA基因，通过分子克隆的方法构建完成PtpA基因原核表达载体．并将表达载体质粒转入感受态细菌BL21（DE3）中，经IPTG诱导表达后进行SDS—PAGE分析，在约35ku处可见特异性蛋白条带。以抗组氨酸标签（HIS）单抗为一抗，对表达产物进行Western—blot检测，结果显示，表达产物可与抗HIS标签单克隆抗体发生特异性结合，证明该蛋白为所需的PtpA蛋白。通过免疫新西兰大白兔获得抗PtpA血清，所得血清用间接ELISA方法测得的血清抗体效价超过1：1000000．具有较好的灵敏性。采用制备的PtpA多克隆抗体检测表达纯化的PtpA融合蛋白及真核表达的PtpA蛋白．结果可在约35ku和约20ku处产生特异性条带，表明该抗体有较强的特异性。本研究为后续PtpA基因调控细胞免疫的具体机制的研究奠定了基础。

To better understand the specific regulatory role of the PtpA gene in cell immunity,The PtpA gene was amplified from the Mycobacterium boris DNAgenome as the template. The prokaryotic ex- pression vector of PtpA gene was constructed by molecular cloning. The expression vector was trans- ferred into competent bacteria BL21（DE3） and induced by IPTG after expression,SDS-PAGE analysis showed the size of expressed protein was about 35 ku. the results showed that the expressed product bands could specifically bind to the anti-HIS-labeled monoclonal antibody. The results showed that the expressed PtpA protein could specifically bind to the anti-HIS-labeled monoclonal antibody. The anti-PtpA serum was obtained by immunizing New Zealand white rabbits, the titer of serum antibody obtained by indirect ELISA was exceeding 1：I 000 O00,which showed good sensitivity. And the prokaryotic expression PtpA protein and the eukaryotic expression PtpA protein can be bind to PtpA polyclonal antibody,which indi- cating that the antibody has high specificity. This study laid the foundation for the study of the par- ticular mechanism of the subsequent PtpA gene regulation of cellular immunity.