空肠弯曲菌粘附蛋白PEB1原核表达、纯化及多克隆抗体制备

【目的】克隆、表达空肠弯曲菌表面蛋白PEB1,并制备针对该蛋白的多克隆抗体。【方法】采用PCR方法从空肠弯曲菌Penner19标准株基因组中扩增出PEB1蛋白基因,用限制性内切酶消化后插入到pUC-19克隆载体中,测序正确后,再亚克隆到融合表达载体pPro-EXHTb中,转化大肠杆菌DH5α,目的基因经IPTG诱导,由T7启动子调控表达了氨基端带6个连续组氨酸残基的PEB1蛋白,并经Western-blotting证实。然后,在非变性条件下用Ni-NTA亲和色谱柱纯化目的蛋白。用纯化的PEB1蛋白作为免疫原免疫BALB/c小鼠,制备多克隆抗体,用ELISA法检测抗体效价。【结果】在非变性条件下用Ni2＋-NTA亲和色谱柱纯化PEB1融合蛋白,纯化获得的蛋白纯度大于90%。用该融合蛋白免疫小鼠后,得到抗PEB1抗体,效价达1：2×105。【结论】成功构建了空肠弯曲菌PEB1基因原核表达载体,并获得了高纯度的PEB1蛋白及其高效价多抗,对进一步制备肠粘膜高亲和力的过敏原重组BCG打下了坚实的基础。

Prokaryotic expression,purification of PEB1 in Campylobacter jejuni and preparation of its polyclonal antibody

Objective]To express Campylobacter jejuni PEB1 protein in prokaryotic cells and prepare polyclonal antibody against it.. Methods]The gene encoding PEB1 was amplified from genome of Campylobacter jejuni by polymerase chain reaction（PCR） and inserted into cloning vector pUC-19. After sequenced,PEB1 gene was subcloned into the expression vector pPro-EXHTb and transformed into E.coli DH5α. After induced by IPTG,the bacteria controlled by T7 promoter expressed the fused PEB1 protein with a hexahistidine tail at its N-terminal and the target protein was identified by Western-blotting. PEB1 protein was purified by Ni-NTA purification system under native condition. Using this protein as antigen to immunize the BALB/c mice and prepare polyclonal antibody against PEB1 was prepared. Then the production of anti-body was confirmed by ELISA. Results]The fusion protein was purified by metal chelate affinity chromatography under native condition. Polyclonal antibody against PEB1 was obtained from mice immunized by PEB1,The titer of which was 1：200 000. Conclusions]The PEB1 prokaryotic expression vector has been successfully constructed. PEB1 protein has been obtained with high purity,and the obtained anti PEB1 polyclonal antibody has a high titer. All these provide useful tools for further study of the construction of anaphylactogen recombinant BCG with high adherence to intestinal epithelium.