空肠弯曲菌粘附蛋白PEB1原核表达、纯化及多克隆抗体制备  

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

作　　者：简文[1] 史皆然[1] 樊爱琳[2] 薛莹[3] 李圣青[1] 柏银兰[4] 戚好文[1] 

机构地区：[1]第四军医大学西京医院呼吸内科,陕西西安710032 [2]第四军医大学西京医院检验科,陕西西安710032 [3]第四军医大学西京医院放疗科,陕西西安710032 [4]第四军医大学基础部微生物教研室,陕西西安710032

出　　处：《武警医学院学报》2010年第4期271-273,F0003,共4页Acta Academiae Medicinae CPAPF

摘　　要：【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。[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.

关 键 词：空肠弯曲菌 粘附蛋白 原核表达 纯化 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]