扩展莫尼茨绦虫vasa基因的原核表达及多克隆抗体的制备

Prokaryotic expression and preparation of polyclonal antibodies of vasa gene in Moniezia expansa

试验旨在表达、纯化扩展莫尼茨绦虫VASA蛋白,并制备兔抗VASA多克隆抗体。根据扩展莫尼茨绦虫全基因核苷酸序列设计特异性引物,利用PCR方法扩增vasa基因片段;将扩增产物与原核表达载体pET-22b(+)连接,获得重组质粒pET-22b-VASA;经Amp抗性筛选阳性菌,双酶切、PCR测序鉴定后,将其转化大肠杆菌(DE3)感受态细胞中,并进行IPTG诱导表达;重组融合蛋白经镍柱纯化后,进行Western blot鉴定;将融合蛋白免疫兔,制备多克隆抗体。结果显示,重组质粒pET-22b-VASA构建正确,通过IPTG诱导获得大小约32200的VASA重组融合蛋白;Western blot分析表明其与鼠抗His单克隆抗体呈阳性反应。纯化的VASA蛋白免疫兔获得了多克隆抗体,ELISA检测其效价为1∶128000。本试验成功制备了具有免疫原性的VASA蛋白及其兔源多克隆抗体,为VASA蛋白生物学功能及该蛋白在绦虫体内的分布等研究奠定基础。

The aim of the experiment was to express and purify porcine VASA protein and prepare rabbit anti-VASA polyclonal antibody.Specific primer was designed according to nucleotide sequences of vasa in Moniezia expansa.vasa gene fragment was amplified by PCR,and the amplified product was connected with prokaryotic expression vector PET-22 b(+)to obtain recombinant plasmid pET-22 b-VASA.After identification,it was transformed into DE3,which expressed by IPTG.The recombinant fusion protein VASA was purified by Ni column,then it was identified by Western blot.The rabbit was immunized by recombinant fusion VASA in order to prepare polyclonal antibodies.The results showed that the recombinant plasmid pET-22 b-VASA was constructed correctly.The recombinant fusion protein VASA was about 32200.Western blot analysis showed that the recombinant protein was positive for mouse anti-His monoclonal antibody.The purified recombinant fusion protein VASA was used to immunize rabbit to obtain polyclonal antibodies.ELISA titer was 1∶128000.In this experiment,the recombinant fusion protein VASA and its rabbit polyclonal antibody were successfully prepared,which laid a foundation for the biological function of VASA protein and the distribution of the protein in Moniezia expansa.