鸡堆型艾美耳球虫Rhomboid蛋白的原核表达及其多克隆抗体的制备和鉴定

Prokaryotic expression of chicken Eimeria acervulina Rhomboid protein and preparation and characterization of its polyclonal antibody

为了研究堆型艾美耳球虫Rhomboid蛋白的生物学功能及其作为潜在免疫抗原蛋白的可能性,根据Gen Bank上柔嫩艾美耳球虫Rhomboid基因序列(DQ323509.1)设计特异性引物,以堆型艾美耳球虫c DNA为模板PCR扩增包含Rhomboid基因ORF的片段。根据测序结果及抗原表位分析,设计表达区域特异性引物,PCR扩增后连接于p GEX-6P-1原核表达载体成功构建p GEX-Rhomboid表达质粒。将此质粒转入大肠杆菌Rosetta感受态细胞中进行IPTG诱导表达,SDS-PAGE分析显示有36 ku的预期蛋白表达。蛋白纯化复性后免疫新西兰大白兔制备兔抗Rhomboid多克隆抗体。ELISA方法检测该多克隆抗体效价达到216,Western-blot及瞬时转染pc DNA-Rhomboid质粒表明,制备的多克隆抗体与堆型艾美耳球虫子孢子目的蛋白和真核质粒瞬时转染BHK21细胞表达蛋白均具有良好的反应性。本试验结果为进一步研究堆型艾美耳球虫Rhomboid蛋白的生物学活性奠定了基础。

In order to study the biological functions of Eimeria acervulina Rhomboid protein and its protential as immunizing antigen, specific primers were designed according to Rhomboid gene sequence（DQ323509.1） retrieved from Gen Bank.Using E.acervulina c DNA as template, interested gene fragment containing the Rhomboid gene ORF was amplified by PCR. After sequencing and epitope analysis, specific primers for protein expression were used to amplify the truncated Rhomboid gene. Then it was ligated into p GEX-6P-1 vector and constructed prokaryotic expression plasmid successfully. The plasmid was transfected into Escherichia coli Rosetta competent cells and inducted with IPTG. SDS-PAGE method showed the expression of expected protein with the size of 36 ku. After purification and renaturation,the recombinant protein was used to immune New Zealand white rabbits for preparation of polyclonal antibody. The antibody titer was up to 216 detected by ELISA. Western-blot and transient tansfection of pc DNA-Rhomboid showed the polyclonal antibody had a good reactivity with Rhomboid protein in E.acervulina sporozoites and transfected BHK21 cells.It laid the foundation for further studies on biological activities of E.acervulina Rhomboid protein.