【摘要】 根据GenBank中鸡pIgR基因序列和蛋白序列,设计引物,利用PCR技术扩增胞外配体结合区片段,构建原核重组表达质粒pET-32a(+)/pIgR,将重组质粒转化到大肠杆菌BL21(DE3),经IPTG的诱导表达融合蛋白,通过镍离子螯合柱纯化后免疫新西兰大白兔,获得兔抗鸡pIgR多克隆抗体,通过酶联免疫吸附试验(ELISA法)和Western Blot法检测抗体的效价和特异性。结果显示,成功制备特异性的鸡pIgR抗体,为研究鸡pIgR的功能提供有力依据。更多还原

【Abstract】 A pair of primers were designed clone the extracellular ligand binding area by RT-PCR on the basis of polymeric immunoglobulin receptor of chicken’s gene sequence and protein sequence from GenBank.The amplified segment was inserted to the expression plasmid pET-32a(+),and then the prokaryotic expression vector pET-32a(+)/pIgR was constructed.The plasmid was transformed into BL21(DE3)and induced by IPTG.The recombinant proteins were purified by using Ni2+-NTA chelating column.The purified proteins were inoculated into New Zealand adult rabbits to develop polyclonal antibody against polymeric immunoglobulin receptor of chicken.Enzyme-linked immunosorbent assay(ELISA) and Western blot were performed to evaluate the specificity of the prepared antibody.The results showed that the prokaryotic expression vector pET-32a(+)/pIgR was successfully constructed in this experiment.The polyclonal antibody of polymeric immunoglobulin receptor in chicken was generated and could be used to study the function of polymeric immunoglobulin receptor in chicken.更多还原