猪源多聚免疫球蛋白受体的表达及抗血清的制备

Expression and antiserum preparation of porcine polymeric immunoglobin receptor

为了制备猪源pIgR蛋白的多克隆抗体,分析NCBI提供的猪源pIgR的胞外区基因序列并设计引物,经过PCR、酶切、连接,将目的序列克隆入原核表达载体pET-30a(+),测序验证成功后,将带有目的片段的重组质粒转化入大肠杆菌(E.coli)BL21(DE3)感受态细胞,经IPTG诱导表达,通过Ni-NTA亲和层析的方法获得了较为纯净的pIgR蛋白。用纯化后的pIgR与佐剂混合后免疫日本大白耳兔,制备兔源的多克隆抗体。结果显示,本研究正确构建了带有pIgR基因的重组质粒,并成功表达pIgR蛋白,预测的分子质量约为82 ku;ELISA测定结果显示,pIgR蛋白免疫日本大白耳兔获得的多克隆抗体效价高,且在稀释至1∶3200时依然具有良好的反应性;Western-blot和IFA检测结果显示,制备的pIgR多克隆抗体特异性高、敏感性好。结果表明,本次研究为后续深入探索猪机体黏膜免疫过程中pIgR相关的分子机制奠定了基础,为后期制备猪源p Ig R的单克隆抗体提供了前期的物质准备。

In preparation for the polyclonal antibodies against porcine pIgR protein,primers were designed according to the extracellular domain of porcine pIgR gene sequence.The target sequence was cloned into the prokaryotic expression vector p ET-30 a(+)via PCR,enzyme digestion and ligation.The successfully constructed recombinant plasmid was transformed into E.coli BL21(DE3)competent cells to express the protein induced by IPTG.Japanese white-eared rabbits were immunized with the mixture of purified pIgR protein obtained using Ni-NTA affinity chromatography and adjuvant to prepare rabbit-derived polyclonal antibody.The results showed that the recombinant plasmid was correctly constructed and pIgR protein was successfully expressed;the prepared antibody could pIgR protein in Western-blot,indirect immunofluorescence assay and ELISA.In conclusion,the prepared polyclonal antibody provides a basis for the study of pIgR in porcine mucosal immunity,and helps to the further preparation of monoclonal antibody against porcine pIgR.