猪源T细胞免疫球蛋白黏蛋白域蛋白-1(TIM-1)多克隆抗体制备与鉴定

Preparation and identification of polyclonal antibody against swine TIM-1 protein

为了探究猪源T细胞免疫球蛋白黏蛋白域蛋白-1(sTIM-1)的生物学功能,制备sTIM-1多克隆抗体,通过软件分析sTIM-1蛋白抗原表位及亲水性,并设计引物,以sTIM-1基因为模板扩增sTIM-1黏蛋白功能域编码基因(sTIM-1-M),成功构建了重组表达质粒pET28a-sTIM-1-M,并在大肠杆菌Rosetta 2中诱导表达。以纯化的重组蛋白sTIM-1-M与佐剂混合免疫新西兰大白兔制备多抗血清。SDS-PAGE及Western blot证实了该重组蛋白在大肠杆菌中以可溶性形式表达,分子量约为36 kDa。经Western blot与免疫荧光试验(IFA)验证sTIM-1多抗血清可特异性识别293T细胞表达的sTIM-1。免疫沉淀(IP)结果显示制备的多抗血清可与sTIM-1蛋白互作。以上数据表明本研究制备的sTIM-1多克隆抗体具有良好的反应性和特异性。sTIM-1多克隆抗体的制备为进一步研究sTIM-1参与病毒入侵细胞的机制奠定了基础。

In order to explore the biological function of sTIM-1,polyclonal antibodies against sTIM-1 were prepared in this study.We analyzed the antigenic epitopes and hydrophilicity of the sTIM-1 protein and designed the primers.The sTIM-1 mucin functional domain encoding gene(sTIM-1-M)was amplified and inserted into pET-28a to obtain the recombinant expression plasmid pET-28a-sTIM-1-M.The recombinant sTIM-1-M protein was successfully expressed in the Escherichia coli strain Rosetta 2.New Zealand white rabbits were immunized with purified sTIM-1-M and adjuvant to prepare polyclonal antibodies.The results of SDS-PAGE and Western blot confirmed that the recombinant protein expressed in a soluble form,with a molecular weight of about 36 kDa.Western blot and IFA verified that the polyclonal antibody serum could specifically recognize sTIM-1 expressed in the 293T cells.The IP results showed that the prepared polyclonal antibody could interact with th esTIM-1 protein.In summary,the polyclonal antibody against sTIM-1 had good reactivity and specificity,which laid a foundation for further study on the function of sTIM-1 during virus invasion of host cells.