鸡异质核糖核蛋白AB单克隆抗体的制备及鉴定

Preparation and Identification of Specific Monoclonal Antibody against Chicken hnRNPAB Protein

为研制鸡异质核糖核蛋白AB(hnRNPAB)单克隆抗体,为其生物学功能研究提供关键试剂,构建重组质粒pET-30a-hnRNPAB,转化感受态细胞E.coli BL21,经IPTG诱导表达,并通过树脂Ni对可溶性表达的鸡hnRNPAB蛋白进行亲和层析纯化之后免疫Balb/c小鼠,利用细胞融合技术制备抗鸡hnRNPAB的单克隆抗体细胞株,最终通过间接酶联免疫吸附试验(iELISA)筛选获得1株单克隆抗体杂交瘤细胞株5H2-H1。间接免疫荧光试验(IFA)和蛋白质免疫印迹(Western blot)分析表明,5H5-H1单克隆抗体可特异性识别鸡胚成纤维细胞(CEF)表达的hnRNPAB蛋白。同时IFA和间接免疫细胞化学试验(ICA)结果还显示,除了特异性识别鸡hnRNPAB蛋白之外,5H5-H1单克隆抗体还可以与源自人、鼠、猴、牛和猪在内的5种常见哺乳动物细胞系表达的hnRNPAB发生特异性反应。

In order to generate the specific monoclonal antibody(m Ab)against chicken heterogeneous nuclear ribonucleoprotein AB(hnRNPAB)for its future function study,the recombinant prokaryotic expression plasmid pET-30a-hnRNPAB was constructed and transformed into E.coli BL21 competent cells for the IPTG-induced protein expression.The soluble recombinant hnRNPAB protein was purified by Ni affinity chromatography resin and used as antigen to immunize Balb/c mice.Cell fusion technology was performed to develop the hybridomas.One m Ab named as 5H5-H1 against chicken hnRNPAB protein was screened by indirect enzyme-linked immunosorbent assay(iELISA).The indirect immunofluorescence assay(IFA)and Western blot analysis indicated that the 5H5-H1 m Ab specifically reacted to hnRNPAB protein expressed in chicken embryo fibroblast(CEF).Furthermore,the IFA and indirect immunocytochemistry assay(ICA)demonstrated that in addition to chicken hnRNPAB protein,the mAb 5H5-H1 also recognized the native hnRNPAB proteins expressed in five commonly used mammalian cell lines derived from human,hamster,monkey,bovine and swine.