鸡LTBP1蛋白单克隆抗体的制备及鉴定

Development of Monoclonal Antibodies against Chicken LTBP1 Protein

为了研究鸡潜在转化生长因子结合蛋白(LTBP1)功能,构建原核表达重组质粒pET-30a-LTBP1,转化E.coli BL21感受态细胞并经IPTG诱导进行表达。对以包涵体形式表达的LTBP1蛋白进行变复性和亲和层析纯化,然后免疫Balb/C小鼠,利用细胞融合技术建立单克隆抗体杂交瘤细胞株,制备抗鸡LTBP1的单克隆抗体。通过间接酶联免疫吸附试验(iELISA)筛选,获得了2株杂交瘤细胞株3C11-A9和2B1-G7。以效价较高的3C11-A9制备单克隆抗体腹水,间接免疫荧光试验(IFA)和蛋白免疫印迹(Western blot)试验结果均表明,单克隆抗体3C11-A9可特异性识别鸡源细胞CEF和DF-1表达的LTBP1蛋白,同时IFA结果还显示,3C11-A9单克隆抗体腹水可与包括人、猴、猪和鼠在内的8种常见哺乳动物细胞发生特异性染色。综上,成功制备了抗鸡LTBP1单克隆抗体,且具有多物种广谱识别特性。

In order to study the function of latent transforming growth factor binding protein 1(LTBP1),the recombinant eukaryotic expression plasmid,pET-30a-LTBP1,was constructed and transformed into E.coli BL21 competent cells for the IPTG-induced protein expression.The recombinant LTBP1 protein was purified by denaturation and affinity chromatography,and used to immunize Balb/C mice.Cell fusion and hybridoma technology were performed to produce hybridomas and develop the monoclonal antibodies(mAbs)against chicken LTBP1 protein.The supernatant of specific antibodies were screened by indirect enzyme-linked immunosorbent assay(iELISA),and two hybridoma cell lines named as 3C11-A9 and 2B1-G7 steadily secreting antibodies against LTBP1 protein of chicken were obtained.The cell line 3C11-A9 with higher titers was chose for the preparation and purification of mAb in mouse ascites.Both of the indirect immunofluorescence assay(IFA)and Western blot analysis indicated that the 3C11-A9 mAb specifically reacted to chicken LTBP1 protein,and the IFA also demonstrated that 3C11-A9 mAb specifically stained the LTBP1 proteins expressed in eight cell lines derived from mammalians,including human being,monkey,pig and mouse.These data indicate that the chicken LTBP1-specific mAbs have been successfully developed,with a wide usage for the LTBP1.