鸡细胞周期蛋白依赖性激酶抑制蛋白p21单克隆抗体的制备

Generation of monoclonal antibody against chicken cyclin-dependent kinase inhibitor p21

为制备特异性的鸡细胞周期蛋白依赖性激酶抑制蛋白p21单克隆抗体(mAb),利用反转录聚合酶链式反应(RT-PCR)扩增鸡p21基因,将其克隆入原核表达载体pET-30a中,随后转化进入BL21(DE3)中,利用异丙基硫代半乳糖苷(IPTG)诱导表达,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离。切胶研磨,将收集的鸡p21蛋白腹腔免疫BALB/c小鼠,三免后,选取抗体阳性的小鼠脾与小鼠骨髓瘤细胞SP2/0融合并筛选单抗。结果显示,经亚克隆纯化,共筛选获得12株阳性杂交瘤细胞株,即1B12、1E12、1H7、2B5、2C5、2C8、2D8、2F2、3C11、3D6、3G7和4G6。利用间接免疫荧光(IFA)、酶联免疫吸附法(ELISA)和免疫印迹检测单抗效价。成功制备了特异性鸡p21单抗。这为进一步研究鸡p21生物学功能奠定了基础。

To prepare monoclonal antibody against chicken p21,the p21 gene was amplified by reverse transcription polymerase chain reaction（Rr-PCR） and subcloned into the prokaryotic expression vector, pET-30a. Therecombinant plasmid,designated as pET-30a-p21,was transformed into E.coli BL21 （DE3）compe- tent cells. Separated by SDS-PAGE,the induced protein was shown to be expressed as inclusionbodies. The positive bands were cut,ground and used as antigens to immunize BALB/c mice intraperitoneally for three times. The spleenwas isolated and fusedwith SP2/0. Hybridoma clones that produced antibodies against p21 were screened by IFA,ELISA and limited dilution. The antibody levels were detected by indire- ct immunofluorescence assay （IFA）,enzyme-linked immunosorbent assay （ELISA） and Western-blot. Twelve positive hybridoma clones were screened and identified as IBI2,1E12,1H7,2B5,2C5,C8,2D8,2F2,3CII,3D6, GTand 4G6. The monoclonalantibody against p21 was prepared as a tool for the study of p21 functions.