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目的制备兔抗MDA-7/IL-24抗体,并鉴定其特异性。方法从经PHA刺激培养的人外周血淋巴细胞中用RT-PCR扩增mda-7/IL-24基因,构建带His6-tag的原核表达载体pET-28a(+)-mda-7/IL-24,并在大肠杆菌中表达。将表达产物进行SDS-PAGE后,切下含有目的蛋白的胶条,免疫新西兰纯种大白兔,收集免疫兔血清,用Westernblot检测抗血清与大肠杆菌表达的重组蛋白His6-MDA-7/IL-24和肺癌细胞A549中重组腺病毒Ad.mda-7/IL-24表达产物的反应性。结果经IPTG诱导表达的His6-MDA-7/IL-24融合蛋白的相对分子质量(Mr)约为28000,主要为包涵体形式。Westernblot证实,表达产物可与抗His6单克隆抗体(mAb)特异性结合。1∶5000的抗血清仍能结合His6-MDA-7/IL-24融合蛋白;稀释为1∶1000时,能与重组腺病毒Ad.mda-7/IL-24在A549细胞中的表达产物结合。结论成功地制备兔抗MDA-7/IL-24抗体,该抗体能够识别体外过表达的MDA-7/IL-24蛋白。
Objective To prepare rabbit anti-MDA-7/IL-24 antibody and identify its specificity. Methods The mda-7/IL-24 gene was amplified by RT-PCR from human peripheral blood lymphocytes stimulated by PHA to construct the prokaryotic expression vector pET-28a(+)-mda-7/IL- with His6-tag. 24, and expressed in E. coli. After the expression product was subjected to SDS-PAGE, the strip containing the target protein was excised, New Zealand purebred white rabbits were immunized, and the immunized rabbit serum was collected, and the recombinant protein His6-MDA-7/IL-expressed by the antiserum and E.coli was detected by Western blot. 24 and reactivity of the recombinant adenovirus Ad.mda-7/IL-24 expression product in lung cancer cell A549. Results The relative molecular mass (Mr) of His6-MDA-7/IL-24 fusion protein induced by IPTG was about 28000, mainly in the form of inclusion bodies. Westernblot confirmed that the expression product can specifically bind to anti-His6 monoclonal antibody (mAb). 1:5000 antisera can still bind His6-MDA-7/IL-24 fusion protein; when diluted 1:1000, it can bind to recombinant adenovirus Ad.mda-7/IL-24 expression products in A549 cells. . Conclusion The rabbit anti-MDA-7/IL-24 antibody was successfully prepared, which can recognize the over-expressed MDA-7/IL-24 protein in vitro.