靶向EGFR免疫毒素BI7D12-PE38KDEL的制备及其体外活性测定

发布时间：2018-01-28 02:54

本文关键词： 表皮生长因子受体纳米抗体D 免疫毒素铜绿假单胞菌外毒素PEKDEL　出处：《中国免疫学杂志》2017年04期 　论文类型：期刊论文

【摘要】：目的:构建基于EGFR纳米抗体的免疫毒素BI7D12-PE38KDEL,并检测其对EGFR肿瘤细胞的细胞毒作用。方法:采用分子克隆技术,将靶向EGFR的纳米抗体7D12以二价的形式,通过柔性连接肽(G4S)4与铜绿假单胞菌外毒素的截断形式PE38KDEL连接,构建原核表达载体p ET28a-BI7D12-PE38KDEL。然后将其转化到BL21(DE3)感受态细胞,用IPTG进行诱导表达。通过镍柱亲和层析法纯化目的蛋白,并经Western blot验证。通过流式细胞技术检测该免疫毒素与EGFR阳性细胞A549、HT29、MCF-7和EGFR阴性细胞CEM、Jurkat的结合能力后,将其按照一定的浓度梯度进行细胞毒实验研究,72 h后,使用WST-1试剂检测BI7D12-PE38KDEL对上述细胞的杀伤效果。结果:通过SDS-PAGE和Western blot实验验证,成功的制备了重组免疫毒素BI7D12-PE38KDEL,该毒素大部分以可溶性的方式表达。BI7D12-PE38KDEL能够与EGFR阳性的A549、HT29、MCF-7肿瘤细胞特异性的结合,并且对上述细胞的细胞毒作用与阴性对照组CEM和Jurkat相比具有极其显著性差异(P0.01)。结论:成功地构建了基于EGFR纳米抗体的重组免疫毒素BI7D12-PE38KDEL,该免疫毒素能特异性的结合并杀伤EGFR阳性的肿瘤细胞。
[Abstract]:Objective: to construct the immunotoxin BI7D12-PE38KDEL based on EGFR nano-antibody and to detect its cytotoxic effect on EGFR tumor cells. Methods: molecular cloning technique was used to detect the cytotoxicity of BI7D12-PE38KDEL to EGFR tumor cells. The nano-antibody 7D12 targeting EGFR was linked with the truncated form of PE38KDEL of Pseudomonas aeruginosa by flexible ligation peptide G4S4 in the form of divalent. The prokaryotic expression vector pET28a-BI7D12-PE38KDELwas constructed and transformed into BL21ODE3) -receptive cells. The expression was induced by IPTG. The target protein was purified by nickel column affinity chromatography. The results were verified by Western blot and detected by flow cytometry. The immunotoxin and EGFR positive cell line A549H T29C MCF-7 and EGFR negative cell CEM were detected by flow cytometry. After the binding ability of Jurkat, the cytotoxicity was studied at a certain concentration gradient for 72 h. WST-1 reagent was used to detect the killing effect of BI7D12-PE38KDEL on the above cells. Results: SDS-PAGE and Western blot experiments were used to verify the effect. Recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared. Most of the toxin expressed in a soluble manner. BI7D12-PE38KDEL can specifically bind to EGFR positive tumor cell line A549 / HT29 / MCF-7. The cytotoxicity of the above cells was significantly different from that of CEM and Jurkat in negative control group (P0.01). Conclusion: recombinant immunotoxin BI7D12-PE38KDEL based on EGFR nano-antibody was successfully constructed. The immunotoxin can specifically bind and kill EGFR positive tumor cells.
【作者单位】：苏州大学唐仲英血液学研究中心江苏省血液学协同创新中心;西安交通大学苏州研究生院;博生吉医药科技(苏州)有限公司;
【基金】：山东省自然科学基金资助项目(ZR2015PH029)
【分类号】：R730.51
【正文快照】： EGFR是HER家族中的一员,在大多数的上皮性肿瘤细胞上过度表达。当EGFR与其配体结合后,受体之间发生同源或异源二聚化,激活下游细胞信号通路,引起肿瘤细胞的增殖、转移等[1]。因此,针对EGFR的治疗具有特别重要的意义。免疫毒素,作为一种新颖的治疗药物已经得到了广泛的研究。它

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