中国免疫学杂志2017，Vol.33Issue(4)：558-562,573,6.DOI:10.3969/j.issn.1000-484X.2017.04.017
靶向EGFR免疫毒素BI7D12-PE38KDEL的制备及其体外活性测定
Preparation of immunotoxin BI7D12-PE38KDEL directed to EGFR and determination of its activity in vitro
摘要
Abstract
Objective:To prepare nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR and to examine its cytotoxicity against EGFR positive tumor cells.Methods:By using molecular cloning strategy,prokaryotic expression construct of pET28a-BI7D12-PE38KDEL was generated which consisted of nanobody 7D12 targeting EGFR in the form of a divalent fused with PE38KDEL,a truncated form of pseudomonas exotoxin A via a flexible peptide(G4S)4,and then transformed into E.coli BL21(DE3).Protein expression was induced by adding IPTG,purified by Ni-affinity column chromatography,and verified by Western blot.The binding capacity of the resulted immunotoxin to EGFR-positive cells A549,HT29,MCF-7 and EGFR-negative cells CEM,Jurkat were determined by flow cytometry assay,and its cytotoxicity against the target cells was examined.Briefly,tumor cells were treated with different dosage of the immunotoxin,and the killing efficacy of BI7D12-PE38KDEL on these cells were assessed by WST-1 assay after 72 hours.Results:The SDS-PAGE and Western blot results showed the recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared,and majority of them was expressed in soluble form.BI7D12-PE38KDEL could selectively bind to EGFR-positive cells of A549,HT29,and MCF-7.More importantly,the immunotoxin exhibited much more significant killing effect on these EGFR positive cells compared to the negative control group of CEM and Jurkat cells(P<0.01).Conclusion:In the current study,the nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR was successfully prepared and exhibited a superior inhibition effect for the growth of EGFR-positive cells.关键词
表皮生长因子受体/纳米抗体7D12/免疫毒素/铜绿假单胞菌外毒素PE38KDELKey words
Epidermal growth factor receptor(EGFR)/Nanobody 7D12/Immunotoxin/Pseudomonas exotoxin PE38KDEL分类
医药卫生引用本文复制引用
毛春燕,安钢力,王祥岭,翟晓晨,孟会敏,游凤涛,杨林..靶向EGFR免疫毒素BI7D12-PE38KDEL的制备及其体外活性测定[J].中国免疫学杂志,2017,33(4):558-562,573,6.基金项目
本文为山东省自然科学基金资助项目(ZR2015PH029). （ZR2015PH029）
