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目的探讨以血管内皮生长因子(VEGF)抗体为导向,白喉毒素突变体(CRM9)作效应部分,构建靶向毒素VEGF抗体-CRM9,为直接杀伤肿瘤细胞寻找新的药物。方法应用异型双功能连接剂甲基丙烯酸甲酯丁二烯苯乙烯三元共聚物(MBS)连接兔抗人VEGF多抗和CRM9,制备成新的构建蛋白,Sephacryl S-300分离纯化后,经聚丙烯酰胺凝胶电泳证明二者的结合情况。对构建蛋白抗体活性采用酶联免疫法检测,生物毒性应用四甲基偶氮唑蓝(MTT)方法检测。结果构建蛋白通过分离并电泳后,出现明显增粗的分子量为116000新条带,位于CRM9和VEGF条带前方。构建的蛋白结合体中抗体活性检测,VEGF抗体与CRM9按1∶1制备的蛋白结合体,抗体活性与VEGF抗体对照差异无统计学意义(P>0·05),按1∶5和1∶8两种比例制备的蛋白结合体,抗体活性降低与VEGF抗体对照差异有统计学意义(P<0·01)。构建的蛋白结合体中CRM9毒性检测,VEGF与CRM9按1∶1比例制备蛋白结合体杀伤效果与空白对照组差异有统计学意义(P<0·01),与CRM9组差异无统计学意义(P>0·05),CRM9组杀伤效果与空白对照组相比差异有统计学意义(P<0·01)。结论MBS能将VEGF抗体与CRM9连接构建成新的蛋白结合体,结合体中CRM9生物毒性和VEGF抗体活性不变。这将为进一步的免疫毒素抗肿瘤的各项研究奠定了基础。
OBJECTIVE: To investigate the effect of anti-diphtheria toxin mutant (CRM9) targeting vascular endothelial growth factor (VEGF) antibody and to construct a targeted toxin VEGF antibody-CRM9 to find a new drug for directly killing tumor cells. Methods Rabbit anti-human VEGF and CRM9 were ligated with methyl methacrylate butadiene styrene terpolymer (MBS) using heterobifunctional linker. The recombinant protein was purified by Sephacryl S-300. Polyacrylamide gel electrophoresis to prove the combination of the two. Enzyme-linked immunosorbent assay (ELISA) was used to detect the antibody activity of the constructed protein, and the toxicity was detected by MTT assay. Results After separation and electrophoresis of the constructed protein, a markedly enlarged 116,000 new band appeared in front of the CRM9 and VEGF bands. The results showed that there was no significant difference between the antibody activity and the VEGF antibody control (P> 0.05), and the ratio of the antibody to VEGF was 1:5 and 1: 8 protein conjugate prepared in two ratios, the decreased antibody activity and VEGF antibody control difference was statistically significant (P <0.01). The results showed that there was a significant difference between the constructed protein conjugates in CRM9 toxicity test and the results that the killing effect of VEGF and CRM9 at a ratio of 1: 1 was significantly different from that of the blank control group (P <0.01), and there was no significant difference between the CRM9 group and the CRM9 group P> 0.05). The killing effect of CRM9 group was significantly different from that of the blank control group (P <0.01). Conclusion MBS could connect VEGF antibody with CRM9 into a new protein conjugate, and the binding activity of CRM9 and VEGF antibody did not change. This will lay the foundation for further studies on antitumor immune toxins.