构建靶向毒素血管内皮生长因子抗体-白喉毒素突变体

Conjuncting on targeting toxin of vascular endothelial growth factor a antibody-diphtheria toxin mutation

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 study the conjuncting of rabbit anti-VEGF as targeting and Diphtheria toxin mutation （CRM9） as domino effect, and to search the new drugs of killing cancer cells with immunotoxin. Methods Anti-VEGF linked with Diphtheria toxin mutation（CRMg） by MBS. The outcomes are eluted through Sephacryl S-300 to purify the linkage-protein. The degree of VEGF and CRM9 linking are observed. The conjuncting conditions are testified by 8% SDS-PAGE. The antibody activity of linkage-protein is tested by ELISA, those proteins are mixed in different ratio. The toxicity of CRM9 from linkage-protein are tested by MTr. Results In image of SDS-PAGE, the linkage-protein, molecular weight 116Mr/103, lags behind VEGF and CRM9. In EUSA, the A450 of five groups have obvious deviation. The activity of linkage-protein＇s antibody have little influence on the group that the ratio of VEGF and CRM9 is 1：1 and have similar activity with anti-VEGF control （P 〉 0. 05）. It is decrease greatly, than those in anti-VEGF control （P 〈 0. 01 ） that the antibody activity from linkage-protein＇s the ratio of VEGF and CRM9 is 1： 5 and 1 ： 8. The toxicity of CRM9 from linkage-protein in which VEGF and CRM9 is 1 ： 1 are markedly higher than those in blank control groups（P 〈0. 01 ） but have not significant differential with the CRM9 control groups （P 〉 0. 05 ）. The toxicity of CRM9 control are markedly higher than those in blank control groups （P 〈0. 01 ）. Conclusion Rabbit anti-VEGF can conjunct CRM9 by MBS. Under this condition, there is little influence on the activity of VEGF antibody and the toxicity of CRM9 from linkage-protein. Based on results in vivo. it will become a useful agent in the studies on treatment cancers with immunotoxin.