【摘要】 目的:用直接偶联法构建新型蛋白兔抗人IgGCRM9.方法:分别应用异型双功能连接剂(m Maleimido benzoyl N hydoxysuccinimideester,MBS)和N琥珀酰亚胺3(2吡啶二巯基)丙酸盐(SPDP)将兔抗人IgG多抗和白喉毒素突变体(CRM9)进行连接,制备出新的蛋白.结合后的蛋白经SephacrylS300分离纯化,用聚丙烯酰胺凝胶电泳和琼脂糖电泳证明二者的结合情况.采用酶联免疫法检测IgG与CRM9按不同比例反应后所构建的蛋白复合体中抗体部分的活性.结果:用MBS连接剂连接IgG与CRM9后的产物经聚丙烯酰胺凝胶电泳显示出的条带明显滞后于IgG所显示的条带,而在琼脂糖凝胶电泳中IgG与CRM9混合反应的样品所显示的两条条带均与IgG和CRM9单独所在的条带一致.酶联免疫实验测得的各组A450nm值表明,IgG与CRM9按1∶1和1∶3两种比例制备的样品对抗体活性影响最小,而按1∶5和1∶两种比例制备的蛋白,抗体活性受到很大的抑制.结论:兔抗人IgG与CRM9可以通过MBS结合,二者在1∶1～1∶3之间的比例时,可以有较好的连接,而且对抗体活性的影响最小.SP DP则不适用于本实验中的两种蛋白的连接.这将为免疫毒素的新型连接提供有效的方法.更多还原

【Abstract】 AIM: To search the method of conjuncting rabbit anti-Hb IgG and diphtheria toxin mutation (CRM9) by chemical crosslinkers MBS (m-Maleimidobenzoyl-N-hydoxysuccinimide ester, NHS-Esters-Maleimide crosslinkers) and SPDP [N-succinidimidyl-3-(2-pyridyldithio) proprionate)] and to study the results achieved by the two methods. METHODS: Rb-anti-Hb Ig-G was linked with diphtheria toxin mutation (CRM9) by MBS and SPDP respectively. The outcomes were eluted through Sephacryl S-300 to purify the linkage-protein. The degree of IgG and CRM9 linking was observed. The conjuncting conditions were testified by 8% SDS-PAGE and agarose electrophoresis. The antibody-part of the linkage-protein, which was mixed in different ratio, was tested by ELISA. RESULTS: In image of SDS-PAGE, the linkage-protein, IgG-MBS-CRM9, conjuncted by MBS lagged behind IgG. In ELISA, the A450 of the five groups had obvious deviation. The activity of linkage-protein’s antibody had little influence on group A and group B, the ratio of IgG and CRM9 being 1∶1 and 1∶3. The antibody’s activities of group C (1∶5) and group D (1∶8) decreased greatly. CONCLUSION: Rabbit anti-Hb IgG can conjunct CRM9 by MBS. The best ratio of IgG to CRM9 is 1∶1-1∶3. Under this condition conjunction is better and has little influence on the activity of antibody. This provides a valid method for the new conjunction of immunity toxin.更多还原