聚乙二醇修饰天花粉蛋白的初步研究

Chemical modification of trichosanthin with activated polyethylene glycol

目的:研究聚乙二醇(PEG)修饰天花粉蛋白(TCS)的条件、纯化方法、生物活性及致过敏反应程度。方法:用SDS-PAGE检测反应条件对产物成分的影响,利用离子交换和分子筛凝胶层析对修饰产物进行分离纯化,通过SDS-PAGE和HPLC检测聚乙二醇化天花粉蛋白(PEG-TCS)的纯度,小鼠引产实验测定PEG-TCS体内生物学活性,用TCS和PEG-TCS作为抗原对豚鼠进行致敏和攻击。结果:在PEG修饰TCS的反应中,PEG与TCS的投料(质量)比和pH值是影响修饰效率的主要因素,修饰反应一般在24～48h内达到平衡;纯化的PEG-TCS经SDS-PAGE检测纯度大于95%,HPLC显示单一峰;小鼠引产实验表明,经过PEG修饰后的TCS,引产活性加强;豚鼠过敏反应实验表明,PEG-TCS具有极低的全身性过敏反应。结论:PEG修饰TCS的最佳反应条件为:TCS与PEG投料比为1:6,pH6．0,反应时间为48h,催化剂氰基硼氢化钠浓度为40mmol·L^(-1);PEG-TCS纯度大于95%; PEG-TCS体内活性能较好的保留,且全身性过敏反应降低。

Objective：To investigate the optimal reaction condition of modifying TCS by methoxy PEG-propionaldehyde, the separation of reaction mixture, the biological activities and anaphylaxis reaction of mono-PEG-TCS. Methods：The influence of various conditions on the components of PEG-TCS was analyzed by SDS-PAGE. The mono-PEG-TCS was purified by ion exchange and molecular size exclusion chromatography. The biological activity in vivo of PEG-TCS was detected by abortifacient activity test. Using TCS and PEG-TCS as antigen, sensitizing and attacking of allergic reaction on guinea pigs were conducted. Results：The mass ratio of PEG to TCS and the pH environment played key roles in the chemical modification of TCS by PEG. The modification was completed within 24 - 48 hours. The optimal reaction of this PEG-TCS formation was conditioned as of 1 ： 6 mass ratio of TCS to PEG; at pH6.0, the reaction balance in 48 hours, and the catalyst concentration of 40 mmol·L^-1. The SDS-PAGE analysis demonstrated that the purity of mono-PEG-TCS was more than 95% , and the size exclusion HPLC appeared one peak only. The PEG-TCS complex significantly enhanced abortifacient activity, and reduced the sys- temic anaphylaxis in guinea pigs. Conclusion：The biological activities of the PEG-TCS produced by this optimal reaction remain better and anaphylaxis was desensitized.