聚乙二醇化海葵神经毒素的修饰反应条件及药效研究

Study on Polyethylene Glycol-Modified Sea Anemone Neurotoxins rhk2a

关键词:  海葵神经毒素rhk2a  聚乙二醇修饰  心力衰竭

Key words:  sea anemone neurotoxins rhk2a  PEG modification  heart failure

摘要:

目的 优化聚乙二醇(PEG)修饰海葵神经毒素rhk2a(rhk2a)的反应条件，并考察修饰后产物PEG化海葵神经毒素rhk2a(mPEG-rhk2a)的药效性质。方法 本研究选用分子量为5 000的PEG丙醛(mPEG-ALD)对rhk2a的N-末端氨基进行修饰；通过单因素考察和正交试验筛选出最佳的修饰反应条件。同时，采用HPLC检测修饰后产物中mPEG-rhk2a的纯度，并通过建立急性心力衰竭豚鼠模型，考察mPEG-rhk2a对豚鼠的强心作用。结果 优选出的mPEG-ALD对rhk2a的N-末端氨基的修饰反应条件是：当pH为5.0、rhk2a与mPEG反应摩尔比1∶20、还原剂氰基硼氢化钠为30 μL、反应时间为12 h时，所得修饰产物mPEG-rhk2a的得率较高。采用SP-650S强阳离子交换层析法对修饰反应后产物进行分离，纯化后产物中mPEG-rhk2a占83.287%。对于心衰豚鼠，给mPEG-rhk2a溶液后，左心室收缩压、左心室压力最大下降速率明显增加，20 min后呈持续的平稳状态。其强心作用虽然不如rhk2a强，但波动起伏较小，而且强心效果明显高于乙酰毛花苷注射液。结论 优选出mPEG修饰rhk2a反应的适宜条件，所得的mPEG-rhk2a强心作用较为理想。

Abstract:

OBJECTIVE To optimize the conditions of polyethylene glycol (PEG)-modified rhk2a reaction and research the pharmacodynamic characteristic of the modified mPEG-rhk2a. METHODS By using mPEG-ALD with weight of 5 000, the rhk2a was modified on its N-terminal amino. Used single factor test and orthogonal experiment to optimize the conditions of modification and established an HPLC detection method to assay the protein concentration of mPEG-rhk2a. The positive inotropic effect of mPEG-rhk2a in guinea pigs was investigated. RESULTS Optimized conditions of reaction were as follows: when the pH was 5.0, the rhk2a/mPEG(molar ratio)=1∶20, the reducing agent was 30 μL, the reaction time was 12 h, the modification was very well. After separation and purification by strong cation exchange column SP-650S, the pure PEG-modified sea anemone neurotoxin rhk2a accounted for 83.287%. For guinea pigs with heart failure, the left ventricular systolic pressure and the maximum change rate of left ventricular systolic pressure fall of the group of mPEG-rhk2a apparently increased and showed a smooth state after 20 min. The positive inotropic effect of mPEG-rhk2a was stronger than the Cedilanid injection but weaker and smoother than the rhk2a injection. CONCLUSION The optimal condition of the modified reaction is successfully found and the cardiac effect of mPEG-rhk2a is very well.