Studying On The Pegylation Of Sea Anemone Neurotoxins Rhk2a

PEG modification technology was used to improve the nature of proteins drug, because that pegylation can reduce the in vivo immunogenicity and antigenicity , and successfully improve the in vivo bioavailability of the proteins drug .In 2000, the lab in School of Life Sciences, Sun Yat-sen University , obtained a sea anemone neurotoxins rhk2a which have cardiovascular activity and specific effect on the sodium ion channel by genetic engineering techniques. The neurotoxins were proved that it have a very specific target on heart ,and the better positive inotropic effect about 3.5 times than cedilanid on the myocardial tissue. In the treatment of congestive heart failure the protein have an excellent prospect.In order to solve the problems which rhk2a encountered in clinical applications (such as poor stability and greater toxicity ).PEG modification technology was used on rhk2a.The activated mPEG reagent mPEG-ALD was selected to modify the amino groups in rhk2a molecule. RP-HPLC was used to determine the concentration of rhk2a, and analysis the composition of PEG-modified products. The optimization of reaction conditions was carried out for the set goal --the highest proportion of single-pegylated rhk2a. Optimized reation conditions were as follows: pH5.0 , rhk2a/mPEG(molar ratio)=1:20, reaction time=12h and the addition amount of reducing agent = 30μl。Among the four influencing factors, pH was the most significant one. while reaction time was the weakest .The pegylated product was separated by cation exchange chromatography (SP-650S).After desalting by ultrafiltration, freeze-dried concentrated we get the final product.The molecular weight, stabilities, purity of the pegylated product were tested. The acute heart failure model was created by pentobarbital in vivo. the three injections as acetyl glycosides eriantha ,rhk2a and mPEG-rhk2a were gave to the heart failure guinea pigs separately, and compared their cardiotonic effects in indices of LVSP and±dp/dtmax.The single-PEGylated rhk2a was 83.287% in the pegylated product. MALDI-TOF-MS proved this product to be single-PEGylated. Compared to natural rhk2a ,the PEGylated protein showed higher stabilities at room temperature and 37℃.In the heart failure guinea pigs, three injections can increase cardiac contractility in heart failure, improving cardiac function, and the effect of rhk2a and mPEG-rhk2a were more obvious the eriantha acetyl glycosides. After delivery, the cardiotonic effect of rhk2a was rapidly and significantly higher than the other two groups. In the mPEG-rhk2a group, the increase of LVSP and- dp/dtmax was slow in 10min , and was the strongest after 20min.