PEG Modification And Stability Of Alpha-conotoxin TxID

Natural bioactive peptides provide a rich library of natural active peptides for the development of peptide drugs,especially peptides and antibiotics derived from animal species such as snake venom,scorpion venom,spider venom,conotoxin,etc.,in analgesic and anti-inflammatory Anti-tumor,neuralgia and other diseases have good effects,but due to their inherent characteristics,such as short half-life,poor stability,low bioavailability,etc.,must be partially or completely solved by means of genetic engineering or chemical modification.Defects in these drugs give them appropriate drug development properties.In this study,the linear peptide of conotoxin TxID was synthesized by using TxID as a model.The TxID was oxidatively folded in vitro by two-step oxidation method to obtain the TxID of the conid toxin with active structure.Obtaining a correctly oxidized folded polypeptide with a purity greater than 95%;The PEG modification reaction was optimized by system,and the conditions affecting the modification reaction were systematically studied.The optimization conditions of the modification reaction system were selected from different conditions such as solvent,ratio,pH and reaction time.The results showed that the prescription for optimizing the PEG modification reaction system was:DMF as solvent,the molar ratio of polypeptide to PEG is 1:2,the pH is adjusted to 8.3 with triethylamine,the reaction time is 120h;The DSPE-PEG-TxlD was synthesized according to the optimal reaction conditions.The PEG was modified by the DSPE-PEG-NHS to the NH2 at the end of the TxID polypeptide,and the desired target compound DSPE-PEG-TxlD was obtained by MADLI-TOF mass spectrometry.The physiological patch clamp technique was used to detect the biological activity of the purified compound,and compared with the bulk polypeptide,the results showed that the modified polypeptide activity was not lost,but the degree of binding to the receptor was closer than that of the unmodified polypeptide;In addition,compared with the bulk polypeptide,the modified peptide was tested for stability in vitro,simulated gastric juice,intestinal fluid and other conditions.The stability in vitro showed that the modified peptide was more stable under different conditions than the bulk.From this we can know that PEG modification of conotoxin TxID can improve its stability,enhance anti-enzymatic ability and prolong half-life.Finally,the PEG modification method of this subject was extended to other different conotoxins for modification.The modification of conotoxin TxIB and conotoxin GeXIVA showed that the same modifier and modification reaction system could not be fully applied.The structural characteristics of the polypeptide are selected differently or modified to modify the reaction system.