The Separation And Purification Of Phospholipase A₂Inhibitors From Snake Serums And The Design Of Simulation Peptide

In the body, phospholipase A2(PLA2) is rate-limiting enzyme of arachidonicacid inflammation pathway, as important marker of early inflammation. Excessiveexpression of PLA2can cause severe acute pancreatitis, acute ischemic brain injury,and a variety of diseases such as severe acute pancreatitis, coronary heart disease, andacute ischemic brain injury; however, PLA2inhibitor anti-inflammatory drugs are lessat clinic. The snake self-protection mechanism research shows that PLI protein ispresent in the blood which can neutralize PLA2toxicity. In addition, snake venomPLA2and human PLA2are both belong to the family of PLA2, showing highly similarspatial structure.Snake PLI proteins including three types, alpha, beta, and gamma asnatural snake venom sPLA2inhibitors, so snake PLI as nature inhibitors of sPLA2worth studying.Objective:Screen out PLA2inhibition protein.According to the amino acid sequence todesign mimetic peptide and evaluate its PLA2inhibition activity.Methods:Collect Elaphe carinata, Bungarus multicinctus, Bungarus fasciatus,Macropisthodon rudis rudis Boulenger, Sinonatrix annularis, Naja naja atra serumsby applying the method of agarose plates and pH dynamic to detect each seruminhibition activity of phospholipase A2, Sinonatrix annularis show strongest activity;Use SourceQ and UnoQ ion exchange chromatography isolation and purification ofSinonatrix annularis serum. The purpose protein by the method of agarose plate andmice hemorrhagic experiments, testing inhibitory activity of the snake venom sPLA2enzyme and hemorrhage activity; Design degenerate primer to cloned Sinonatrixannularis PLI gamma sequence by RT-PCR; in vivo, injection venom into Sinonatrixannularis test the PLI gamma expression level; on the basis of Sinonatrix annularisPLI gamma amino acid sequence to design mimetic peptide, and use autodockmolecular docking software for virtual screening, as above method to detectsimulated peptide inhibition effect of sPLA2enzyme and hemorrhage activity; With lipopolysaccharide (LPS) induced raw264.7cell to construct inflammation model,detecting sPLA2gene expression level. Detection of arachidonic acid levels toevaluate the anti-inflammatory activity of mimetic peptide by ELISA.Results:In the screen of6kinds of snake serums, Sinonatrix annularis has the strongestsPLA2inhibition activity. Successfully isolated from PLI gamma protein fromSinonatrix annularis serum, the protein inhibitory activity of sPLA2was85%, andsignificantly inhibit Deinagkistrodon acutus venom hemorrhagic toxicity of sPLA2.By RT-PCR and sequencing got PLI gamma gene from Sinonatrix annularis liver, itscode length is about550bp. In addition, Sinonatrix annularis PLI gamma expressionlevel increased significantly after injection of venom PLA2that further verify theSinonatrix annularis PLI gamma antivenoms protection.Design a length of19aa mimetic peptide sequence PGLPLSYPNGGGGSVAFRS based on Sinonatrix annularis active region. Autodock molecular dockinganalysis shows that the19aa mimetic peptide interact with sPLA2hydrophobic activecenter. The19peptide has good sPLA2inhibitory activity on enzyme and hemorrhage.In addition,19aa mimetic peptide inhibited the generation of arachidonic acid onLPS induced Raw264.7inflammation cells, IC50was86.3μmlo/LConclusion:The isolated sPLA2inhibiting protein was PLI gamma from Sinonatrix annularis,and it is a new protein which anti-sPLA2enzyme and anti-hemorrhage, and will beuseful for the treatment of snake bite.19aa mimetic peptide has similar activity andcertain anti-inflammatory effect which was designed based on Sinonatrix annularisPLI gamma.