The Expression,Purification And Biological Activity Determination Of Water Snake Phospholipase A₂ Inhibitor ? In Pichia Pastoris

Phospholipase A₂ is a kind of enzyme which can catalyze the hydrolysis of the Sn-2 position of membrane glycerophospholipids,leading to production of free fatty acids and lysophospholipids.Phospholipase A₂ involved in the arachidonic acid pathway and further promote large amounts of inflammatory factor production which leads to the body injury.Phospholipase A₂ distributed widely in the individual organisms,a variety of snake venom have been found generally rich in many types of PLA₂.They have abundant and various toxicity,like cytotoxicity,neurotoxicity,muscle toxicity and bleeding.So PLA₂ may be emphasized as a new therapy target for snake bite.Studies showed that a kind of protein called PLA₂ inhibitors?PLI?from liver of some species of snakes can protect themselves from damage by their venom,which can neutralize the toxicity of PLA₂.Previously,we separated a new PLI? from Chinese Sinonatrix annularis and found it had good control effect on enzyme activity and bleeding toxicity of D.acutus.sv PLA₂.Due to the lack of the snake serum,it become difficult to extract natural PLI from this limited resource.Our lab constructed a recombinant vector to achieve the prokaryotic expression of Sinonatrix annularis PLI?.But the PLI?existed in the form of inclusion bodies which need to refolding,due to the low efficiency of refolding and bad impact on the activity of enzyme,we will use a eukaryotic expression to produce PLI? of Sinonatrix annularis.Objective:In order to make it more convinient to obtain higher active Sinonatrix annularis PLI?,we constructed the p PICZ?A-PLI? recombinant expression vector,transfored into Pichia pastoris and then detected the activity of PLI?.This provided the foundation of exploiting new anti-venom and anti-inflammatory drugs.Method:Using agarose diffuse method,according to the area of the formative transparent zone,testing the inhibitory activity of water snake serum PLI? to the enzyme activity of three kinds of snake venom;next the mice were injected with three kinds of snake venom which leads to subcutaneous bleeding.We injecte another mice with a mixture of snake venom and Sinonatrix annularis serum,compared the size of bleeding spot to test the activity of the Sinonatrix annularis serum PLI? against snake venom.Clone the PLI? gene about full-length of 570 bp from recombinant plasmid pET28c?+?-PLI? with the primer that contains EcoR?and Not?restriction enzymes sites,then connect it to the yeast expression vector p PICZ?A after double enymze digestion.Amplify the p PICZ?A-PLI? vector in E.coli DH5?.Extract the p PICZ?A-PLI? recombinant plasmid and transferred into Pichia pastoris GS115 through electrotransformded,the condition was:1.5 KV,25 ?F,5 ms.Screen out the positive transformants by zeocin and extract the genome DNA of yeast,then identify it by PCR.Continue the cultivation at 30? and 250 prm for 120 h,the expression of PLI? was induced by the final concentration of 0.5 % methanol,collect the fermentation broth every 24 hours and use the anti-His antibody to detected the PLI? by western blot.Finally,we determined the optimum inducement time.The tagged PLI? was purified on Ni-NTA column with imidazole elution,to examined the purity of PLI? through SDS-PAGE.The inhibitory activity of the PLI? against D.acutus venom was tested by agarose diffuse method,to measure the diameter of transparent zone and compute the inhibition rate.The anti-hemorrhagic effect was valued by subcutaneous inject of PLI? and crude D.acutus venom into mice back skin,compare the area of hemorrhagic plaques.Results:Acroding to the agarose diffuse method,we found the Sinonatrix annularis serum PLI can inhibite the enzyme activity effect of D.acutus and A.halys venom,the diameter of transparent zone was reduced.Compare with the positive control,the diameter of transparent zone has no significant change after the mice were jected with serum PLI and N.atra venom.In subcutaneous injection of mice,we found injected with the PLI can decrease the area of hemorrhagic plaques compared with injecting D.acutus and A.halys crude venom.The Sinonatrix annularis serum PLI can extend the times of mice infected with N.atra venom survival time from 30 minutes to an hour.The target gene was 570 bp cloned from the pET28c?+?-PLI? plasmid by PCR successfully and constructed pPICZ?A-PLI? recombinant plasmids through the double enzymes digestion.Gene sequencing showed that there was no mutation in the PLI? gene.Transferred the plasmid dealed with the restriction endonuclease Sca I into GS115 yeast cells by electrotransformded successfully.The recombinant yeast cells could grow in independent colony on the solid medium with zeocin.Analysis of 7 clones with PCR demonstrated that all clones were positive.Methanol was used to induced the expression of PLI?,collect the fermentation broth to determine the expression of PLI? by Western bolt.The result showed its molecular weight was about 23 k Da and the amount of protein expression reached highest when cultivated for 72 h.The tagged PLI? was purified by Ni-NTA column,there is only one purified protein by SDS-PAGE.PLI? decreased the diameter of transparent zone,it showed a inhibition rate of 84 % to D.acutus venom enzymatic activity.At the same time,PLI? aslo significantly relieved the hemorrhage symptom of D.acutus venom on subcutaneous skin,it can help relieve vascular compromise and decrease the area of hemorrhagic plaques.Conclusion:The Sinonatrix annularis serum PLI? showed a good inhibitory effect of enzymatic activities and hemorrhagic toxicity,neurotoxic to snake crude venom.,it has a wide anti-venom potential.The pPICZ?A-PLI? recombinant plasmid in yeast GS115 cells can be induced by methanol to produce PLI? protein,and the PLI? showed a good inhibitory effect of enzymatic activities and hemorrhagic toxicity to D.acutus venom,but the culture parameters of recombinant Pichia pastoris should be optimized.