Study On The Expression Of Prostaglandin Synthase Related Gene

Prostaglandins are a group of physiologically active lipid compounds called eicosanoids,which has multiply hormonal effects in animals.Prostaglandin E1,also known as alprostadil.As a member of the Prostaglandin family,Prostaglandin E1 is recognized as an endogenous and physiological substance,widely existing in the mammalian body.It has the ability to expand blood vessels,inhibit aggregation of platelets and secretion of gastric acid,stimulate smooth muscle of Intestines and uterus.At present,the main synthesis method of prostaglandin E1 is to make fresh COX-1 and mPGES-1 enzyme separated from living body and Dohomo-?-Linolenic Acid(DGLA)react.This method needs a large amount of fresh enzyme,and it is easy to cause system pollution and waste.Therefore,study on the expression of prostaglandin synthetases COX-1 and mPGES-1 has economic and social benefits.In this study,COX-1 and mPGES-1 belong to prostaglandin synthetases gene were used as research objects to construct pET-28a-COX-1/mPGES-1 expression vectors;Genetically engineered enzymes COX-1 and mPGES-1 were expressed by E.coli BL21(DE3)which is the host;HPLC and MS are used to identify the reaction of the enzyme COX-1 and mPGES-1 with DGLA in order to verify enzyme activity.Determine the effects of various reaction conditions on enzyme activity,and determine the conversion rate from DGLA to PGE1.The results showed that the recombinant strain BL21(DE3)-pET-28a-COX-1/mPGES-1 were successfully constructed,determined by double enzyme digestion and gene sequencing.SDS-PAGE results show that COX-1 and mPGES-1 are successfully expressed in the supernatant of E.coli,the induction conditions are optimized.Western Blot results showed that histidine labeled antibody was specific to COX-1 and mPGES-1 proteins,indicating that COX-1 and mPGES-1 get active expression.HPLC and MS are used to determine the successful synthesis of prostaglandin E1,confirm that COX-1 and mPGES-1 are active.and determine the optimal enzymatic reaction conditions: the pH was 7.8,the concentration of EDTA-2Na was 6?10 ?mol/mL and the reaction temperature was 37?.Meanwhile,the concentration of prostaglandin E1 was 0.15199 mg/mL,and the conversion rate was1.49329%.In this study,the active COX-1 and mPGES-1 enzymes were successfully expressed,and prostaglandin E1 was synthesized,which has opened a new path for the production of prostaglandin E1 and has important effect in protein expression and drug production.