The Expression And Purification Of PON1and The Preliminary Research Of Its Ativity And Function

PON1is a member of PON gene family, it is a broad-spectrum and non-specific esteraseexisting in vivo of the organisms who can degrade the carbamic acid ester,organophosphate andthe aromatic carboxylic acid ester through catalyzing the hydrolysis of phosphate bond. Thus，Itmay play an improtant role in catalyzing the hydrolysis of organophosphorus compound and thedegradation of the nerve agent.The name PON1is just coming from its special toxicologysignificance.Nowdays,more and more evidences show that PON1plays an importent role notonly in catalyzing the hydrolysis of the organophosphorus compound,but also in lipidmetabolism of mammals. Therefore,PON1has an intinate connection with the occurrence anddevelopment of organophosphorus intoxication, atherosclerosis, oronary heart disease and typeⅡ diabetes mellitus.In this research, a recombinant prokaryotic expression vector pET-32a-PON1has beenconstructed, and after induce it with IPTG, interest protein PON1has been expressed. Through aseries of purification methods like Ni-NTA affinity chromatography,anion exchangechromatography and gel filtration chromatography, more than90%purity of PON1has beenacquired.Then immune New Zealand male rabbit to produce polyclonal antibody with thepurified PON1to lay a foundation of purifying PON1expressed in silkworm bioreactor.At the same time,we have successfully constructed a recombinant baculovirus vBmPON1with bombyx mori baculovirus expression system(BMBEVS).And then we inoculated it intosilkworm fifth stadium larvae and chrysalis.Waiting until they appears morbidity, collect thehemolymph to construct SDS-PAGE and Western blotting analysis.The identification resultshows that the target protein PON1has been successfully expressed in BmN cells,silkworm fifthstadium larvae and chrysalis.Using paraoxon as the substrate,We make an in vitro activity detection to the PON1expressing in silkworm bioreactor and E.coli,as well as the PON1natural existing in rabbitserum. Besides,we detected and compared the DDVP hydrolyzing effect in mice,which lays afoundation for the further research of the biological activity and the function mechanism ofPON1expressing in silkworm bioreactor.