The Experimental Study Of Protective Effect Of The Recombinant Human Serum Paraoxonase 1 Subtype Isozyme Against Organophosphate Poisoning

Objective:Organophosphrorus pesticide is a kind of common agriculture capital widely used around the world,especially in the developing countries.But in the process of production,transportation and application of organophosphrus chemicals,acute and chronic poisoning cases occurrs ocassionally.The statistics show that millions of people suffered from organophosphrus poisoning every year.And the organophosphrus poisoning cases are even among the top ones of suicide cases in some developing countries.We can also find the contents about organophosphrus neurotoxic angents such as sarin,soman,tabun and propylamine ethyl methylthiophosphonate（VX）in the news report related to the Gulf War and terrorism attack.Even so,the use of organophosphrus pesticide is still extensive for its lower price and availibility.Therefore,to meet the need of society security and defence,the research on detoxification of organophosphrus poisoning is very urgent.Paraoxonase 1（PON 1）is a non-specific esterase exists in the mammalian liver,blood,kidney and brain so on,which can hydrolyze a broad spectrum of substrates such as organophosphate,carbamate and aromtic carboxylate,which is named for its toxicological significance.The molecule of PON1 is combined with high density lipoprotein（HDL）in the blood,protecting the cells from atteck of toxin and peroxide.PON 1 has attracted wide attention because it has some efficient and non-toxic hydrolysis effect on organophosphrus pesticides,even including G type neurotoxic agents such as sarin,soman,tabun and VX,in vivo and vitro studies,which make it the focus of research on detoxication of organophosphrus poisoning.Although PON1 exists in the body of everyone,the difference of the PON1 activity is very significant,ranging from 10 to 40 times,among the individuals.Some researches have shown that the difference is determined by its genetic polymorphism.The polymorphism sites are dozens in the sequence of PON1 DNA,but most of them are introns which can not be translated.The phenotypes of the PON1 gene are determined by the exons primarily.There are two exons,having polymorphism in 55 and 192 locus of the PON1 DNA sequence,which are reported to affect the concentration and activity of PON1 in vivo respectively.The polymorphism of the 55 site is Leu/Met（L/M）mutation and the polymorphism of the 192 site is Gln/Arg（Q/R）mutation.The polymorphism of the 192 site is reported to affect people?s susceptibility to the different organophosphrus chemicals,which make the people having Q subtype PON1 susceptible to chlorpyrifos,dichlorvos and paraoxon,while the others having R subtype PON1 are susceptible to diazinon and sarin.In the earlier experiment,we used the Escherichia coli to express the recombinant PON1 gene.But we found that the prokaryotic expression system lack of post-translation modification,which may affect the acivity of expressed eukaryotic protein.And the expressed protein aggregated and formed inclusion bodies,which may undermine the output.Bac-to-Bac system is a kind of insect cell-baculovirus protein expression system,which can express eukaryotic genes better for its post-translational modification such as phosphorylation and glycosylation,making the expressed protein closer to the natural one in functions.In this study,we plan to use the Bac-to-Bac system to express the Q/R subtype PON1 of polymorphism in the 192 site and explore the difference of the two recombinant subtype isozymes as antidote againt organophosphrus poisoning.At the same time,we investigated the functions of glycosylation in keeping the activity of PON1.Method:1 Identification and preparation of subtype isozyme of PON1 by gene engineering.We get the subtype DNA sequence of PON1 from the GeneBank.Then we use the gene synthesizer to produce the subtype gene sequence with a bit modulation artificially.The target gene was carried to the receptive IPLD-SF 21 cells by the vector pBacPAK8.We get the Bacmid DNA containing the subtype gene sequence of PON1after the transposition.We used the bacmid virus to infect the SF21 cells to acquire the stains with higher level of target DNA after cultures.Then the virus strains of higher titer were transfected to the SF21 cells in order to express the subtype isozyme of PON1 by suspension culture.We could acquire the purified expressed PON1 by the cobalt agarose affinity chromatography and test its activity by spectrophotometric method.2 Investigate the effect of glycosglation on the PON1 activity.The recombinant PON1 subtype and mixed human PON1 were deglycoslated by endoglycosidase Endo F2 to test the function of post-transcription glycosylation.We use paraoxon,chlorpyrifos,diazine and trithion as the substrate to test and compare the activity of the purified recombinant subtype isozyme before and after deglycosylation.3 Explore the protective effect of rhPON1subtype isozyme on the central nervous system of the rats exposed to organophosphrus chemicals.108 waister rats were chosen and divided into four groups randomly according to the exposed poisons,named control group,chlorpyrifos group,diazinon group and trithion group.Then each group was divided into three subgroups according to the treatment they received,including normal saline subgroup,rhPON1_R192 subgroup and rhPON1_Q192 subgroup.All the rats were exposed to organophosphrus pesticides at a dose of two times LD50 by intragestric administration.The rhPON1_R192 and rhPON1_Q192 were administered by injection vena caudalis as antidote at a dose of 10U/kg to the rhPON1_R192 subgroup and rhPON1_Q192 subgroup respectively within 1 minute after the exposure.To the blank subgroup,the exposed rats received the normal saline as the same amount to the treatment group.The onset time of death and signs such as salivation,muscle fibrilation,dyspnea and muscle strength above 3 grades were recorded.The experiment was terminated after continuous observation for 12 hours.All the rats were decapitated to collect the serum and brain tissue at the termination point.The cholinesterase activity of the serum and brain tissue was detected by spectrophotometry among the groups.Use the optical microscope and transmission electron microscope to observe and compare the ultrastructure change of the hippocampal regaion in the brain among the groups.The change of cholinesterase was compared among the groups by means of Western Blot and immunohistochemity.Investigate the substrate speciality of the subtype isozyme of PON1 through these comparations to find the accute antidote of higher efficient hydrolysis to the different organophosphrus poisoning.Conclusion:1 The Bac-to-Bac system can be used to express the subtype isozyme of PON1 with great efficiency and output.The positive vectors containing the DNA of target subtype isozymes with polymorphism in 192 loci could be expressed in the SF21cells sucessfully.The product with a molecular weight about 43 KD was detemined by Western Blot to be the taget protein.And a great amount of enzyme was acquired after suspension culture and purified by colbalt argarose affinity chromatograhpy.The specific activity of purified rhPON1_R192 was incresed 435 fold over the crude extract,up to152U/mL,with the yield of 8.5%.And the specific activity of the purified rhPON1_Q192was incresed 597 fold over the crude extract,up to 74.1U/L,with the yeild of 12.5%.The purified rhPON1_R192 and rhPON1_Q192 were tested to have the phosphatase activity.2.Deglycosylation of the rhPON1 will affect its phosphatase activity.（1）We found that rhPON1_R/Q192 and hPON1_mix of the same amount have different hydrolysis activity toward different organophosphrus insecticides such as chlorpyrifos,diazinon and trithion.The hydrolysis efficency on chlorpyrifos of the different PON1 were showed in such seuqency that rhPON1_R192>hPON1_mix>rhPON1_Q192;while the hydrolysis efficiency on diazinon was shown in opposite sequence that rhPON1_R192<hPON1_mix<rhPON1_Q192;whether rhPON1 or hPON1 had shown lower hydrolysis efficiency on the trithion.（2）We found the hydrolysis activity of rhPON1 and hPON1 toward organophosphrus poisoning and the substrate speciality decreased obviously after deglycosglation.（3）The molecular weight of the rhPON1_R192,rhPON1_Q192 and hPON1_mix decreased to the same extent after deglycosglation.3 The protective effects of rhPON1_R/Q192 on the nerve system exposed to organophosphrus insecticides are different.In the treatment subgroups,the results of observation about onset time of poisoning signs and symptoms are better than that of NS subgroups.The cholinesteras avtivity in the brain tissue of the treatment subgroups are higher than that of the blank control subgroups as well.The results of the rats exposed to chlorpifros were better than that to the diazinon and trithion among the rhPON1_R192192 subgroups in these comparations,while the results of the rats exposed to diazinon were better than that to the chlorpifros and trithion among the rhPON1_Q192subgroups.The results of the rats exposed to trithion showed the worst.The differences are of stastical significance.The ultrastructual changes of rat?s brain hippocampus cells were observed by light and electron microscope,which shows that the injury cause by the organophosphrus poisoning in the exposed group led to neuron cell necrosis and cavitation,emergence of heterocromatin,the breaking or disappearance of the cell zone under light microscope.The neurons become deformity,pyknotic nucleolus disappeared,the quantity of ribosome declining,the nuclear structure unclear and nucleus disaggreated under the electron microscope observation.In the treatment subgroups,the damaged to the tissue and neurons shows slight.The injury to the brain cell of the rats exposed to the chlorpyfros is lest among the rhPON1_R192 subgroups,while the injury in the rats exposed to diazinon is lest among the rhPON1_Q192 subgroups.The damage to the neurons is the worst among the three subgroups of trithion group,approximate to the NS subgroup.Conclusion:1 rhPON1 subtype isozymes with higher activity and yield can be expressed by the Bac-to-Bac system.The product has the similar biological chracteristics as natural hPON1 that the PON1 of R type has higher hydrolitic activity toward paraoxon than the Q type.The difference,similar to the human susceptibility to organophosphrus pestcides,can be correlated to the molecular structure of the isozyme 2 Glycosylation play an important role in keep the activity of PON1.The rhPON1 and natural hPON1 show the same substrate specifity before deglycosylation.However,the specifity and the molecular weight decreased to the same extnet after deglycosylation.3 Because the P-S bond is stronger than the P-O bond,PON1 can hydorlyze the P-O bond better than P-S,which make the organophosphrus chemicals with P-S bond harder to be hydrolyzed by the PON1.4 The rhPON1 R/Q192 can protect the rat?s never system exposed to the organophosphrus pesticides,because it can debate the inhibition on the cholinesterase caused by the organophosphrus poisoning.The effects of rhPON1R192 and rhPON1Q192 as antidote against different organophosphrus pesticides are different...