幽门螺杆菌超氧化物歧化酶基因克隆 表达及多克隆抗体制备

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1、郑州大学2009届硕士研究生论文幽门螺杆菌sod基因克隆表达及多克隆抗体制备Clone-exPressionandPreParationofPolyelonalantibodyof石几价口吞acter刀)lorisodgenePostgraduate劝angJianguangSuPervisorProf.DuanGuangeaiDePartmentofEPidemiologyCollegeofPublicHealthZhengZhouUulversityZhengZhou，Henan，450051ABSTRACTIt15kon

2、wnthatCytotoxin一associatedgeneA(CagA)15oneofthemostimportantvirulencefaetorofH.Pylori.TdfortherstudyonthebiologiealfunetionofCagA，TheresearehgrouPofH.PyloriinZhengZhouuniversityeonstruetedCagAnegativemutants一HP27△eagAviahomologousreeombination.Somedifferent允netional

3、ProteinofH.PylorirelatedwithCagAwassereenedoutandidentifiedbycomParingthebiologicalProPertyofwildstrainsandmutanswiththeaidofproteomiesmethods.OneofSomedifl七rentfunctionalProtein15suPeroxidedismutase(SOD)Protein.ThestudyontherelationshiPofSODwithCagA15notrePortedyet

4、.ThefirstimPortantthing15toeonstruetPolyclonalantibodyofSOD，andtheneonfirmthatit15differentoftheestablishmentofSODbetweenwildandCagAdeletedmutants.Methods:1.于叮getgenewaselonedbyPCRanditwasthroughPrimerPrimier5.0systernthatthePrimerswasdesigend.2.TheamPlifiedgenefrag

5、llentwereconnectedwithvectorPMD19一T，andtransfonlledinioDH5a.3.DoubledigestionwithNdel、乃01restrictionendonuelease，thetargetfraglnentWerecotmectedwithPET30(a)andtransformedintoBLZI.4.肠吧以proteinwasPurifiedbyNi一NTAeolun匡naccordingtotheofthefusionProteinwithwith6xHis一tag

6、affinity.ThePurifiedfusionSODProteinasantigenwasinjeCtedintorabbit.ThetiterofthePolyelonalantiserumwasmeasuredbythemethodsofELISA.犷郑州大学2以)9届硕士研究生论文幽门螺杆菌sod基因克隆一表达及多克隆抗体制备ReSUlts:1.SodgenefragmentwasamPlifiedbyusingtotalDNAofHP27asthetel旧Plate.‘2.Afteral刀Plifi目produc

7、tswasPurifiedandconeded，colmect曰withvectorPMD19一T，andthetral招伪彻edinioDHS屯therecombinantPlasrnidPMD19一sodwascons七ucted.3.M阳gottheherecombinantPlasmidPET30一sodconfirmedbyDNAsequenceanalysis.4.ThefusionProtdnwith6xHis一tagaffinitywasgotviaaffinitychromatograPhy.5.Thetit

8、erofthepolyclonalantiseru幻。wasrnorethanl:3200，itmeantthesueeessofthePr印肚滋ionofpolyelonalantisel”rn.ConcIUSion:1.Sodgenefra罗明ntcouldbeelone