A型肉毒毒素重組抗原的克隆表達(dá)及其ELISA檢測試劑盒的制備.pdf

1、湖南農(nóng)業(yè)大學(xué)碩士學(xué)位論文A型肉毒毒素重組抗原的克隆表達(dá)及其ELISA檢測試劑盒的制備姓名：李秀山申請學(xué)位級別：碩士專業(yè)：動物醫(yī)學(xué)指導(dǎo)教師：陳鐵橋王景林20050501Cloning，ExpressionofRecombinantBotulinumneurotoxinserotypeAanditsPreparationofELISAKitAbstractBotulinumneurotoxin(BoNDproducedbyClostridi

2、umbotulinum，isamultimericmoleculecomposedofsevenserotypesthathavebeenassignedthelettersAthroughGaccordingtoitsantigenicityandBoNT／AB，EareresponsibleformostofhumanbotulismBoNTmoleculeisdividedintotwopartssLructurally：ligh

3、tchain(to)isthecatalyticdomainaszincdependentmetalloprotease；Heavychain(Hc)isthebindingandtranslecationdomainThecarboxyterminalportionofHCplaysamajorroleininteractionwiththereceptorlocatedontargetcellsmembraneAtpresent，B

4、oNTisoneoftheimportanttoxinsinbiologicalwarfareagents；therefore，itissignificanttoperformaninvestigationintotherecombinantBoNTantigenicfragmentsfordevelopingELlSAKitandpreventivevaccinecandidateThepurposeofthisstudywastod

5、evelopcloningandexpressionofthegeneofBoNT／AinEcolibyusingPCRandDNArecombinanttechnologyandthentopreparemonoclonalantibodiesandpolyclonalantibodyWepurifiedandlabeledantibodiesfordevelopingELISAKit，whichwereappliedinthedet

6、ectionofBoNTtoxinTheexperimentalresultsasfollow：RecombinantbotulinumneurotoxinserotypeAbindingdomain[BoNT／AHeC]，expressedinEscherichiacolicellsToimprovethelevelsofexpressioninEscherichiacoli，thegeneencodingBoNT／AHc—Cwaso

7、ptimizedbyreplacingrarecodonswithhighfrequencyonesandadjustingATcontents，thensynthesizedusingoverlappingPCRTheoptimizedBoNTAHcCwassubelonedintothepETHisvectorA714bpfragmentofpETHiscloningvectorwascutbyBamHIandHindllI，and

8、BoNTAHcwasone—steppurifiedandrefoldedbyusingNi“affinitygelcolumnchromatographyThepurifiedrecombinantBoNTAHcCwasbeusedtoimmunizetheBp連BfcmiceThreehybridomacelllines，whichsecretedmonoclonalantibodies(mAbs)againstBoHTAconsi

9、stentlywereobtainedthroughcellfusion，screeningandcloning，lsotypeofthesethreemAbsareallIgGlThetitresoftheasceticfluidsofthreemAbsmeasurebyindirectELISAwere1110000TheirspecificreactionistoBoNT／AonlynottoBoNT／B，andSEBAntise