29525.家蠶bmch基因的原核表達和亞細胞定位研究

1、浙江理工大學碩士學位論文家蠶BmCH基因的原核表達和亞細胞定位研究姓名：梁莉惠申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：張耀洲20091201浙江理工大學碩士學位論文AbStractAcDNAsequencewasscreenedfromaBombyxmoricDNAlibraryconstructedbyourlaboratory，whichcontainsaCHCHdomainidentifiedafterblastba

2、sedonNCBIdatabaseWepreliminarynominatedthisnovelgeneasBmCH(BombyxmoriBraCt1)(GenBankaccessionnumberisABF515141、ItcontainsallORFof435bpandencodes144aminoacidswiththepredictedmolecularweightof1482kDaandisoelectricpointof85

3、49AccordingtotheORFofBmCHtwoprimersweredesignedtOobtainthecodingregionofBmCHgeneTheORFofthegeneWasclonedintopET28a()vector、析thEcoRIandXhoIdigestTherecombinantplasmidwastransformedintoEcoliRosetta(DE3)PCRanddigestionwithE

4、coRI／XhoIshowedthatthedesignedfragmentWasinsertedcorrectlyRecombinantproteinwasexpressedandsuccessfullyinducedbyIPTGTheanalysisofSDS—PAGEshowedthatthefusionproteinHisBmCHWasexpressed11iglllyinRosettawithabout20kDaThefusi

5、onproteinformedtheinclusionbodiesandthenwaspurifiedwimmetal—chelatingaffinitychromatographyPolyclonalantibodyWasgeneratedbyimmunizationofNewZealandrabbitwithfusionproteinThetiteroftheantiserumcanreach1：12800withahighspec

6、ificitybyexperimemofELISAWeuseWesternblotandreal—timefluorescentquantitativePCRtoanalyzethetranscriptionallevelandtheexpressionlevelofBmCHindevelopmentalstagesanddifferenttissuesofthefifthinstarlarvaofBombyxmoriOurresult

7、ssuggestedthatBmCHWasexpressedinthefourstages，wimhighestinlarva；infifthinstarlarvathepositivesignalofBmCHwasdetectedobviouslyinepidermis，gut，testis，Malpighiantubule，ovary，thesignalinblood，silkglands，fatbody，butnotdetecte

8、dintracheaandheadTheresultsweresimilarteal—timefluorescentquantitativePCRFinallywecarriedoutthesubcellularlocalizationaboutBmCHinBmNcellandpositivesignalofBmCHwasmostlyfoundinthecytoplasmTheseresultslaidagoodfoundationof