29529.家蠶bme(spl)like基因的原核表達、亞細胞定位及功能分析

1、浙江理工大學碩士學位論文家蠶BmE（spl）like基因的原核表達、亞細胞定位及功能分析姓名：劉穎碩申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：張耀洲20091201AbstractWescreenedanovelgenewitharlopenreadingframe(oRF)of606bpandencoding201aminoacidswithapredictedmolecularweightof2213kD，isoelec

2、tricpointwas970fromthesilkwormpupaecDNAlibraryconstructedbyourlaboratoryAfterblastingthesequencehomologyintheNCBldatabase，weidentifieditwasanovelgeneintheBombyxmori(GenBankaccessionnumberisGU238288)Theproteinpresemedhere

3、exhibitahighdegreeofsequencesimilaritytoproteinsencodedbyE(spOE(spi)WaSfoundtoencodehighlyconservedhelixloophelix(HLH)proteinswhichcontainedthebHLHmotiforangedomainandtryptophanarginineprolinetryptophan(WR—PmdomainWename

4、ditBmE(spl)like(BombyxmoriEnhancerofspinlike)geneTheORFofthegeneWasclonedintotheprokaryoticexpressionvectorpET28aandtherecombinantplasmidwastransformedintoEcoliBL21(DE3)AfterinducedwithIPTGthisgenewassuccessfullyexpresse

5、dininsolubleinclusionbodyTheanalysisofSDSPAQEshowedthatthereWasaspecificproteinstrapWedissolvedtheinsolubleinclusionbodybythesolutionwhichcontained8mol／Lureal，andthenthefusionproteinWaspurifiedbyaffinitychromatographand1

6、0RIFPLCThemass。spectrumanalysisindicatedthattherealmolecularweightofthefusionproteinwas2628kD，whichWasconcordancewithpredictedThepurifiedfusionproteinWasusedtoimmunizeamaleNewZealandwhiterabbitandthentheantiserumWaSpurif

7、iedbyproteinAgelcolumnThetiteroftheantiserumCanarriveto1：25600withahighspecificityidentifiedbyELISAandWesternblottingWeextractedthetotalproteinandRNAfromdifferenttissuesanddevelopmentalstagesofBombyxmoriandfoundthatthele

8、velsoftranscriptionandexpressionweregreatdifferenceinthedifferenttissuesanddevelopmentalstagesofBombyxmoribyWesternblottingandrealtimePCRMeanwhile，welocatedtheproteinintheBmNcellsandtheresultindicatedthatitWasdistributed

9、inthecytoplasmThefusionprotein(HisBmE(spi)一like)wasusedtoinvestigatethebindingproteinofBmE(spi)1ikebyHispulldownElectrophoresisresultsshowedthattherewereatleasttwoBmE(spl)likebindingcomponents，andwhichmolecularweightofwe