人MASP1N端片段原核表達載體的構(gòu)建及其表達

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1、?論著?文章編號：1007-8738(2008)06-0546-04人MASPlN端片段原核表達載體的構(gòu)建及其表達蔡學(xué)敏，趙挪，左大明，張麗蕓，陳政良收積日期：2007-07?16；接受日期：2007?08-13墓金項目：Bl家自怎料學(xué)屋金竇助巧目(30371310)；廣東省自然科學(xué)雇金研究團隊責(zé)助頊目(015003)作者簡介：＜tM(1970-).另.廣東潮州人，主管技呻，博士生Corraipondingauthor9Tel：020-61648477E-mail：zhlchen@fimmu?com(南方醫(yī)科大學(xué)免疫學(xué)教研室，廣東廣州510515)Prokaryoticexpression

2、ofNterminalfragmentofmannan-bindinglectinassociatedserineprotease-1CAIXue-min,ZHAONa,ZUODa-ming,ZHANG“yun,CHENZheng-liang*DepartmentofImmunologytSouthernMedicalUniversitytGuangzhou510515,China[Abstract]AIM：ToexpresstheN-terminalfragmentofhumanmannan-bindinglectin(MBL)associatedserinepro-teases-1

3、(MASP1-N)inE.coli.METHODS：ThetargetsequenceinpGEM-MASPlplasmidthatcontainshumanMBL-MASP1cDNAwasamplifiedbyPCRtinsertedintoprokaryoticexpressionvectorpGEX4T-landidentifiedbyrestrictionmappingandsequencing.TherecombinantexpressionvectorwastransformedintoE.coliBL21(DE3)cells.Theexpressedproductwa

4、spurifiedbyGSTrapImmobilizedMetalAffinityChromatography(IMAC)andidentifiedbySDS-PAGEandWesternblotassaytitsbinding-activitywiththecollagen-likeregionofhumanMBL(MBL-CLR)andwithrecombinanthumanMBLwasanalyzedbyanindirectenzyme-linkedimmunosorbentassay(ELISA).RESULTS：TheDNAfragmentof860bp,whichencod

5、etheN-terminalregionofhumanMASP1,wasamplifiedfrompGEM-MASPlplasmidandtherecombinantexpressionvector,pGEX4T-MASP1-N,wasconstructed,whoserestrictionmapsandsequencewereconsistentwiththoseexpected?ThecomponentofMr60000jnthepurifiedrecombinantproductwasfoundbySDS-PAGEandcouldberecognizedbyanti*GSTanti

6、bodyinWesternblotassay.ThepurifiedrecombinantproductcouldreactwithhumanMBL-CLRandhumanMBLintheindirectELISA.CONCLUSION:TheprokaryoticcellstrainthatexpressesefficienttyrecombinanthumanMASP1-N(rhMASPl-N)proteinandthepurifiedrhMASPl-Nproteinweresuccessfullyobtained.whichprovidesthebasisforfurtherres

7、earchofMASP1molecule?［Keywords］mannan-bindinglectinassociatedserineprote?ase-1；N?terminalfragment；prokaryoticexpression［摘要］目的：在大腸桿菌中表達人甘露聚糖結(jié)合凝集素(MBL)相關(guān)絲氨酸蛋白酶l(MASPl)N端片段。方法：采用PCR技術(shù)從含人MASP1cDNA的質(zhì)粒pGEM-MASPl中擴增MAS