人MASP1N端片段原核表达载体的构建及其表达

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1、•论著•文章编号：1007-8738(2008)06-0546-04人MASPlN端片段原核表达载体的构建及其表达蔡学敏，赵挪，左大明，张丽芸，陈政良收積日期：2007-07・16；接受日期：2007・08-13墓金项目：Bl家自怎料学屋金竇助巧目(30371310)；广东省自然科学雇金研究团队责助頊目(015003)作者简介：＜tM(1970-).另.广东潮州人，主管技呻，博士生Corraipondingauthor9Tel：020-61648477E-mail：zhlchen@fimmu・com(南方医科大学免疫学教研室，广东广州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：在大肠杆菌中表达人甘露聚糖结合凝集素(MBL)相关丝氨酸蛋白酶l(MASPl)N端片段。方法：采用PCR技术从含人MASP1cDNA的质粒pGEM-MASPl中扩增MAS