人masp1 n端片段原核表达载体的构建及其表达

《人masp1 n端片段原核表达载体的构建及其表达》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、人MASP1N端片段原核表达载体的构建及其表达作者：蔡学敏,赵娜,左大明,张丽芸,陈政良【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1（MASP1）N端片段。方法:采用PCR技术从含人MASP1cDNA的质粒pGEMMASP1中扩增MASP1N端基因片段,将其插入原核表达载体pGEX4T1,转化BL21(DE3)感受态菌诱导表达MASP1N端蛋白,通过GSTrap亲合层析柱纯化目的蛋白,以SDSPAGE和Westernblot进行鉴定,并以ELISA分析了目的蛋白与重组MBLCLR、重组MBL的结合活性。结果:从pGEMMASP

2、1中扩增得到约860bp的基因片段,构建成重组载体经酶切出现约4900bp和860bp片段,测序结果与预期的完全一致。纯化蛋白经SDSPAGE可见Mr60000蛋白带,该蛋白可与抗GST抗体反应并能与重组人MBLCLR、重组人MBL蛋白结合。结论:获得了表达人MASP1N端片段的大肠杆菌菌株和重组人MASP1N端片段/GST融合蛋白,为MASP1分子的进一步研究提供了条件。【关键词】甘露聚糖结合凝集素相关丝氨酸蛋白酶1N端片段原核表达　　[Abstract]AIM:ToexpresstheNterminalfragmentof13humanmannanbindingle

3、ctin(MBL)associatedserineproteases1(MASP1N)inE.coli.METHODS:ThetargetsequenceinpGEMMASP1plasmidthatcontainshumanMBLMASP1cDNAwasamplifiedbyPCR,insertedintoprokaryoticexpressionvectorpGEX4T1andidentifiedbyrestrictionmappingandsequencing.TherecombinantexpressionvectorwastransformedintoE.co

4、liBL21(DE3)cells.TheexpressedproductwaspurifiedbyGSTrapImmobilizedMetalAffinityChromatography(IMAC)andidentifiedbySDSPAGEandWesternblotassay,itsbindingactivitywiththecollagenlikeregionofhumanMBL(MBLCLR)andwithrecombinanthumanMBLwasanalyzedbyanindirectenzymelinkedimmunosorbentassay（ELISA

5、）.RESULTS:TheDNAfragmentof860bp,whichencodetheNterminalregionofhumanMASP1,wasamplifiedfrompGEMMASP1plasmidandtherecombinantexpressionvector,pGEX4TMASP1N,wasconstructed,whoserestrictionmapsandsequencewereconsistentwiththoseexpected.ThecomponentofMr60000inthepurifiedrecombinantproductwasfo

6、undbySDSPAGEandcouldberecognizedbyantiGSTantibodyinWesternblotassay.ThepurifiedrecombinantproductcouldreactwithhumanMBLCLRandhumanMBLintheindirectELISA.CONCLUSION:Theprokaryoticcellstrainthatexpressesefficientlyrecombinant13humanMASP1N(rhMASP1N)proteinandthepurifiedrhMASP1Nproteinweres

7、uccessfullyobtained,whichprovidesthebasisforfurtherresearchofMASP1molecule.　　[Keywords]mannanbindinglectinassociatedserineprotease1;Nterminalfragment;prokaryoticexpression　　甘露聚糖结合凝集素（mannanbindinglectin,MBL）是由肝细胞分泌的血浆蛋白,在血循环中与MBL相关丝氨酸蛋白酶（MBLas