人masp1 n端片段原核表達(dá)載體的構(gòu)建及其表達(dá)

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1、人MASP1N端片段原核表達(dá)載體的構(gòu)建及其表達(dá)作者：蔡學(xué)敏,趙娜,左大明,張麗蕓,陳政良【摘要】　　目的:在大腸桿菌中表達(dá)人甘露聚糖結(jié)合凝集素（MBL）相關(guān)絲氨酸蛋白酶1（MASP1）N端片段。方法:采用PCR技術(shù)從含人MASP1cDNA的質(zhì)粒pGEMMASP1中擴(kuò)增MASP1N端基因片段,將其插入原核表達(dá)載體pGEX4T1,轉(zhuǎn)化BL21(DE3)感受態(tài)菌誘導(dǎo)表達(dá)MASP1N端蛋白,通過(guò)GSTrap親合層析柱純化目的蛋白,以SDSPAGE和Westernblot進(jìn)行鑒定,并以ELISA分析了目的蛋白與重組MBLCLR、重組MBL的結(jié)合活性。結(jié)果:從pGEMMASP

2、1中擴(kuò)增得到約860bp的基因片段,構(gòu)建成重組載體經(jīng)酶切出現(xiàn)約4900bp和860bp片段,測(cè)序結(jié)果與預(yù)期的完全一致。純化蛋白經(jīng)SDSPAGE可見(jiàn)Mr60000蛋白帶,該蛋白可與抗GST抗體反應(yīng)并能與重組人MBLCLR、重組人MBL蛋白結(jié)合。結(jié)論:獲得了表達(dá)人MASP1N端片段的大腸桿菌菌株和重組人MASP1N端片段/GST融合蛋白,為MASP1分子的進(jìn)一步研究提供了條件。【關(guān)鍵詞】甘露聚糖結(jié)合凝集素相關(guān)絲氨酸蛋白酶1N端片段原核表達(dá)　　[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　　甘露聚糖結(jié)合凝集素（mannanbindinglectin,MBL）是由肝細(xì)胞分泌的血漿蛋白,在血循環(huán)中與MBL相關(guān)絲氨酸蛋白酶（MBLas