masp2+egf和sp功能区抗体制备及初步鉴定

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1、———————————塑塑L一一。fEGFandSPfragmentswereconnectedt。thedigestedpGEx．6p．2Vectorw油T4DNAligasetoconstructrecombinantplasmidrespectivelv．。IheEGF，SPrecombinantplasmidsweretransformedintoEcD疗competentBL21respectivelybytheHeatShockmethod，thenthetr觚s士omedbacteriaweresmeareddirectly

2、ontoLBplateandthenculturedoVemight·Thirdly，singlecolonygrownontheLBplatewasscreenedbvPCRwiththeuniversalprimersforpGEX．6p．2；t11enthep。sitivesing二colonywasinoculatedinLBliquidselectivemediumandculturedintheshaI(eroVemightat37。C·Lastly，theplasmidswereextracted丹ommepurecul2啪

3、as‘emplate，cross_PCR(withuniversalprimerandspecificprimer)anddlgestlonmethod(withBamHIandNotI)wereusedt0identifiedtheirconectne鹊preliminarily。Theplasmidswithbothpositiveresultswere8equencedto缸rifleridentifythecorrectresultsofrecombinantplasmids．TheresuitsofDNASequencewere

4、consistentwith黝垆．2EGFandSPstand甜d∞quencefromGenBankentirely，andnodriftwasfoulld．Itmeanttne心combinantpGEX-6P-2-EGFandpGEX一6P．2．SPweresuccess如11vconstructed。’ARerid训矗cationbyrestrictiveenzymesdigestionandsequencing，BL21withtherecombinantplasmidpGEX⋯6P2EGF，pGEX．6P一2．SPwascul

5、turedOVemightrespectively．Thesecondday，IPTGwasaddedimotheenlargedcukivationofbacteriatoinduceproteinexpression，thenthecuJtI鹏wascollectedbycentrifugationat4℃．ThesupematantwasdlsPosedof，andthepellet(bacteria)waswashedwithTris．HCLbuffer．agaln．Afterultrasonicdegradationofbact

6、eria，thesupematantwass印aratedfrompelletbycentrifugationrespectively，andbothofthemweretakena1ittle。titassampleforSDS．PAGE，thenSDS．PAGEgelWasstainedwithCoomassiebrillianttoanalyzeexpressionoffusionprotein．GST_EGFfusionproteinasasolubleproteinwasexpressedinthesupematantwaspu

7、rifiedfromthesupernatant；andSPfusionpmteinexistedonlyintheformofinclusionbody；aRerureatreatment。GST．SP4英文摘要fusionproteinwaspurified．The6~7agedBALB／cmicewereimmunizedwithpurifiedGST-EGFandGST-SPfusionproteinrespectively．Micewereimmunizedviaintraperitonealinjectionthreetime

8、s，0，21，35daysrespectively．Serumwascollectedin7daysafterthelastinjection．Thespecificityofthepolyc