1f-COI上海交通大学学报(医学版)1000IJOURNALOFSHANGHAIJIAOTONGUNIVERSITY(MEDICALSCIENCE)论著-基础研究人源骨架蛋白FN3展示脑钠肽前体N端抗原表位研究李梦阳I,杨春光'，孙慎侠②，徐炜晔②，汪轩羽⑵，胡学军2,T宁九曲鹏'大连大学I.医学院人体解剖学教研室；2.医学研究中心，大连116622［摘要】目的•应用人源骨架很白FN3展示脑钠肽前体N端(NT-proBNP)抗原表位，采用大肠杆菌表达系统高效表达具有脑钠肽前体N端抗原表位活性的结构稳定的重组蛋白。方法•将编码脑钠肽前体N端第12~21位和第13~20位氛基酸残基的抗原表位序列分别取代骨架蛋白FN3FGk)op区，并克隆到大肠杆菌表达载体pET28(a)+上，由大肠杆菌BL21(DE3)plySs表达后纯化，检测其抗原活性并进行理化性质分析。结果•成功构建骨架蛋白FN3展示脑钠肽前体第12~21位和第13~20位敏基酸残基抗原表位的表达栽体，获得高效表达骨架蛋白FN3展示脑钠肽前体、端抗原表位的大肠杆菌表达菌株。镣柱纯化后获得及纯度响组蛋白，经Westernblotting与ELISA签定R有脑钠肽前体N端抗原免疫活性。通过差式热仙扫描法检测显示重组贺白保持FN3骨架堂白原有结构稳定性；血浆稳定性检测显示重组蛋白是与抗原蛋白等效的高稳定的抗原赏白替代物，可明显延长降解周期(血浆半衰期)。结论•成功利用了FN3骨架蛋白展示脑钠肽N端前体，采用大肠杆菌表达系统可以简单，快速、高效制备具有与脑钠肽前体的抗原性相当的脑钠肽前体抗原替代物。［关键词】骨架蛋白：脑钠肽前体；衣位展示(DOI］10.3969/j.issn.1674-8115.2016.11.004［中图分类号］Q245［文献标志码1AStudyonthehumanscaffoldproteinFN3fordisplayingNT-proBNPepitopesLIMeng-yang',YANGChun-guang2,SUNShen-xia1,XUWei-ye?,WANGXuan-Yu',HUXue-jun2,DINGNing5,QUPeng'1.DepartmentofAnatomy.MedicalCollege,2.MedicalResearchCentre.DaLianUniversity.Dalian116622,China［Abstract］Objective-TodisplayNT-proBNPepitopesusinghumanscaffoldproteinFN3andtoefficientlyexpressastablerecombinantproteinwithactiveNT-proBNPepitopesviaE.cotiexpressionsystem.Methods-TheFGloopsequenceofscaffoldproteinFN3wasreplacedbyNT-proBNPepitope12-2!and13-20sequencesataminoacidresidue,respectively,andwasclonedinto(heE.coliexpressionvectorpET28(a)+.TherecombinantFN3proteinsdisplayingNT-proBNPepitopeswereexpressedinE.coliBL2KDE3)plySsandwerepurified.Theantigenactivitywasdetectedandphysiochemicalpropertieswereanalyzed.Results,TheexpressionvectorforrecombinantproteinFN3displayingNT-proBNPepicope12-21and13-20sequencesataminoacidresiduewassuccessfullyconstructed.TheE.colistrainwithefficientexpressionofscaffoldproteinFN3displayingNT-proBNPepitopeswasobtained.HighlypurifiedrecombinantproteinwithantigenimmuneactivityagainstNT-proBNPwasobtainedwithnickelcolumnpurificationandwasverifiedwithWesternblottingandELISA.DifferentialthermalscanningmethodshowedthattherecombinantproteinmaintainedthestabilityofscaffoldproteinFN3.Plasmastabilitytestindicatedthattherecombinantproteinwasahighlystablesubstituteforantigenproteinwiththesameeffectandcouldsignificantlyextendthedegradationcycle(seroushalf-life).Conclusion•NT-proBNPwasdisplayedsuccessfullyviascaffoldproteinFN3.AsubstituteforantigenproteinofNT-proBNPwiththesameantigenicitycanbesimply,rapidly,andefficientlypreparedwithE.coliexpressionsystem.［Keywords］FN3;NTproBNP;antigenicepitope脑钠肽（brainnatriureticpeptide,BNP）是利钠肽家族成员之一，乂称B型利钠肽（B-typenatriureticpeptide,BNP）,具.有利钠排水的作用。在心脏负荷加重时，由心肌细胞分泌的BNP可有效抑制肾素一血管紧张素一醛固狷系统，扩张血管，改善心功能。临床研究奶表明，BNP是反映心脏功能障碍的特异敏感性指标。在多种心脏疾病如心力衰竭、急性冠状动脉综合征、心肌...