wwpdf - 柑橘溃疡病菌二硫键稳定fv 抗体基因的构建及表达产物的生物学VIP专享VIP免费

中国农业科学2009,42(9):3123-3130ScientiaAgriculturaSinicadoi:10.3864/j.issn.0578-1752.2009.09.013收稿日期：2008-12-11；接受日期：2009-03-09基金项目：国家“十一五”科技支撑计划项目（2007BAD47B03-5）、国家“863”计划项目（2006AA10Z434）作者简介：王中康（1956－），男，四川雅安人，教授，研究方向为微生物分子生物学。Tel：023-65120489；Fax：023-65120489；E-mail：zkwang646@sina.com。通信作者殷幼平（1955－），女，重庆长寿人，教授，研究方向为微生物分子生物学。Tel：023-65120489；Fax：023-65120489；E-mail：ypy128@vip.sina.com柑橘溃疡病菌二硫键稳定Fv抗体基因的构建及表达产物的生物学活性分析王中康1，李泮志1，袁青2，于红1，李蒙1，殷幼平1（1重庆大学生物工程学院/重庆大学基因工程研究中心/重庆市基因功能与调控重点实验室，重庆400030；2四川省泸州医学院免疫学教研室，四川泸州646000）摘要：【目的】构建鼠源抗柑橘溃疡病菌二硫键稳定Fv抗体(dsFv）片段基因，原核表达并将其复性为具有免疫活性的dsFv抗体。【方法】采用PCR定点突变的方法构建dsFv重链（VH）及轻链可变区（VL）基因，分别将其连接入表达载体中，于E.coliBL21（DE3）中诱导表达，表达产物溶解后稀释入复性缓冲液中使其重折叠为dsFv抗体并纯化。SDS-PAGE及Western-blot分析表达及复性产物，BIAcore检测dsFv与Xac-LPS的亲和力，ELISA验证dsFv的特异性及稳定性。【结果】测序结果表明在VH的44位和VL的100位成功引入了半胱氨酸突变位点，实现了高效的原核表达，表达产物主要以包涵体的形式存在。SDS-PAGE分析显示VH和VL的大小为23kD，并将其成功复性，复性后大小为46kD。BIAcore分析表明dsFv对Xac-LPS保持了很高的亲和力，亲和常数（KD）达到3.40×10-10M。ELISA证明dsFv具有较高的抗原特异性并且热稳定性较scFv提高近20℃。【结论】成功表达并复性了dsFv抗体，为柑橘溃疡病菌的免疫诊断提供了高效优质的重组抗体。关键词:柑橘溃疡病菌；二硫键稳定Fv抗体；包涵体；复性；BIAcore分析GeneConstruction,ExpressionandBiologicalActivityAnalysesofDisulfideStabilizedFvFragmentAgainstXacWANGZhong-kang1,LIPan-zhi1,YUANQing2,YUHong1,LIMeng1,YINYou-ping1(1BioengineeringCollegeofChongqingUniversity/GeneEngineeringResearchCenter/KeyLaboratoryofGeneFunctionandRegulationofChongqing,Chongqing400030;2DepartmentofImmunology,LuzhouMedicalCollege,Luzhou646000,Sichuan)Abstract:【Objective】Toconstructthegeneofmouseanti-XacdisulfidestabilizedFvfragments(dsFv),expressandrefoldtotheformofdsFvwithbiologicalactivity.【Method】ThegenesofVHandVLraisedagainstXacweremutatedbythemethodofPCR-basedmutagenesis,andthenclonedintoexpressionplasmid.TherecombinantplasmidsweretransformedintoE.coliBL21(DE3)strainandthesetwogeneswereexpressed.TheinclusionbodyproteinsofVHandVLwereharvestedanddissolved,andthendilutedintorefoldingsolutionproratatoformdsFvantibodywhichwasfurtherpurifiedbyHisTrapHPcolumn.TheproductsofexpressionandrenaturationwereanalyzedbySDS-PAGEandwestern-blot.TheaffinityofdsFvtoXac-LPSwasdeterminedbyBIAcore.ThespecificityandstabilityweredetectedbyELISA.【Result】SequenceanalysisprovedthatcysteineswereintroducedintoVH44andVL100,andthegenesexpressedinE.coliBL21(DE3).Mostoftheproteinexistedintheformofinclusionbody,theexpressionproductswererefoldedsuccessfully.SDS-PAGEshowedthatthemolecularmasseswere23kDforVHandVL,and46kDfordsFv.BIAcoreanalysisshowedthatdsFvretainedhighaffinitytoXac-LPSwithanaffinityconstant(KD)of3.40×10-10M.ELISAindicatedthatdsFvhadahighspecificitytoXac.ComparedwithscFv’s,thethermalstabilityofdsFvelevatednearly20℃.【Conclusion】dsFvwasexpressedandrefoldedsuccessfully,whichmaintainedhighaffinity,specificityandstability.Thisresear...