.论著.人呼吸道合胞病毒截短F1重组蛋白包涵体的制备、纯化和复性傅生芳，朱传凤，姜英，安静，余黎，周旭兰州生物制品研究所有限责任公司甘肃省疫苗工程研究中心，甘肃兰州730046摘要：目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法37T诱导重:组菌体pET42b-FlJ/R°ssata,诱导完毕后离心收集曲体，高压破碎菌体并收集包涵体后用不同浓度的TritonX-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8moI/L尿素进行溶解并用镣离子亲和层析方法进行初步纯化，用阳高子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果37勾诱导5000mL重组pET-42b-FIJ/Rossata共收获37g湿菌体，经过不同浓度TritonX-100洗涤包涵体后纯度可达75%。包涵体用8mol/L尿素溶解后经镣离子亲和层析纯化纯度约为40%，再用阳离子交换层析介质SPHP进一步纯化样品后纯度可达90%o纯化蛋白以3种不同的复性液都能得到复性，其中复性液3的复性效果相对较好。结论实臆中探索r人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。关键词:呼吸道合胞病毒截断短F1蛋白；包涵体;纯化;复性中图分类号：R373.9文献标志码：A文章编号：1005-5673(2015)03-0021-05DOI：10.13309/j.cnki.pmi.2015.03.005Preparation,purificationandrenaturationofinclusionbodyoftruncatedFlrecombinantproteinofhumanrespiratorysyncytialvirusFUSheng-fang,ZHUChuan-fen,JIANGYing.ANJing,YULi,ZHOUXuLanzhouInstituteofBiologicalProductsCo.lid,CenterforGansuProvincialVaccineEngineeringResearch,Lanzhou730046,GansuProvince,ChinaCorrespondingauthor：YULi,E-mail:lyu000@sina.comAbstract：ObjectiveThetruncatedFlrecombinantproteinfromsubtypeLanzhouisolateofhumanrespiratorysyncytialvirus,whichwaspreviouslyexpressedinEscherichiacoli,waspurifiedandcompletedofrenaturationinpreparationantigenforimmunizationofanimal.MethodsRecombinantbacterialpET-42b-FlJ/Rossatawasinducedat37YandIhencentrifugedandcollected.Inclusionbodywasobtainedbypressurecrushandcentrifugation,andwashedwithdifferentconcen-trationofTritonX-100forthreetimes.Thewashedinclusionbodywaslysisedwith8Mureasolutionandpurifiedwithaneckelionaffinitychromatography,thenthetargetproteinwasfurtherpurifiedwithacationexchangechromatography.TheafGnitypurifiedrecombinantproteinwasrefoldedwiththreedifferentrenaturationliquidbydilution.Results37gwetbacteriawasgainedfrom5000mLre(*ombinantbacteriapET-42b-F1J/Rossataat37T.Thepurityofinclusionbodywasupto75%afterbeingwashedwithdifferentconcentrationTritonX-100.Hiepurityofrecombinantproteinwasabout40%bytheneckelionaHinitychromatography,whilethepuritycouldreach90%withthecationexchangechromatographySPHP.Purifiedproteincouldberefoldedbyusingthreedifferentrenaturationliquid.Therenaturationliquid3isrelativelybetteramongthem.ConclusionThepurificationmethodandproceduresfortruncatedFlrecombinantproteinofhumanrespiratorysyncytialviruscontainedininclusionbodywereperformedinthisstuidy,onwhichafoundationwassetupinpreparationofproteinandimmunizationofanimalinlaterstage.Keywords：Fltruncatedproteinofhumanrespiratorysyncytialvirus;Inclusionbody;Purification;Renaturation作者简介:傅生芳，副研究员，主要从事病毒性疫苗研发工作通讯作者:余黎.E-mail：lyu000@sina.com人呼吸道合胞病毒(Humanrespiratorysyncytialvirus,HRSV),属副粘病毒科，是引起婴幼儿病毒性肺炎最常见的病原'F,可引起间质性肺炎及毛细支气管炎。在中国多数地区，RSV相关疾病流行高峰均在1-2月。2岁以下婴幼儿、免疫缺陷及年老体弱者容易感染RSV,流行时间与地理气候有关，温带地...