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时间:2020-05-04
《两种嵌合乙型肝炎病毒preSl中和表位的乙肝核心病毒样颗粒的制备与抗原性分析-论文.pdf》由会员上传分享,免费在线阅读,更多相关内容在应用文档-天天文库。
1、·336·中华实验和临床病毒学杂志2013年lO月第27卷第5期ChineseJExpClinVirol,October2013,Vo1.27,No.5.论著两种嵌合乙型肝炎病毒preS1中和表位的乙肝核心病毒样颗粒的制备与抗原性分析苏秋东,郭敏卓,伊瑶,陈斯勇,贾志远,卢学新,邱丰,毕胜利【摘要】目的构建嵌合preS12147位氨基酸(aa)、以全长乙肝病毒(HBV)核心抗原(HBcAg)为骨架的病毒样颗粒H2和嵌合preS1aa3745、以截断HBcAg为骨架的病毒样颗粒H3,并对比其抗原性。方法将HBVadr血清型preS1中和表位aa2147和aa3745分别插入到全长
2、HBc(aa1.183)和截断HBc(aa1.144)的第78位天冬氨酸和第79位脯氨酸之间,密码子优化之后全基因合成,目的片段经双酶切(NdeI和XhoI)回收后连接到同样处理的pET43.1a载体上,在大肠埃希菌E.coliBL21(DE3)中表达,精细纯化后通过电镜观察蛋白形态、ELISA以及WesternBlott检测其抗原性。结果成功构建表达质粒H2和H3,并在BL21(DE3)中高效表达和自发组装成病毒样颗粒。纯化后电镜观察H2和H3形态为二十面体立体对称样病毒样颗粒,H2为实心颗粒,直径为(31.614-1.27)nm,H3为空心颗粒,直径为(28.46±1.16
3、)nm。统计分析可得H2直径要比H3直径大(P<0.01)。ELISA和WesternBlott检测结果证实其插入表位preS1以及载体蛋白HBe的抗原性。结论成功获得两种嵌合HBVpreS1中和表位的病毒样颗粒,为其在疫苗研究中的应用探索奠定基础。【主题词】肝炎病毒,乙型;表位;病毒核心蛋白质类;病毒样颗粒AntigenicanalysisoftwochimerichepatitisBcoreparticlespresentingthepreS1neutralizingepitopesSUQiu—dong‘,GUOMin—zhuo,YIYao,CHENSi—yong,flAZ
4、hi—yuan,LUXue—xin,QIUFeng,BISheng—li.NationalInstituteforViralDiseaseandPrevention,ChinaCDC,Beng102206,ChinaCorrespondingauthor:BISheng—li,Email:shengli—hi@163.corn【Abstract】0bjectiveToconstructfull—lengthhepatitisBcoreparticlespresentingpreS1aa2147epitopeandtruncatedcoreparticlespresentingp
5、reS1aa3745epitopeontheirsurfaceandcomparetheirantigenicity.MethodsPreS1aa21-47epitopeandaa3745epitopewereinsertedrespectivelyintofull—lengthhepatitisBcore(aa1—183)andtruncatedHBcAg(aa1—144),betweenthe78(Asp)and79(Pro).ThegenessynthesizedafterthecodonoptimizationwereligatedtothepET43.1avector
6、withthesamecohesiveterminal(NdeIandXhoI)andexpressedintheE.coliexpressionsystem.ThemorphologyoftheproteinsofinterestwereobservedbyelectronmicroscopeandcharacterizedbyELISAandWesternBlotting.ResultsThemorphologyofthevirus—likeparticleswereconfirmedbyelectronmicroscope.H2weresolidparticleswith
7、adiameterof(31.61±1.27)nm,whileH3werehollowparticleswithadiameterof(28.464-1.16)/lm.StatisticalanalysisshowedthatH2islargerthanH3inthediameter(P<0.01)。TheantigenicityoftheinsertedepitopesandcarrierproteinwereidentifiedbyELISAandWesternBlotting.Conc
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