Genetic variation in HBV infection genotypes and mutants

Introduction to PhysiologyIntroductionPhysiology is the study of the functions of living matter. It is concerned with how an organism performs its varied activities: how it feeds, how it moves, how it adapts to changing circumstances, how it spawns new generations. The subject is vast and embraces the whole of life. The success of physiology in explaining how organisms perform their daily tasks is based on the notion that they are intricate and exquisite machines whose operation is governed by the laws of physics and chemistry.Although some processes are similar across the whole spectrum of biology—the replication of the genetic code for or example—many are specific to particular groups of organisms. For this reason it is necessary to divide the subject into various parts such as bacterial physiology, plant physiology, and animal physiology.To study how an animal works it is first necessary to know how it is built. A full appreciation of the physiology of an organism must therefore be based on a sound knowledge of its anatomy. Experiments can then be carried out to establish how particular parts perform their functions. Although there have been many important physiological investigations on human volunteers, the need for precise control over the experimental conditions has meant that much of our present physiological knowledge has been derived from studies on other animals such as frogs, rabbits, cats, and dogs. When it is clear that a specific physiological process has a common basis in a wide variety of animal species, it is reasonable to assume that the same principles will apply to humans. The knowledge gained from this approach has given us a great insight into human physiology and endowed us with a solid foundation for the effective treatment of many diseases.The building blocks of the body are the cells, which are grouped together to form tissues. The principal types of tissue are epithelial, connective, nervous, and muscular, each with its own characteristics. Many connective tissues have relatively few cells but have an extensive extracellular matrix. In contrast, smooth muscle consists of densely packed layers of muscle cells linked together via specific cell junctions. Organs such as the brain, the heart, the lungs, the intestines, and the liver are formed by the aggregation of different kinds of tissues. The organs are themselves parts of distinct physiological systems. The heart and blood vessels form the cardiovascular system; the lungs, trachea, and bronchi together with the chest wall and diaphragm form the respiratory system; the skeleton and skeletal muscles form the musculoskeletal system; the brain, spinal cord, autonomic nerves and ganglia, and peripheral somatic nerves form the nervous system, and so on.Cells differ widely in form and function but they all have certain common characteristics. Firstly, they are bounded by a limiting membrane, the plasma membrane. Secondly, they have the ability to break down large molecules to smaller ones to liberate energy for their activities.生理学简介介绍生理学是研究生物体功能的科学。

!N"!慢性乙型肝炎信号转导及转录激活因子３、５与Ｔｒｅｇ／Ｔｈ１７平衡的关系李敏癑１，杨红菊１，李　静２，宋　瑞１，游　晶１１国家卫健委毒品依赖和戒治重点实验室，昆明医科大学第一附属医院老年消化内科，昆明６５００３２；２昆明医科大学公共卫生学院，昆明６５０５００摘要：慢性乙型肝炎持续感染的免疫机制与Ｔ淋巴细胞密切相关，Ｔ淋巴细胞的发育需要多种细胞因子的协调作用，而信号转导及转录激活因子家族蛋白主要参与细胞因子的信号转导，尤其是ＳＴＡＴ５ａ／ｂ和ＳＴＡＴ３在调节性Ｔ淋巴细胞（Ｔｒｅｇ）和辅助性Ｔ淋巴细胞１７（Ｔｈ１７）的分化、发育过程中有着重要作用。

本文主要就在慢性乙型肝炎中，对信号转导及转录激活因子３、５与Ｔｒｅｇ／Ｔｈ１７平衡的关系进行分析，研究ＨＢＶ感染的慢性化及其引起的肝脏炎症调控机制。

关键词：乙型肝炎，慢性；ＳＴＡＴ３转录因子；ＳＴＡＴ５转录因子；Ｔ淋巴细胞，调节性；Ｔｈ１７细胞基金项目：国家自然科学基金项目（８１７６０１１１，８１７６０６１７）；国家卫生健康委毒品依赖和戒治重点项目（２０２０ＤＡＭＡＲＡ－００３）Ａｓｓｏｃｉａｔｉｏｎｏｆｓｉｇｎａｌｔｒａｎｓｄｕｃｅｒａｎｄａｃｔｉｖａｔｏｒｏｆｔｒａｎｓｃｒｉｐｔｉｏｎ３ａｎｄｓｉｇｎａｌｔｒａｎｓｄｕｃｅｒａｎｄａｃｔｉｖａｔｏｒｏｆｔｒａｎｓｃｒｉｐｔｉｏｎ５ｗｉｔｈｒｅｇｕｌａｔｏｒｙＴｃｅｌｌ／Ｔｈｅｌｐｅｒ１７ｃｅｌｌｂａｌａｎｃｅｉｎｃｈｒｏｎｉｃｈｅｐａｔｉｔｉｓＢＬＩＭｉｎｙｕｅ１，ＹＡＮＧＨｏｎｇｊｕ１，ＬＩＪｉｎｇ２，ＳＯＮＧＲｕｉ１，ＹＯＵＪｉｎｇ１．（１．ＮＨＣＫｅｙＬａｂｏｒａｔｏｒｙｏｆＤｒｕｇＡｄｄｉｃｔｉｏｎＭｅｄｉｃｉｎｅ，ＤｅｐａｒｔｍｅｎｔｏｆＧｅｒｉ ａｔｒｉｃＧａｓｔｒｏｅｎｔｅｒｏｌｏｇｙ，ＴｈｅＦｉｒｓｔＡｆｆｉｌｉａｔｅｄＨｏｓｐｉｔａｌｏｆＫｕｎｍｉｎｇＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｋｕｎｍｉｎｇ６５００３２，Ｃｈｉｎａ；２．ＳｃｈｏｏｌｏｆＰｕｂｌｉｃＨｅａｌｔｈ，ＫｕｎｍｉｎｇＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｋｕｎｍｉｎｇ６５０５００，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＹＯＵＪｉｎｇ，ｊｉｎｇｙｏｕｋｍ＠１２６．ｃｏｍ（ＯＲＣＩＤ：００００－０００２－５９１９－５８６４）Ａｂｓｔｒａｃｔ：ＴｈｅｉｍｍｕｎｅｍｅｃｈａｎｉｓｍｏｆｃｈｒｏｎｉｃｈｅｐａｔｉｔｉｓＢ（ＣＨＢ）ｐｅｒｓｉｓｔｅｎｔｉｎｆｅｃｔｉｏｎｉｓｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｗｉｔｈＴｃｅｌｌｓ，ａｎｄｔｈｅｄｅｖｅｌｏｐｍｅｎｔｏｆＴｃｅｌｌｓｒｅｑｕｉｒｅｓｔｈｅｃｏｏｒｄｉｎａｔｉｏｎｏｆａｖａｒｉｅｔｙｏｆｃｙｔｏｋｉｎｅｓ．Ｔｈｅｐｒｏｔｅｉｎｓｏｆｔｈｅｓｉｇｎａｌｔｒａｎｓｄｕｃｅｒａｎｄａｃｔｉｖａｔｏｒｏｆｔｒａｎｓｃｒｉｐｔｉｏｎ（ＳＴＡＴ）ｆａｍｉｌｙａｒｅｍａｉｎｌｙｉｎｖｏｌｖｅｄｉｎｔｈｅｓｉｇｎａｌｔｒａｎｓｄｕｃｔｉｏｎｏｆｃｙｔｏｋｉｎｅｓ，ａｎｄＳＴＡＴ５ａ／ｂａｎｄＳＴＡＴ３ｐｌａｙａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｔｈｅｄｉｆｆｅｒｅｎｔｉａｔｉｏｎａｎｄｄｅ ｖｅｌｏｐｍｅｎｔｏｆｒｅｇｕｌａｔｏｒｙＴｃｅｌｌｓ（Ｔｒｅｇ）ａｎｄＴｈｅｌｐｅｒ１７ｃｅｌｌｓ（Ｔｈ１７）．ＴｈｉｓａｒｔｉｃｌｅａｎａｌｙｚｅｓｔｈｅａｓｓｏｃｉａｔｉｏｎｏｆＳＴＡＴ３ａｎｄＳＴＡＴ５ｗｉｔｈＴｒｅｇ／Ｔｈ１７ｂａｌａｎｃｅｉｎＣＨＢａｎｄｉｎｖｅｓｔｉｇａｔｅｓｔｈｅｃｈｒｏｎｉｃｉｔｙｏｆｈｅｐａｔｉｔｉｓＢｖｉｒｕｓｉｎｆｅｃｔｉｏｎａｎｄｔｈｅｒｅｇｕｌａｔｏｒｙｍｅｃｈａｎｉｓｍｏｆｌｉｖｅｒｉｎｆｌａｍｍａｔｉｏｎ．Ｋｅｙｗｏｒｄｓ：ＨｅｐａｔｉｔｉｓＢ，Ｃｈｒｏｎｉｃ；ＳＴＡＴ３ＴｒａｎｓｃｒｉｐｔｉｏｎＦａｃｔｏｒ；ＳＴＡＴ５ＴｒａｎｓｃｒｉｐｔｉｏｎＦａｃｔｏｒ；Ｔ－Ｌｙｍｐｈｏｃｙｔｅｓ，Ｒｅｇｕｌａｔｏｒｙ；Ｔｈ１７ＣｅｌｌｓＲｅｓｅａｒｃｈｆｕｎｄｉｎｇ：ＮａｔｉｏｎａｌＮａｔｕｒａｌＳｃｉｅｎｃｅＦｏｕｎｄａｔｉｏｎｏｆＣｈｉｎａ（８１７６０１１１，８１７６０６１７）；ＮａｔｉｏｎａｌＨｅａｌｔｈＣｏｍｍｉｓｓｉｏｎＫｅｙＰｒｏｊｅｃｔｏｆＤｒｕｇＤｅｐｅｎｄｅｎｃｅａｎｄＲｅｈａｂｉｌｉｔａｔｉｏｎ（２０２０ＤＡＭＡＲＡ－００３）ＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－５２５６．２０２２．０６．０３６收稿日期：２０２１－０９－２６；录用日期：２０２１－１１－０２通信作者：游晶，ｊｉｎｇｙｏｕｋｍ＠１２６．ｃｏｍ 慢性乙型肝炎（ＣＨＢ）已经是全球严重公共卫生事件之一，虽然现在乙型肝炎疫苗已经问世多年，但是全球仍然有超过２ ４亿的人群感染ＨＢＶ，并逐渐往肝硬化、肝癌方向发展［１］。

不结球白菜（Brassica campestris ssp．chinensis Makino）种质资源SRAP遗传分化分析作物ACTAAGRONOMICASINICA2007,33(11):1862—1868ISSN0496—3490;CODENTSHPA9/zwxb/E-mail:*********************.cn不结球白菜(Brassicacampestrisssp.chinensisMakino)种质资源SRAP遗传分化分析韩建明,侯喜林,徐海明史公军耿建峰邓晓辉(南京农,l大学作物遗传i种质创新旧家再点实验窜,江苏南京210095;洛阳农业科学研究院,河南洛阳471022;浙汀大学农业与生物技术学院农学系,浙江杭州310029)摘要:利用SRAP分子标记分析了国内外64份不结球白菜种质资源的DNA遗传多样性和遗传分化.21对引物组合共检测出215个位点,其中112个为多态性位点,多态性比率达52.09%,平均每对引物组合产生10.24个位点和5.33个多态性位点.不结球白菜各类型中普通白菜的Nei's基因多样性指数(0.1410)和遗传丰富度[(190)88.37%]最高;各生态区域[fI汀淮流域不结球白菜的Nei's基因多样性指数(0.1451)和遗传丰富度[(185)86.05%]最高;国内的Nei's基因多样性指数(0.1293)和遗传丰富度[(188)87.44%]分别高于国外.遗传变异估算表明,不结球白菜遗传分化系数58.22%,大部分变异存在丁种群问;基因流为0.4031,说明群体问基因流动较少,遗传分化程度较高.以遗传相似系数0.872为截值,口『把不结球球白菜分为Ⅵ个类群.关键词:不结球白菜(Brassicacampestrisssp.chinensisMakino);种质资源;SRAP;遗传多样性;遗传分化GeneticDifferentiationofNon-HeadingChineseCabbage(Brassicacampestrisssp. chinensisMakino)GermplasmBasedonSRAPMarkersHANJian—Ming,,H0UXi—Iin,,XUHai—Ming,SHIGong—Jun,GENGJian—Feng,andDENGXiao—Hui (NationalKeyLaboratoryofCropGeneticsandGermplasmEnhancement,Agricuhur',dSciences,Luoyang471022,Henan;DepartmentofAgronomy, NamingAgriculturalUniversity,Nanjing210095,Jiangsu;LuoyangAcademyofZh~iangUniversity,Hangzhou310029,Zh~iang,China)Abstract:Thegeneticdiversityanddifferentiationwereassessedin64accessionsofnon—headingChinesecabbagefromallthewoadbySRAP(Sequence—relatedamplifiedpolymorphism)markers.Twohundredandfifteenlociweredetectedby21 pairsofSRAPprimers.Amongthem,112werepolymorphie,thepolymorphieratewas52.09 %,and10.24lociand5.33 polymorphielociwereamplifiedbyeachpairprimeranaverage.TheNei'sgenediversityinde xandGeneticrichnessofB.muniswere0.1410and(190)88.37%respectively,thehighestamongdif ferenttypesofnon—headingChinesecabbage,and0.1451and(185)86.05%respectivelyinJianghuaiPlain,thehighesta mongalltheregionsecologicalregions.Thesetwoindiceswererespectively0.1293and(188)87.44%higherindo mesticcabbagethaninthefbreigncabbage.Themeasurementofgeneticvariationshowedthatthecoefficientofgene tic differentiationwas58.22%. Mostofthegeneticvariationexistedamongpopulations.Thegeneflow(0.4031)waslessamo ngpopulations,indicatingthat thedegreeofgeneticdifferentiationwashigher.Sixgroupswereclusteredaccordingtogeneti csimilaritycoefficientof0.872.Keywords:Non—headingChinesecabbage(Brassicacampestrisssp.chinensisMakino);Germplasm;SRAP; Geneticdiversity;Geneticdifierentiation不结球白菜(Brassicacampestrisssp.chinensisMakino)原产f{1国,具有适应性广,生长期短,高产,省工,易种,品质柔嫩,营养丰富以及鲜食腌债皆宜等特点.不结球白菜占长江中下游各大中城市蔬菜年总产量的30%～40%,占秋,冬,春季蔬菜复种面积的40%～60%,在蔬菜周年生产和供应上占有重要地位.近年来不仪北方大量引种栽培,东南亚,日本,美国及欧洲一些国家也广泛引种,不结球白菜已逐渐成为世界性的蔬菜.种质资源是遗传改良的基础,只有了解种质资基金项目:周家自然科学基金项目(30671420);南京农q大学作物遗传j种质创新闻家重点实验室开放课题(ZW2005007)作者简介:韩建明(1966一),男,副研究员,主要从事蔬菜遗传育种.Tel:025—84395332;E—mail:*********************通汛作者(Correspondingauthor):侯喜林,男,教授,博f生导师.TeI:025—84395917;E-mail:**************Received(收稿F1期):2006—10—12;Accepted(接受日期):2006.12—19.第11期韩建明等:不结球白菜(Brassicacampestrisssp.chinensisMakino)种质资源SRAP遗传分化分析源遗传变异信息及亲缘关系的疏远,才能有目的地选配亲本,培育出优良品种.目前小麦,玉米,水稻,大豆等世界性主要作物种质资源的研究日新月异J.巾尉作为不结球白菜遗传多样性巾心和起源中心,曾在不结球白菜种质资源形态学,生物学特性,分类及品质鉴定等方面进行了研究',并通过研究其起源,构建了包括不结球白菜的中同白菜演化关系图,但利用分子标记对国内外不结球白菜种质资源的遗传分析报道较少.近几年,DNA分子标记被广泛应用于辅助选择育种,遗传多样性研究,其中应用较多的有RAPD, AFLP和SSR等,RAPD方法简单,成本低,但重复性较差,检测位点不多;SSR多为共显性,重复性好,但位点较少,引物开发成本高;AFLP谱带多,但分析程序复杂,成本高.2001年Li和Quiros12]开发了一种新型分子标记SRAP(Sequence—relatedamplified polymorphism),可通过独特的引物设计优先扩增基因组内的开放阅读框(ORF),对基因组内龠子区域, 启动子区域进行特异扩增.因不同个体,物种的内含子,启动子及问隔区长度不同而产生多态性.该标记具有简便,稳定,兼有RAPD的操作简单和AFLP重复性好,多态性强的优点,目前已应用于图谱构建n,比较基因组学¨和遗传多样性分析..本研究将SRAP分子标记应用于不结球白菜的遗传分析,旨在了解不结球白菜的遗传背景,为不结球白菜杂种优势利用中的亲本选配提供理论基础,以解决生产中由于少数高产品种大面积应用而使品种遗传脆弱性加剧的问题.1材料与方法1.1材料为保证不同类型和地理来源的遗传代表性,从5个国家和国内14个省(市)选取有代表性的不结球白菜共64份,其中同内47份,国外17份;包含了普通白菜,乌塌菜,菜心(薹),分蘖菜和薹荣等类型(表1)表1供试材料及来源Table1Thecodeandoriginofthematedal 编出种O来ri源gin生Ec态olo区gic域alCodeAccession编号●品种O米ri源gin生Ec态ologi:c域alCodeAccession1二青ErqingJiangsu,China江淮JH27雅抗1号F】Y akang1F】Jiangsu,China江淮JH2高自梗GaobaigengJiangsu,China江淮JH28特矮青梗TeaiQinggengZhejiang,China东南sE3大头荣DatoucaiJiangsu,China江淮JH29强华F】QianghuaFlZhejiang,China东南sE4中自梗ZhongbaigengJiangsu,China江淮JH3O山西白ShanxibaiFujian,China东南sE5油冬儿Y oudongerJiangsu,China江淮JH31超群优CaoqunyouFujian,China东南SE6老鸟子LaowuziJiangsu,China江淮JH32四季中箕SijizhongqiHubei,China华中MC7白叶苏州青BaiyesuzhouqingJiangsu,China汀淮JH33白王子BaiWangziHunan,China华中MC8短白梗DuanbaigengJiangsu,China江淮JH34黄邦HuangbangLiaoning,China华北NC9亮白叶LiangbaiyeJiangsu,China江淮-lH35绿青菜FlL{iqingcaiFlJapan东,IAE1O黑叶}==1菜HeiyebaicaiJiangsu,China江淮JH36小青翠XiaoqingcuiJapan东亚AE11矮脚黄AijiaohuangJiangsu,China2I淮JH37黄心莱HuanxincaiHebei,China华北NC12奶白莱NaibaicaiJiangsu,China江淮JH38紫罗兰F】ZiluolanF1Beijing,China华』匕Nc13苏州青SuzhouqingJiangsu,China江淮JH39绿生1号Liisheng1Henan,China华中MCl4五月慢WuyuemanJiangsu,China江淮JH4f】上海青ShanghaiqingShanghai,China 汀淮JH15夏冬青XiadongqingJiangsu,China江淮JH41正旺达ZhengwangdaShaanxi.China 华北NC16新矮青XinaiqingJiangsu,China江淮JH42乌塌菜WutacaiJiangsu,China汀淮JH 17镇青梗ZhenqinggengJiangsu,China江淮JH43黄心乌HuangxinwuAnhui,China 江淮JH18早生京华ZhaoshenjinghuaJapan东亚EA44小八叶XiaobayeShanghai,China江淮JH19暑绿ShultiJiangsu,China江淮JH45马耳头MaertouJiangsu,China江淮JH2O寒笑HanxiaoJiangsu,China江淮JH46四九菜心SijiucaixinGuangdong,China两广GG21矮抗5号Aikang5Jiangsu,China江淮lIH4745天菜心45daycaixinGuangxi,China 两广GG22青优1号Qingyou1Jiangsu,China江淮JH48南蔬极品6O天Guangxi,China两广GG23f'月柳ShiyueliuJiangsu,China江淮JH60daynanshujipin24香青菜XiangqingcaiJiangsu,China江淮JH49油绿5O天50dayyoultiGuangdong,China两广GG25绿星青菜LiixingqingcaiJiangsu,China江淮JH5O油丰18Y oufeng18Guangxi,China两广GG26热优2号ReY ou2Jiangsu,China江淮lIH51香港油菜HongKongyoucaiJapan东EA作物第33卷续表编f1种O来rig源in4Ec态olo区gic域al CodeAccession 编号^种O来ri源ginEEc态oh)p(gic域alC odeAccession region52菏邦油菜QingbangyoucaiJapan东,】EA58好吃FlHaoehiFTJapan】EA53徐州花叶摹荣Jiangsu,China江淮JH59VS一40120FtJapanl身,】EAXuzht)uhuavetait,aj60KugiyaJapan,】EA54AsiawhiteFIKorea东,】EA61泰抗TaisankangThailat.1东南,SEA55Hagye1hoFIKorea东EA62EthopicIndia东南,匝SEA56冬女FIDongfeiFlJapan力世EA63EthopiccabbageIndia东带SEA57夏帝FlXiadiFIJapan东亚EA64金品21FlJinpin21FTNewZealand大洋洲OC编42～44属呜塌菜,编45分蘖菜s,编46～52属菜心,编号53属壤菜,其他属普通】J荣.Cabbagecoded42—44areBcampestrisvarrosularis,45isB.campestrisvar.muhiceps,46—52areB.campestrisvat.parachinen,53isBcampestrisvartai-tsai,andtheothersareB.campestrisvatf0r,lr,l"n.JH:JianghuaiPlain,SE:S outheast,GG:GuangdongandGuangxiProvinces,NCNorthChina,MC:MiddleChina,EA:EastAsia,SEA:SoutheastAsia,OC:Oceaina.1.2DNA提取将供试材料播种于八衙中,待长出2片真叶时,每份材料随机选取幼嫩叶片l～2g,于研钵中用液氮速冻,迅速研磨.采用CTAB法提取叶片总DNA,并保存于100ITE中.1.3SRAP反应体系及扩增程序参照"和Quiros已发表的序列没计SRAP—PCR引物,由J:海博公司合成.PCR总反应体积为l0I,含lL10×buffer,2.5mmolIMgCl2,0.2mmolL—dNTPs,2.5UTaq酶(购自TAKARA公司),模板DNA30ng,止向和反向引物各0.3/~molL,不足部分用ddH,O补足.PCR于MastercycleEP扩增仪上进行.热循环程序为94℃变性5min;94℃变性lmin,35℃复性lmin,72℃延伸1.5min,5个循环;94℃变性lmin,50%复性lmin,72℃延伸1.5min,33个循环;72℃延伸7min;4℃保存.1.4扩增产物检测扩增产物用6%聚内烯酰胺凝胶电泳,采用JY—SCZ6电泳槽和JY—ECP3000电泳仪.l×TAE电泳缓冲液,Marker为TaKaRa公一J的50bpLadder,电压120V(5vcm),电泳结束后进行银染,显色后拍照保存.1.5统计分析每对SRAP引物检测1个位点,视每条多态性带为1个等佗基因.将观察到的每条带视为1个性状,记录100～800bpDNA带的有或无,条带清晰可辨的记为"l",缺失或模糊不清的记为"0".用PopGenV ersion1.3l软件计算观察等位基因数(ⅣA)16],有效等位基因数(NE)¨】,Nei's基因多样性指数(日)¨,遗传分化系数(G.),基因流(Nm)h,遗传丰富度(G尺).=l一∑∑P;G=(一H)/H;11Nm=(1一G)/2G式中,P为第i个性状第个表现型的频率,日为总群体的基L大]多样性指数,为种群的基因多样性指数.1.6聚类分析用Jaccard遗传牛H似系数进行聚类分析,计算方法为GS=N/(N+Ⅳ+),其中Ⅳ为材料i和材料/址有条带数,Ⅳ为i材料特有的条带数,Ⅳ,为i材料特有的条带数.用NTSYSpc2.1Oe软件巾的SHAN程序进行聚类,并作Ⅲ聚类.2结果与分析2.1不结球白菜SRAP引物扩增多态性用筛选出的2l对引物组合在64份种质中共扩增出215个位点,其中JJ2个位点为多态性位点,多态性比率达52.09%,平均每个引物组合产生l0.24个位点和5.33个多态件位点.不旧引物扩增出的总位点数和多态性位点数的差异较大,产牛位点数最多的是mel—em2引物组合,扩增出l5个位点;产生位点数最少的是me2一em5,me2一em6和me3一em2引物组合,分别扩增出8个位点;多态性比率最高的是me5一em3,me5一em4引物组合,达77.78%;多态性比率最低的是me3一em4,me4一em4,me4一em6引物组合,仅为30.00%(表2).2.2不同类型不结球白菜SRAP遗传多样性由2l对引物检测的结果表明,不结球白菜各类型遗传多样性高低不同(表3).普通白菜的观察等位基因数(1.5302),有效等位基因数(1.2395),Nei'S基因多样性指数(1.2395)和遗传丰富度(190)第11期韩建明等:不结球白菜(Brassicacampestrisssp.chinensisMakino)种质资源SRAP遗传分化分析88.37%都以普通白菜最高;此外菜心的观察等位基因数(1.1721)和遗传丰富度(166)77.21%略高于乌塌菜,而乌塌菜的有效等化基囚数(1.1097)和Nei'S基因多样性指数(0.0618)高于菜心,这说明普通白菜不仅遗传离散度较高,而且遗传丰富度也较高,不结球白菜的遗传多样性主要分布在普通白菜中.表2SRAP引物组合扩增不结球白菜DNA的多态位点数Table2ThenumberofpolymorphiclociamplifiedbySRAPprimers TotalnumbeofpolymocP引rim物eTotalnumher多Polymorph数ie胁eofpolymorcmet—em49666.67me2一em2t4750.OOme2一em49666.67me2一em68337.5Ome2em58450.O0me3em28562.50me3一emt9666.67me3em3t3646.15me3一em4t0330.OOme3一em5t0440.OOme4一em2tt545.45me4一eu14lO55O.OOme5一era2tt327.27me4一era5lO33O.OOme5一em39777.78me4一em6lO33O.o0me5一em49777.78me5一emt8450.OOmet—em2t5t066.67me5一em5t4857.t4met—em3t0770.00总tl-/-T.均Total/Mean2t5tt252.09表3不同类型不结球白菜SRAP标记的遗传多样性指数Table3ThegeneticdiversityindexbasedonSRAPmarkerfordifferenttypesofnon—beadingChinesecabbageNA:Observednumberofallele;NE:Effectivenumberofallele;H:Nei'Sgenediversity;GR: Geneticrichness2.3不同生态区域不结球白菜SRAP遗传多样性用不同的引物估算的不结球白菜种质资源在各牛态区域的遗传多样性也不同(表4).国内各生态区域内不结球白菜观察等位基因数(1.4526),有效等位基因数(1.2460),Nei's基因多样性指数(0.1451)和遗传丰富度(185)86.05%都以江淮流域最高;华北地区和两广地区其次.国外各生态区域内不结球白菜观察等位基凶数(1.3105),有效等位基因数(1.1714),Nei'S基因多样性指数(0.1014)和遗传丰富度(170)79.07%均以东亚地区最高;东南亚地区其次.国内种质的上述指标均高于国外种质,说明不结球白菜的遗传多样性主要分布于国内.表4不同生态区域不结球白菜SRAP标记的遗传多样性指数Table4啦geneticdiversityindexbasedonSRAPmarkerfordifferentecologicalregionsofnon-beadin gChinesecabbage作物第33卷2.4不结球白菜的SRAP遗传分化遗传分化系数反映了总群体的遗传多样性与各种群遗传多样性的差异,代表种群问遗传不一致或分化程度.由21对引物对不结球白菜各种群(普通白菜,乌塌菜,菜心,分蘖菜和薹菜)遗传多样性检测的结果表明,不结球白菜总群体平均遗传多样性指数为0.1209,种群内平均遗传多样指数为0.0466.在总变异中有41.78%存在于种群内,种群问遗传变异占总变异的58.22%,即不结球白菜5个变种群间的遗传分化为58.22%,说明不结球白菜的遗传变异主要存在于种群问.种群问的基因流平均为0.4013,说明不结球白菜各种群问基因交流较少,种群间遗传分化程度较高(表5).表5不结球白菜SRAP遗传分化Table5ThegeneticdifferentiationbasedonSRAPmarkeramongnon?headingChinesecabb age2.5不结球白菜SRAP聚类分析利用SRAP谱带数据对64份不结球白菜进行聚类(图1).以遗传相似系数0.872为截值,可分为Ⅵ个类群.第l类9个品种,为普通白菜地方常规晶种;第Ⅱ类44个品种,为混合类群;第Ⅲ类1个品种,编号为53,为薹菜品种;第Ⅳ类5个品种,为国内普通白菜白扁梗品种;第V类2个品种,编号为62,63,全部来自印度;第Ⅵ类1个品种,编号为24, 为古老的普通白菜地方品种.在第Ⅱ类群里,以遗传相似0.916为截值,除编号52,61,64的品种外,编号为47,48,50,49,46的5个菜心品种聚为1类,编号为51,59,58,56,57的来自日本的5个品种聚为1 类,编号为54,55的来自韩国的2个品种聚为一类; 以遗传相似系数0.910为截值,编号为43,44的2个塌菜品种聚为一类.3讨论SRAP是一种新的基于PCR的DNA分子标记,多态性和重现性好,与AFLP接近,但操作步骤比AFLP简单,引物易于获得,成本低廉,适于进行品种多态性分析.Ferriol等.在西葫芦种质研究中平均每对引物组合扩增的多态性位点数为5.8个,林忠旭等在棉花研究巾每对引物组合获得的多态性位点数为5.14个,本研究中每对引物组合的多态性带数为5.33个,与前人结果基本一致.但多态性位点比率差异较大,Ferriol等¨.在西葫芦和笋瓜的遗传多样性研究中多态性位点比率为72.7%, Budak等在野牛草的研究中多态性位点比率为95%,而本研究中仅为52.09%.这可能与供试类型有关,本研究使用的材料为遗传关系比较近的白菜第11期韩建明等:不结球白菜(Brassicacampestrisssp.chinensisMakino)种质资源SRAP遗传分化分析088091094Coefncient图l基于SRAP标记产生的不结球白菜聚类图Fig.1Dendrogramof64non-headingChinesecabbageaccessionsbasedonSRAP亚种中的不结球白菜变种,而Budak等和Ferriol等".的所用的材料为遗传关系较远的种间材料及多倍性种质材料.揭示不结球白菜遗传多样性是研究种问亲缘关系的基础.李家文认为普通白菜(青梗类)最早,然后向乌塌菜,菜薹等进化.本研究结果表明,普通白菜的Nei'S基因多样性指数(0.1410)和遗传丰富度(190)88.37%都高于其他类型的不结球白菜,而遗传分化系数却较低,说明乌塌菜,菜心是由普通白菜进化而来,这与前人的研究结果相一致.曹家树等..认为不结球白菜是在太湖地区优越的自然条件和栽培条件下形成株型直立,叶片发达的普通白菜和株型塌地的耐寒性强的塌菜,并分别在华东沿海,长江流域和华南两广(广东,广西)的生态条件下演化形成分蘖菜,紫菜薹和菜心.本研究结果表明,国内的Nei'S基因多样性指数和遗传丰富度分别高于国外,江淮流域的这些指标又高于东南地区,两广地区,华北地区和华中地区.而遗传分化系数则是国外高于国内,东南地区,两广地区,华北地区和华中地区及东亚和东南亚地区高于江淮流域.根据62,47∞=呈"如∞嚣强卯卯鸦如刚如8¨9他∞1868作物第33卷V avilov和Harlan关于"每一种作物都有1个独特的多样性初生中心,即分化中心,这种中心往往在进化的起始阶段就扩散到较大的区域,其分布是地理学上的连续一体"的理论,并根据作物一般由生物多样性丰富区向次丰富区传播的趋势,说明了中国作为不结球白菜原产地的地位,也说明了江淮流域为不结球白菜的多样性中心和分化中心,这与前人的研究结果也是一致的.曹寿椿等根据白菜品种的发育特性,生态特征把不结球白菜分为普通白菜,乌塌菜,菜心(薹),分蘖菜,薹菜和油菜等6类.本研究的聚类分析中, 有的类群按园艺学分类聚在一起,如第工类,第Ⅲ类和第Ⅳ类;有的按生态区域聚在一起,如来自印度的第V类.第Ⅱ类群依不同的遗传相似系数归类.这与前人的分类结果基本一致,也与Ferriol等"的观点一致,认为SRAP标记所提供的遗传信息更接近于农艺性状的差异和历史演变的结果.4结论用SRAP分子标记对不结球白菜各变种类型和生态区域的遗传多样性检测及遗传结构分析表明, 不结球白菜多态性位点比率达52.09%,遗传分化系数达58.22%,基因流为0.4031,大部分变异存在于种群间,各类型遗传分化明显,普通白菜的Nei's 基因多样性指数(0.1410)和遗传丰富度(190)88.37%都高于其他类型的不结球白菜,而遗传分化系数较低,说明乌塌菜,菜心是由普通白菜进化而来.国内的Nei's基因多样性指数(0.1293)和遗传丰富度(188)87.44%分别高于国外;江淮流域的Nei's基因多样性指数(0.1451)和遗传丰富度(185)86.05%高于东南地区,两广地区,华北地区和华中地区,说明江淮流域为不结球白菜的多样性中心和分化中心[1]CaoS-C(曹寿椿),bs—J(李式军).Apreliminarystudyonthelocal varietyofChinesecabbage(BrassicachinensisL):I.Morphological observationandstudyJNanfingAgricCoil(南京农学院),1980,(2):3238(inChinesewithEnglishabstract)[2]HouX—L(侯喜林).Advancesinbreedingofnon.headingChinese cabbage.JNamingAgricUniv(南京农业大学),2003,26(4):111—115(inChinesewithEnglishabstract)[3]Jinw.L(金文林),WenZ.X(文自翔),Pus—J(濮绍京),ZhaoB(赵波).GeneticdiversityandevolutionofAdzukiBean(m angularis)germplasmresourcesbasedonRAPDmarkers.SciAgricSin (中国农业科学),2005,38(2):241249(inChinesewithEnglish 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引用格式：张艳琼，晏泽辉，石新星，等．ＳＬＣ１０Ａ１基因变异与ＨＢＶ母婴传播易感性的相关性研究［Ｊ］．中华临床感染病杂志，２０１６，９（２）：１６８－１７２，１７９．・论著・ＳＬＣ１０Ａ１基因变异与ＨＢＶ母婴传播易感性的相关性研究 张艳琼　晏泽辉　石新星　吴全新　黄鸿菲　王宇明４０００３８重庆，第三军医大学西南医院感染科通信作者：王宇明，Ｅｍａｉｌ：ｗｙｍ４１７＠１６３．ｃｏｍＤＯＩ：１０．３７６０／ｃｍａ．ｊ．ｉｓｓｎ．１６７４－２３９７．２０１６．０２．０１４ 【摘要】　目的　在群体水平探讨乙型肝炎病毒（ＨＢＶ）功能性受体ＳＬＣ１０Ａ１基因ｃ．８００Ｇ＞Ａ变异和ｃ．３５６＋１０９８Ｃ＞Ｔ变异对ＨＢＶ母婴传播易感性的影响。

方法　连续收集２０１１年５月至２０１５年７月西南医院感染科收治的未经核苷（酸）类药物干预的ＨＢＶｅ抗原（ＨＢｅＡｇ）阳性且ＨＢＶ高病毒载量母亲所生子代３０６例，其中包括感染组２４７例（疫苗干预５５例）和未感染组５９例（疫苗干预５６例）。

采集两组人群的血液样本，提取基因组ＤＮＡ，对ｃ．８００Ｇ＞Ａ和ｃ畅３５６＋１０９８Ｃ＞Ｔ进行基因分型，并进行病例－对照关联研究分析。

组间基因型比较采用卡方检验（Ｐｅａｒｓｏｎ卡方或连续校正卡方）。

结果　在经疫苗干预的人群中，未感染组ｃ畅８００Ｇ＞ＡＧＡ基因型频率为１４．３％（８／５６），高于感染组ＧＡ基因型频率（５畅５％，３／５５），但差异无统计学意义（χ２＝２畅４２４，Ｐ＝０畅１１９）；感染组ｃ畅３５６＋１０９８Ｃ＞Ｔ位点ＣＣ、ＣＴ和ＴＴ基因型频率分别为２０畅０％（１１／５５）、４７畅３％（２６／５５）和３２畅７％（１８／５５），未感染组分别为１２畅５％（７／５６）、６９畅６％（３９／５６）和１７畅９％（１０／５６），两组基因型分布差异无统计学意义（χ２＝５畅７６６，Ｐ＝０畅０５６）。

在总人群中，未感染组ｃ畅８００Ｇ＞ＡＧＡ基因型频率为１３畅６％（８／５９），高于感染组ＧＡ基因型频率（６畅９％，１７／２４７），但差异无统计学意义（χ２＝２畅０１０，Ｐ＝０畅１５６）；感染组ｃ畅３５６＋１０９８Ｃ＞Ｔ位点ＣＣ、ＣＴ和ＴＴ基因型频率分别为２０畅２％（５０／２４７）、４９畅８％（１２３／２４７）和３０畅０％（７４／２４７），未感染组为１１畅９％（７／５９）、６９畅５％（４１／５９）和１８畅６％（１１／５９），两组基因型分布差异有统计学意义（χ２＝７畅４３６，Ｐ＝０畅０２４）。

352017.04基础研究核酸检测结果的符合率均为0，单试剂阳性标本与核酸检测结果符合率较低，故建议：（1）取消酶免检测灰区值的设定。

（2）对单试剂检测阳性标本因再进行确认试验。

核酸检测57例阳性标本中50例为隐匿性乙肝感染，建议增加抗-HBc 检测以降低乙肝病毒漏检的风险。

（通讯作者：王琦）参考文献[1]Bush MP.Transfusion-transmitledviral infections:building bridges to transfusion medicine to reduce risks and understand epidemiology a n d p a r h o g e n e s i s.T r a n s f u s i on,2006,46(09):1624-1640.[2]D e l w a r t E ,K u h n s M C ,B u s c hMP.Surveillance of the genetic variation in incident HIV,HCV,and HBV infections in blood and plasma donors；implications for blood safety,diagnostics,treatment,an d molecular epidemiology.J Med Virol,2006,78(Suppl1):S30-35.[3]LapercheS.Blood safety and nucleicacid testing in Europe.Euro Surveill,2005,10(02):3-4.[4]张锋,张琼,林碧,等.血清学联合核酸检测在献血者血液筛查中的互补性[J].中国艾滋病性病,2016,22(03):197-199.[5]梁浩坚,汪传喜,许结仪,等.2011-2014年广州地区无偿献血者核酸检测结果分析[J].中国医药科学,2016,6(10):138-141.[6]赵红娜,王艺芳,李俊英,等.核酸检测与酶免检测在郑州地区血液筛查中的联合应用[J].中国输血杂志,2015,28(10):1269-1271.[7]邓雪莲,安万新,梁小华.大连市血液中心血清学检测与核酸检测并行的效果观察[J].中国输血杂志,2012,25(01):38-40.[8]Yoshikawa A,GotandaY,ItabashiM,etal.Hepatitis B NAT viruspositivebolld donors in the early and tate stages of HBV infection:analyses of the window period and kinetics of HBV DNA.Vox Sang,2005,88(02):77-86.[9]KleinmanSH,KuhnsMC,ToddDS,etal.Frequency of HBV DNA detection in US blood donors testing positivef o r t h e p r e s e n c e o f a n t i -HBc:implications for transtusion transmission and donor screening.Transfusion,2003,43(06):696-704.[10]曾劲峰，郑欣，许晓绚.ELISA检测与NAT 在血液筛查应用中的互补性研究[J].中国输血杂志,2012,25(10):1012-1014.[11]陈显,胡文佳,黄成埂，等.献血者ELISA 检测为灰区标本的确证试验与核酸检测情况分析[J].中国输血杂志,2015,28(02):198-199.[12]颜秀娟,陆祝选，邱昌文,等.核酸检测技术应用于血清学筛查合格的献血者标本检测[J].中国输血杂志,2012,25(12):1322-1324.[13]L l u i s i r i r o t c h a n a k u l S .OotaS,KhuponsardK,etal,Occult hepatitis B virus infection in Thai blood donors[J].Transfusi on,2011,51(07):1532-1540.[14]R e n F R ,W a n g J X ,H u a n g Y ,e t a l.Hepatitis B virus nucleic acid testing in Chinese blood donors with normal and elevated alanine aminotransferase[J].Transfusi on,2011,51(12):2588-2595.FOCUS-PDCA 程序在血液透析患者钙磷水平的效果研究卢璧云　李　彦　张耀宣广州市番禺区中心医院 广东省广州市 511400【摘　要】目的：探讨FOCUS-PDCA 程序在血液透析患者钙磷水平的实践与效果。

肝细胞癌中真核延伸因子1A2的表达及意义黄燕;易纯;李琳子;刘莉莉;陆世旬;周璇;谢国斌;云径平【期刊名称】《中山大学学报（医学科学版）》【年(卷),期】2012(033)003【摘要】[目的]探讨真核延伸因子1A2(eEF1A2)在肝细胞癌(HCC)中的表达及其临床病理意义.[方法]收集104例HCC患者的临床病理资料,利用组织芯片对其癌及癌旁肝组织进行免疫组化检测,应用统计学方法分析eEF1A2在这些组织中的表达水平及与患者临床病理特征的关系.[结果]免疫组化结果显示:56.7％肝癌组织中eEF1A2高表达(eEF1A2++,eEF1A2+++),而仅13.5％的癌旁肝组织呈高表达,差异具有统计学意义(P=0.014).eEF1A2的表达与乙肝病毒感染(P=0.030)和复发(P=0.012)相关.Kaplan-Meier分析表明:eEF1A2高表达患者的总体生存时间(P=0.024)和无复发生存时间(P=0.023)较eEF1A2低表达患者更长.多因素Cox回归分析提示:eEF1A2表达为影响HCC患者总体生存的独立危险因素(P=0.038).[结论]Hcc中eEF1A2蛋白表达增高,与肝癌患者术后复发和预后相关,eEF1A2可以作为判断肝癌患者预后的潜在分子标志物.%[Objective] To investigate the expression of eukaryotic elongation factor (eEF) 1A2 and its clinicopathologic characters in hepatocellular carcinoma (HCC). [Methods] Clinical pathological data of 104 HCC patients were collected. Immunohistochemical analysis for eEFlA2 was performed on tissue microarrays for these HCC and adjacent liver tissues. The expression of eEFlA2 and its clinicopathologic and prognostic significance was analyzed. [Results] Immunohistochemical analysis revealed eEFlA2 was significantlyexpressed (eEFlA2 ++, eEFlA2 +++) in 56.7% of HCC tissues, while it only high expressed in 13.5% adjacent liver tissues (P=0.014). Expression of eEFlA2 were associated with hepatitis B virus infection (P = 0.030) and clinical recurrence (P = 0.012). Kaplan-Meier analysis showed that patients with high expression of eEFlA2 had both longer overall survival time (P= 0.024) and recurrence-free survival time (P= 0.023) as compared with those with low expression of eEFlA2. Logistic regression analysis demonstrated eEFlA2 expression may be an independent prognostic parameter for overall survival for patients with HCC (P = 0.038). [Conclusion] EEF1A2, which is high expressed in HCC tissues, is associated with recurrence and prognosis. EEF1A2 may be a potential prognostic marker for patients with HCC.【总页数】6页(P293-298)【作者】黄燕;易纯;李琳子;刘莉莉;陆世旬;周璇;谢国斌;云径平【作者单位】华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060;华南肿瘤学国家重点实验室//中山大学肿瘤防治中心病理科,广东广州510060【正文语种】中文【中图分类】R735.7【相关文献】1.肝细胞癌患者真核生物翻译起始因子－5 A2、P53和上皮型黏附素表达及意义[J], 吴葆华2.溃疡性结肠炎中真核细胞翻译起始因子A1和血管内皮生长因子表达及意义 [J], 廖如奕;姚萍;张艳3.真核延伸因子-1A2基因在宫颈癌侵袭和迁移中的作用研究 [J], 靳馥源;潘泽民;辛慧珍;者湘漪;张春贺;李洪涛;潘贞贞;李冬妹;郑威楠4.人类真核延伸因子1A2与生长因子受体结合蛋白2在胰腺癌组织中的表达及临床病理意义 [J], 周维霞;蒋晓红;杨勇;陈瑞东;胡端敏5.真核翻译延伸因子1A2及真核延长因子1A2在肿瘤中的作用 [J], 张愫;诸琦因版权原因，仅展示原文概要，查看原文内容请购买。

Genetic Variation in Plant Species Genetic variation in plant species is a crucial aspect of their survival and adaptation to changing environments. This variation is the result of genetic mutations, genetic recombination, and gene flow between different populations. In this essay, we will explore the importance of genetic variation in plant species and the factors that contribute to it. First and foremost, genetic variation in plant species plays a vital role in their ability to adapt to environmental changes. When the environment undergoes changes, such as shifts in temperature or the introduction of new pathogens, plants with genetic variation are more likely to have individuals with traits that allow them to survive and reproduce. This, in turn, ensures the continued existence of the species. Additionally, genetic variation in plant species is essential for the long-term sustainability of ecosystems. It allows for the maintenance of diverse plant populations, which in turn supports a wide range of other organisms that depend on these plants for food and habitat. Without genetic variation, plant species would be more susceptible to diseases and environmental stressors, which could have cascading effects on the entire ecosystem. Furthermore, genetic variation in plant species is influenced by several factors. One of the primary factors is natural selection, which acts on the variation present in a population and allows individuals with advantageous traits to survive and reproduce. Another factor is genetic drift, which can lead to random changes in the frequency of certain genes within a population. Additionally, gene flow, which is the transfer of genes between different populations, can also contribute to genetic variation in plant species. In conclusion, genetic variation in plant species is of utmost importance for their adaptation to changing environments, the sustainability of ecosystems, and the overall diversity of life on Earth. Understanding the factors that contribute to genetic variation, such as natural selection, genetic drift, and gene flow, is crucial for conservation efforts and the preservation of plant species in the face of ongoing environmental changes. It is imperative that we continue to study and protect the genetic variation present in plant species to ensure their continued existence and the health of our planet.。

中国畜牧兽医 2022,49(8):3140-3150C h i n aA n i m a lH u s b a n d r y &V e t e r i n a r y Me d i c i n e猪流行性腹泻病毒H B /H E B E U /2020株全基因组遗传变异与重组分析翟新国1,郑培培1,苏金辉2,李庆阳2,焦贺静2,张若冰2,李朋辉1,2(1.河北工程大学生命科学与食品工程学院,邯郸056038;2.河北大北农农牧食品有限公司,衡水053900)摘 要:ʌ目的ɔ了解猪流行性腹泻病毒(P o r c i n ee p i d e m i cd i a r r h e av i r u s ,P E D V )的基因组特征及变异规律㊂ʌ方法ɔ以3只发病仔猪小肠病料作为模板,采用R T -P C R 技术检测病原㊂以检测阳性的肝脏组织R N A 为模板,进行R T -P C R ,分段扩增P E D V 全基因组,使用D N A S t a r 软件对P E D V 基因组测序结果进行剪辑㊁拼接和相似性分析,利用M e g aX10.0.5软件对P E D V 基因组和S 基因进行遗传进化分析;利用R D P 4软件对P E D V 基因组进行重组分析㊂ʌ结果ɔP C R 检测结果表明,3只病仔猪的小肠组织均检测到P E D V 阳性㊂采用R T -P C R 分段扩增成功获得1株P E D V 全基因组序列,命名为H B /H E B E U /2020株,基因组大小为28038b p ,含有7个开放阅读框,从5'到3'依次为复制酶1a 基因㊁复制酶1b 基因㊁S 基因㊁O R F 3基因㊁E 基因㊁M 基因和N 基因㊂相似性分析结果显示,H B /H E B E U /2020与S N J -P ㊁U S A /C o l o r a d o /2013和H B 2018等变异毒株全基因组和S 基因的相似性均较高,分别为97.8%~99.3%和95.7%~98.8%,在所有市售疫苗株中,与变异型疫苗株A J 1102毒株全基因组和S 基因的相似性分别为98.3%和97.3%;遗传进化分析结果显示,21株P E D V 可划分为G 1群和G 2群2个分支,G 1群进一步分化为G 1a 亚群和G 1b 亚群,G 2群进一步分化为G 2a 亚群和G 2b 亚群,H B /H E B E U /2020属于G 2a 亚群㊂在G 2群内,所有流行株均为2010年后出现,H B /H E B E U /2020与市售疫苗株A J 1102遗传距离较近,其次是L W /L ,与C V 777和a t t e n u a t e dC V 777株遗传距离较远;经重组分析,H B /H E B E U /2020基因组可能为重组毒株,存在3个潜在的重组事件,重组事件1~3的断点分别为15918 22119㊁100 734和2214 2729b p ,其中重组事件1和2发生概率较大㊂ʌ结论ɔH B /H E B E U /2020为1株变异型P E D V ,与以C V 777为代表的经典毒株亲缘关系较远,与2010年后中国发生的变异型毒株亲缘性较近,并且存在潜在重组事件㊂本研究结果可为调研中国当前P E D V 进化和变异提供重要资料,为遏制P E D V 蔓延与进化㊁制定合理防控方案提供理论和现实依据㊂关键词:猪流行性腹泻病毒;全基因组;遗传变异;重组分析中图分类号:S 855.3文献标识码:AD o i :10.16431/j.c n k i .1671-7236.2022.08.030 开放科学(资源服务)标识码(O S I D ):收稿日期:2022-02-08基金项目:河北工程大学创新基金-博士专项基金联系方式:翟新国,E -m a i l :z h a i x g 1966@163.c o m ㊂通信作者李朋辉,E -m a i l :l i p e n gh u i 101@126.c o m G e n e t i cV a r i a t i o na n dR e c o m b i n a t i o nA n a l ys i s o fW h o l eG e n o m e o f P o r c i n eE pi d e m i cD i a r r h e aV i r u sH B /H E B E U /2020S t r a i n Z HA IX i n g u o 1,Z H E N GP e i p e i 1,S UJ i n h u i 2,L IQ i n g y a n g 2,J I A O H e j i n g 2,Z H A N G R u o b i n g 2,L IP e n gh u i 1,2(1.C o l l e g e o f L i f eS c i e n c e a n dF o o dE n g i n e e r i n g ,H e b e i U n i v e r s i t y o f E n g i n e e r i n g ,H a n d a n 056038,C h i n a ;2.H e b e i D a b e i n o n g F a r m i n g a n dA n i m a lH u s b a n d r yF o o dC o .,L t d .,H e n gs h u i 053900,C h i n a )A b s t r a c t :ʌO b j e c t i v e ɔT h i ss t u d y w a s a i m e d t oi n v e s t i ga t et h e g e n o m e c h a r a c t e r i s t i c s a n d v a r i a t i o no f P o r c i n e e p i d e m i cd i a r r h e av i r u s (P E D V ).ʌM e t h o d ɔT h es m a l l i n t e s t i n e t i s s u e so f 3d i a r r h e a p i g l e t sw e r eu s e da s t e m p l a t e s ,t h e p a t h o g e nw a sd e t e c t e db y R T -P C R .U s i n g th eR N A8期翟新国等:猪流行性腹泻病毒H B/H E B E U/2020株全基因组遗传变异与重组分析o f t h e p o s i t i v e i n t e s t i n et i s s u e,R T-P C Rt e c h n o l o g y w a su s e dt oa m p l i f y t h e w h o l e g e n o m eo f P E D V.D N A S t a rs o f t w a r e w a su s e dt oe d i ta n ds p l i c eo ft h ea m p l i f i e ds e q u e n c ea n d p e r f o r m s i m i l a r i t y a l i g n m e n t.M e g a X10.0.5s o f t w a r e w a su s e dt oc o n s t r u c t p h y l o g e n e t i ct r e e so ft h e w h o l e g e n o m ea n d S g e n eo fP E D V s t r a i n.R D P4s o f t w a r e w a su s e dt oa n a l y z et h e p o t e n t i a l r e c o m b i n a t i o ne v e n t s o f P E D Vs t r a i n.ʌR e s u l tɔP C Rr e s u l t s s h o w e dt h a tP E D V w a sd e t e c t e d i n a l l t h e t h r e es i c k p i g l e t s.T h ew h o l e g e n o m eo fP E D Vs t r a i n w a ss u c c e s s f u l l y o b t a i n e db y R T-P C Rf r a g m e n t e da m p l i f i c a t i o n,n a m e d H B/H E B E U/2020.T h ev i r u s g e n o m es i z ew a s28038b p a n d i t c o n t a i n e d7o p e nr e a d i n g f r a m e s(O R F)w h i c he n c o d e dr e p l i c a s e p o l y p r o t e i n1a,r e p l i c a s e p o l y p r o t e i n1b,s p i k e(S),O R F3,e n v e l o p e(E),m e m b r a n e(M),a n dn u c l e o p r o t e i n(N)g e n e i n5' t o3'o r i e n t a t i o n.T h e s i m i l a r i t y a l i g n m e n t o f t h e c o m p l e t e g e n o m e a sw e l l a s S g e n e b e t w e e nH B/H E B E U/2020a n dP E D Vv a r i a n t s t r a i n s,s u c ha sS N J-P,U S A/C o l o r a d o/2013a n d H B2018w e r e97.8%-99.3%a n d95.7%-98.8%,r e s p e c t i v e l y.A m o n g a l l t h ec o m m e r c i a l l y a v a i l a b l ev a c c i n e s,A J1102s h a r e dt h eh i g h e s ts i m i l a r i t y w i t h H B/H EB E U/2020s t r a i n,t h en u c l e o t i d es i m i l a r i t y b e t w e e n t h e c o m p l e t e g e n o m ea sw e l l a s S g e n eo fH B/H E B E U/2020a n dA J1102w e r e98.3% a n d97.3%,r e s p e c t i v e l y.G e n o m e-w i d ee v o l u t i o nr e s u l t ss h o w e dt h a t21P E D V s t r a i n s w e r e c l a s s i f i e d i n t oG1g e n o g r o u p a n dG2g e n o g r o u p,G1g e n o g r o u p w a s d i v i d e d i n t oG1a s u b g r o u p a n d G1b s u b g r o u p,G2g e n o g r o u p w a sd i v i d e d i n t oG2as u b g r o u p a n dG2bs u b g r o u p,H B/H E B E U/ 2020b e l o n g e d t oG2a s u b g r o u p.A l l t h e P E D Vs t r a i n s t h a t b e l o n g e d t oG2g e n o g r o u p i n t h e s t u d y w e r e e m e r g e d a f t e r2010.A m o n g a l l t h e c o m m e r c i a l l y a v a i l a b l ev a c c i n e s,A J1102h a d t h en e a r e s t g e n e t i c r e l a t i o nw i t h H B/H E B E U/2020,L W/L w a ss e c o n d,t h e g e n e t i cd i s t a n c e sb e t w e e n H B/ H E B E U/2020a n dC V777a sw e l l a s a t t e n u a t e dC V777w e r e f a r.3p o s s i b l e r e c o m b i n a t i o ne v e n t s w e r e i d e n t i f i e d i n t h e g e n o m eo fH B/H E B E U/2020s t r a i n.T h e r e c o m b i n a n t r e g i o n so f t h e t h r e e p o s s i b l e r e c o m b i n a t i o ne v e n t sw e r e15918-22119,100-734a n d2214-2729b p,r e s p e c t i v e l y.T h e f i r s t t w or e c o m b i n a t i o ne v e n t sh a dh i g h p r o b a b i l i t y o fo c c u r r e n c e.ʌC o n c l u s i o nɔH B/H E B E U/ 2020w a sav a r i a n tr e c o m b i n a n tP ED V,w h i c hh a r b o r e dd i s t a n t p h y l o g e n e t i cr e l a t i o n s h i p sw i t h c l a s s i cPE D Vs t r a i nC V777,b u tw a s c l o s e dr e l a t e d t o t h ev a r i a n t s t r a i n so c c u r r e d i nC h i n aa f t e r 2010.T h er e s u l t s w o u l d p r o v i d e t h e o r e t i c a l a n d p r a c t i c a lr e f e r e n c e f o r p o s t p o n i n g n a t u r a l s e l e c t i o na n d e v o l u t i o na sw e l l a s f o r m u l a t i n g t h e v a c c i n a t i o n p r o c e d u r e o f P E D V.K e y w o r d s:P o r c i n e e p i d e m i c d i a r r h e a v i r u s;w h o l e g e n o m e;g e n e t i c v a r i a t i o n;r e c o m b i n a t i o n a n a l y s i s猪流行性腹泻病毒(P o r c i n ee p i d e m i cd i a r r h e a v i r u s,P E D V)属于冠状病毒科(C o r o n a v i r i d a e)病毒大家族[1]㊂P E D V是一种单股正链具有囊膜的R N A病毒,基因组长约28000b p[1-2]㊂P E D V基因组编码3个非结构蛋白(复制酶1a㊁复制酶1b以及附属蛋白(O R F3))和4个结构蛋白(刺突糖蛋白(S)㊁囊膜蛋白(E)㊁核衣壳蛋白(N)和膜蛋白(M))[3-5]㊂在所有P E D V蛋白中,S蛋白主要参与病毒吸附和入侵,决定病毒的毒力和细胞嗜性,并诱导宿主产生中和抗体,在分子流行病学上具有重要意义[6-10]㊂P E D V感染各年龄段猪均可导致猪流行性腹泻(p o r c i n e e p i d e m i c d i a r r h e a,P E D),但仔猪往往更为严重,表现为厌食,呕吐,肠绒毛萎缩,水样腹泻,恶性脱水,体重减轻,病死率极高(最高可达100%)[11-13]㊂1971年英国报道首例P E D疫情,但P E D V却直到1977年才被比利时科学家首次鉴定出来,命名为C V777株[13-15],随后P E D V广泛传播至欧洲和亚洲诸多生猪养殖国家[16]㊂1984年,中国学者利用免疫荧光试验和血清中和试验首次证实P E D V在中国的存在[17]㊂由于中国广泛使用传染性胃肠炎(t r a n s m i s s i b l e g a s t r o e n t e r i t i s,T G E)和P E D的腹泻二联疫苗,P E D疫情一直维持在较低水平[16,18]㊂2008 2010年间,韩国和中国暴发了由高毒力变异型P E D V毒株引起的P E D,随后疫情迅速席卷亚洲㊁北美洲和欧洲,对全球养猪业造成巨大的损失[19-20]㊂为更好防控变异型P E D V毒株,中国基1413中国畜牧兽医49卷于A J1102和L W/L毒株研发并上市两款新的变异毒株疫苗㊂尽管如此,近些年P E D仍是中国最常见危害性最大的猪病之一㊂T i a n等[19]报道2014 2018年四川省和贵州省有47.66%的猪场呈P E D V 阳性,对52株P E D V S基因进行遗传变异分析,发现变异型P E D V达82.7%㊂C u i等[16]报道2015 2019年许多河南省和山西省的经典弱毒疫苗免疫的猪场仍会暴发腹泻,P E D V阳性率为63.7%,对32株P E D V S基因进行遗传变异分析,发现其均属于变异型P E D V㊂因此,变异P E D V毒株已经发展为流行并循环于中国猪场的最主要毒株[15-16,19]㊂作为一种重要的猪冠状病毒,P E D V仍在不断进化和变异,免疫逃逸能力和传染性在不断加强,中国P E D V已呈现出较强的流行性和基因异质性,亟需加强对P E D V毒株的遗传变异监测并发展更有效的防控方案[19]㊂2020年底,河北省某大型猪场暴发P E D,大量新生仔猪水样腹泻,迅速死亡,损失惨重㊂为了了解河北地区遗传变异情况,掌握本地区P E D V的流行情况,本研究对该猪场病仔猪进行检测,成功获得1株P E D V全基因组序列,并进行了遗传变异和重组分析,以期了解该P E D V毒株的遗传进化和重组规律,为P E D V疫苗的研制和该病的合理防控提供新思路和理论依据,也为进一步研究P E D V的致病机制以及病毒与宿主的相互作用奠定基础㊂1材料与方法1.1样品采集与处理急性仔猪腹泻样品于2020年底采集于河北某大型种猪场,产房内出生仔猪水样腹泻,无菌条件下采集3份濒死仔猪新鲜小肠组织,利用无菌生理盐水冲洗,剪取约1g组织,充分剪碎并放入灭菌2m LE P管中,加入1m L无菌P B S,再加入2~4粒灭菌玻璃匀浆珠,提前30m i n预冷组织研磨仪的适配器,按照标准研磨程序,将小肠组织研磨成组织悬液,在4ħ下,10000ˑg离心3m i n,取上清于-80ħ保存备用㊂1.2主要试剂离心柱法D N A/R N A提取试剂盒购自I D E X X 公司;琼脂糖购自B I OW E S T公司;D L5000D N A M a r k e r㊁大肠杆菌D H5α感受态细胞和p M D18-T V e c t o r均购自T a K a R a公司;快速P C R试剂盒(1.1ˑT3S u p e rP C R M i x)购自北京擎科生物技术有限公司;T I A N p r e p M i n iP l a s m i d K i t和T I A N g e lM i d i P u r i f i c a t i o nK i t均购自天根生化科技(北京)有限公司;T r a n s S c r i p tF i r s t-S t r a n d c D N AS y n t h e s i sS u p e r M i x 购自北京全式金生物技术有限公司㊂1.3引物与探针基于L W/L(MK392335.1)㊁U S A/C o l o r a d o/ 2013(K F272920.1)㊁S14(MH891585.1)㊁P E D V-W S(KM609213.1)㊁P E D V1842/2016I T A (K Y111278.1)㊁S D2014(K X064280.1)㊁J S X2014 (MH056658.1)㊁G D-A(J X112709.1)㊁C H/S (J N547228.1)㊁C H/H N Y F/14(K P890336.1)㊁B J-2011-1株(J N825712.1)和A J1102(J X188454.1)等设计P E D V引物,分别为:P E D V-5'-F:A C T T A A A-A A G A T T T T C T A T C T A C G G A T A G T T A G C T C; P E D V-3'-R:G T G T A T C C A T A T C A A C A C C;用于扩增P E D V基因组内部序列的引物参考文献[21],引物由北京擎科生物技术有限公司合成㊂1.4总R N A提取与R T-P C R扩增参考翟新国等[22]方法进行总R N A提取和R T-P C R㊂取适量肠组织研磨液上清按照核酸提取试剂盒说明书提取总R N A;取950n g总R N A按照T r a n s S c r i p tF i r s t-S t r a n d c D N AS y n t h e s i s S u p e r M i x说明书(A T301)合成c D N A;以c D N A为模板参照1.1ˑT3S u p e rP C R M i x说明书进行P C R扩增, P C R反应体系25μL:1.1ˑT3S u p e rP C R M i x 20μL,上㊁下游引物(10μm o l/L)各1μL,c D N A模板1μL,补d d H2O至25μL㊂P C R反应条件:98ħ预变性3m i n;98ħ变性10s,56ħ退火10s,72ħ延伸10s,共31个循环;72ħ延伸2.5m i n㊂1.5序列测定与基因组拼接将获得的各P E D V基因片段的R T-P C R产物进行1.0%琼脂糖凝胶电泳,按照T I A N g e l M i d i P u r i f i c a t i o nK i t说明书对各P E D V片段进行切胶纯化,送北京擎科生物技术有限公司测序;利用D N A S t a r软件对测序结果进行拼接和整理,组装成完整的P E D V基因组㊂1.6遗传变异和重组分析从G e n B a n k中选取20条P E D V基因组序列作为参考序列(表1),利用D N A S t a r软件对所获的P E D V毒株基因组和S基因序列与参考毒株相应序列进行相似性分析;利用N C B I在线B L A S T程序对所获得的P E D V毒株基因组与参考毒株S N J-P株进行核苷酸变异性比对;利用M e g a X 10.0.5软件对所获的P E D V毒株基因组和S基因24138期翟新国等:猪流行性腹泻病毒H B/H E B E U/2020株全基因组遗传变异与重组分析序列与参考毒株相应序列进行拟合对比和最大似然法(M L)遗传进化分析[23];利用R D P4软件对所获的P E D V毒株基因组序列与参考毒株基因组序列进行重组分析[24-25]㊂表1参考毒株信息T a b l e1T h e i n f o r m a t i o no fP E D Vr e f e r e n c e s t r a i n s毒株S t r a i n sG e n B a n k登录号G e n B a n ka c c e s s i o nN o.来源S o u r c e s年份Y e a rS N J-P MK702008.1中国2018 A J1102J X188454.1中国2011 L W/L MK392335.1中国2010 G D-X L-2019MN759311.1中国2019 P E D V-Y Z MK841495.1中国2016B J-2011-1J N825712.1中国2011C H/H L J/18MW561264.1中国2018 H B2018M T166307.1中国2018 S D2014K X064280.1中国2014 C H/HN Y F/14K P890336.1中国2014 U S A/C o l o r a d o/2013K F272920.1美国2013 G E R/L03208/2019L R812930.1德国2019 S14MH891585.1韩国2018 J S2008K C210146.1中国2008 v i r u l e n tD R13J Q023161.1韩国2009 a t t e n u a t e dD R13J Q023162.1韩国2011 a t t e n u a t e dC V777K T323979.1中国1998 S C1402K P162057.1中国2014 C H/S J N547228.1中国1986 C V777A F353511.1比利时19782结果2.1P E D V基因组扩增与测序R T-P C R扩增产物经1.0%琼脂糖凝胶电泳分析,产生1条清晰条带,大小约1816b p(图1),说明小肠病料为P E D V阳性,将该毒株命名为H B/ H E B E U/2020㊂成功克隆了覆盖P E D V全基因组的目的片段,经测序和拼接,获得H B/H E B E U/ 2020全基因组序列,并提交G e n B a n k,获得的登录号为:MW413556㊂2.2H B/H E B E U/2020基因组相似性分析由表2可知,H B/H E B E U/2020基因组全长28038b p,其中5'-非编码区(U T R)的1 36b p和3'-U T R的28020 28038b p为引物序列,其余27983b p均为本研究测序所得㊂H B/H E B E U/2020基因组编码区全长27412b p,含有7个开放阅读框,从5'到3'依次为复制酶1a基因(12309b p)㊁复M,D L5000D N A M a r k e r;1~3,样本M,D L5000D N A M a r k e r;1-3,S a m p l e s图1P E D V基因组R T-P C R扩增结果F i g.1R T-P C Ra m p l i f i c a t i o n r e s u l t s o fP E D V g e n o m e3413中 国 畜 牧 兽 医49卷制酶1b 基因(8037b p )㊁S 基因(4161b p )㊁O R F 3基因(675b p )㊁E 基因(231b p )㊁M 基因(681b p )和N 基因(1326b p)㊂与S N J -P 株(MK 702008.1)相比,H B /H E B E U /2020基因组包含207个核苷酸点突变,但不存在插入或者缺失㊂由图2可知,H B/H E B E U /2020与P E D V 流行株株的相似性在96.5%~99.3%之间,H B /H E B E U /2020株与分离自中国的S N J -P 株相似性最高,为99.3%,S D 2014㊁U S A /C o l o r a d o /2013㊁B J -2011-1㊁S 14㊁H B 2018㊁C H /H L J /18㊁P E D V -Y Z ㊁G E R /L 03208/2019㊁A J 1102㊁C H /H N Y F /14㊁G D -X L -2019和L W /L 与H B /H E B E U /2020相似性均>97.5%,S C 1402与H B /H E B E U /2020相似性为96.6%;v i r u l e n tD R 13㊁C H /S 和J S 2008与H B /H E B E U/2020相似性分别为97.3%㊁97.0%和96.7%㊂在所有市售疫苗株中,变异疫苗株A J 1102与H B /H E B E U /2020相似性最高,为98.3%,其次是变异疫苗株L W /L ,与H B /H E B E U /2020相似性为97.8%;经典疫苗株a t t e n u a t e dC V 777和C V 777则与H B /H E B E U /2020的相似性稍低,均为96.5%㊂表2 H B /H E B E U /2020株与S N J -P 株基因组的变异性比对T a b l e 2 C o m p a r i s o no f g e n o m i c v a r i a b i l i t i e s o fH B /H E B E U /2020a n dS N J -P 基因G e n e s位置P o s i t i o n /b p 核苷酸突变位点A m i n o a c i dm u t a t i o n s i t e s5'-U T R 1 292T 5C ㊁C 51T ㊁T 251C ㊁C 255T复制酶1aR e pl i c a s e 1a 293 12601G 466T ㊁C 508T ㊁C 610T ㊁A 631G ㊁T 832A ㊁G 1316T ㊁G 1612T ㊁A 1704T ㊁C 1757T ㊁C 1781T ㊁C 1888T ㊁A 1930T ㊁C 2006T ㊁C 2014T ㊁C 2188T ㊁A 2207T ㊁A 2212C ㊁A 2232C ㊁C 2301T ㊁T 2379C ㊁T 2480C ㊁G 2519A ㊁C 2740T ㊁C 2749T ㊁C 2773T ㊁G 2809T ㊁T 2869C ㊁C 2896T ㊁T 2959A ㊁C 3236A ㊁T 3238C ㊁T 3240C ㊁T 3253C ㊁A 3292G ㊁T 3361C ㊁C 3439T ㊁T 3457A ㊁C 3466T ㊁C 3529T ㊁C 3572G ㊁T 3613C ㊁C 3655T ㊁C C 3685 3686T T ㊁G 3697T ㊁G 3704A ㊁T 3763C ㊁T 3766C ㊁T 3967A ㊁T 3982C ㊁T 4054C ㊁T 4132G ㊁T 4171C ㊁G 4175A ㊁T 4201C ㊁T 4303C ㊁G 4369C ㊁C 4549T ㊁C 4650T ㊁A 4701C ㊁C 4993T ㊁C 5052T ㊁G 5099A ㊁A 5108G ㊁G 5121A ㊁C 5146T ㊁T 5167C ㊁C 5344T ㊁C 5504T ㊁C 5803T ㊁T 5878C ㊁C 5881T ㊁A 5964G ㊁G 5977T ㊁T 6199C ㊁G 6271T ㊁C 6290T ㊁C 6605T ㊁T 6644C ㊁C 6766T ㊁G 6883A ㊁C 6913T ㊁C 7522T ㊁A 7727G ㊁C 8011T ㊁T 8026C ㊁C 8047T ㊁A 8059G ㊁G 8368T ㊁C 8485A ㊁C 8896T ㊁C 9416A ㊁C 9472T ㊁C 9586T ㊁T 9616C ㊁T 10049C ㊁C 10054T ㊁C 10355T ㊁T 10608C ㊁T 11194C ㊁T 11611C ㊁G 12226A ㊁C 12570T 复制酶1bR e pl i c a s e 1b 12601 20637C 12765T ㊁A 12809C ㊁C 12954T ㊁T 13351C ㊁C 13401T ㊁G 13635A ㊁T 13827C ㊁C 13935T ㊁C 14475T ㊁T 14544C ㊁T 14838C ㊁T 15021C ㊁T 15027A ㊁C 15033T ㊁T 15210C ㊁C 15216T ㊁A 15333G ㊁C 15394T ㊁C 15468T ㊁T 15537A ㊁C 15588T ㊁C 15609T ㊁A 15648G ㊁G 15717A ㊁G 15732A ㊁G 15855A ㊁C 15861T ㊁T 16896C ㊁C 17595T ㊁G 18665A ㊁C 19890T ㊁G 20028A ㊁T 20091C ㊁A 20193G ㊁C 20319TS20634 24794A 20774G ㊁T 21238C ㊁C 21283T ㊁C 21300A ㊁T 21326C ㊁T 21334C ㊁G 21383A ㊁T 21515C ㊁C 21572T ㊁G 21597T ㊁C 21663T ㊁T 21759C ㊁C 22093T ㊁T 22107C ㊁T 22139C ㊁C 22232T ㊁C 22265T ㊁C 22304T ㊁C 22334T ㊁22344T ㊁A 22445G ㊁T 22525C ㊁T 22574C ㊁T 22652C ㊁G 22762A ㊁T 22802C ㊁C 22927T ㊁C 22943T ㊁A 22965G ㊁C 23077A ㊁T 23189C ㊁T 23249C ㊁G 23253T ㊁C 23369T ㊁C 23492T ㊁T 23589A ㊁A 23653G ㊁G 23717A ㊁C 23834T ㊁T 23892G ㊁C 24023T ㊁T 24341C O R F 324794 25468C 24922T ㊁C 24997T ㊁C 25096G E 25449 25679T 25502C ㊁C 25553TM 25687 26367G 25851A ㊁C 25941T ㊁G 26035A ㊁T 26357CN 26379 27704T 26393C ㊁C 26528T ㊁C 26702T ㊁T 26705C ㊁C 26858T ㊁T 27085C ㊁T 27401C ㊁G 27566㊁A 27611T ㊁C 27656T ㊁T 27670C ㊁T 27689C 3'-U T R27705 28038G 27709T ㊁T 27733G44138期翟新国等:猪流行性腹泻病毒H B /H E B E U /2020株全基因组遗传变异与重组分析五角星表示本研究的目标毒株;黑圆表示疫苗毒株㊂下同P e n t a g r a mr e p r e s e n t s t h e t a r g e t s t r a i no f t h i s s t u d y ;T h eb l a c kc i r c l e s r e p r e s e n t v a c c i n e s t r a i n s .T h e s a m e a sb e l o w 图2 H B /H E B E U /2020全基因组与各P E D V 参考毒株的相似性比对F i g .2 S i m i l a r i t y a l i gn m e n t o fw h o l e g e n o m e o fH B /H E B E U /2020a n d t h eP E D Vr e f e r e n c e s t r a i n s 2.3 H B /H E B E U /2020S 基因相似性分析通过对H B /H E B E U /2020全基因组测序结果进行分析,可知其S 基因全长4161b p ,编码1386个氨基酸㊂由图3可知,H B /H E B E U /2020S 基因与比利时C V 777标准株的相似性为93.5%,共存在248个核苷酸突变,在167b p 处存在G 插入,在175 185b p处存在C A G G G T G T T A A 插入,在416 418b p 处存在A T A 插入,在475 480b p 处存在C G T G A T 缺失,推导的氨基酸相似性为92.8%,共存在93个氨基酸突变,在56 59和140位氨基酸处分别存在G E N Q 和N 插入,在163 164位氨基酸处存在D I缺失㊂H B /H E B E U /2020S 基因与所选参考株相似性在93.2%~98.8%之间,其中与H B 2018的相似性最高,为98.8%㊂H B 2018㊁C H /H L J /18㊁G E R /L 03208/2019㊁C H /H N Y F /14㊁G D -X L -2019㊁S 14㊁U S A /C o l o r a d o /2013㊁A J 1102㊁S N J -P ㊁S D 2014㊁B J -2011-1㊁L W /L ㊁P E D V Y Z 株S 基因与H B/H E B E U /2020相似性均超过95.5%,S C 1402相似性为93.3%㊂变异疫苗株A J 1102和L W /L S 基因与H B /H E B E U /2020相似性分别为97.3%和96.9%,相比之下,经典疫苗株a t t e n u a t e dC V 777和C V 777则与H B /H E B E U /2020的相似性稍低,分别为93.3%和93.5%㊂2.4 H B /H E B E U /2020基因组遗传进化分析由图4可知,21株P E D V 毒株进化形成了G 1群和G 2群,G 1群包含G 1a 和G 1b 亚群,G 1a 亚群以经典株C V 777为代表,还包含v i r u l e n tD R 13和C H /S ,G 1b 亚群以J S 2008为代表,还包含a t t e n u a t e dD R 13㊁S C 1402和a t t e n u a t e dC V 777毒株;G 2群包含G 2a 和G 2b 亚群,G 2a 亚群以变异株B J -2011-1为代表,还包括S N J -P ㊁S D 2014㊁U S A/C o l o r a d o /2013㊁S 14㊁H B 2018和C H /H L J /18等毒株,G 2b 亚群以变异株A J 1102为代表,还包括G D -X L -2019和L W /L 毒株㊂H B /H E B E U /2020属于G 2a 群,该群中所有14株P E D V 均分离于2010年之后,包括11株中国株S N J -P ㊁S D 2014㊁B J -2011-1㊁H B 2018㊁C H /H L J /18㊁P E D V -Y Z ㊁A J 1102㊁C H/H N Y F /14㊁H B /H E B E U /2020㊁G D -X L -2019和L W /L ,1株美国株U S A /C o l o r a d o /2013,1株韩国株S 14和1株德国株G E R /L 03208/2019㊂H B /H E B E U /2020与市售的经典疫苗株a t t e n u a t e dC V 777和C V 777遗传距离(亲缘关系)较远,而与变异疫苗株A J 1102和L W /L 遗传距离较近㊂5413中 国 畜 牧 兽 医49卷图3 H B /H E B E U /2020S 基因与各P E D V 参考毒株的相似性对比F i g .3 S i m i l a r i t y a l i gn m e n t o f S g e n e o fH B /H E B E U /2020a n d t h eP E D Vr e f e r e n c e s t r a i ns 图4 H B /H E B E U /2020基因组遗传进化分析F i g .4 P h y l o g e n e t i c a n a l ys i s o fw h o l e g e n o m e o fH B /H E B E U /20202.5 H B /H E B E U /2020S 基因遗传进化分析由图5可知,S 基因遗传进化分析结果与全基因组结果基本一致,H B /H E B E U /2020S 基因与6株中国株P E D V -Y Z ㊁B J -2011-1㊁C H /H L J /18㊁H B 2018㊁S D 2014和C H /H N Y F /14,1株美国株U S A /C o l o r a d o /2013和1株韩国株S 14均属于G 2a 亚群,其中与H B 2018的遗传距离最近,具有共同的起源,而A J 1102与S N J -P ㊁L W /L 和G D -X L -2019形成了G 2b 亚群,C V 777与C H /S ㊁v i r u l e n t D R 13㊁a t t e n u a t e dD R 13和G E R /L 03208/2019形成了G 1a亚群,J S 2008与a t t e n u a t e d D R 13㊁a t t e n u a t e d C V 777和S C 1402形成了G 1b 亚群㊂值得关注的是,基于S 基因遗传进化分析,德国株G E R /L 03208/2019和中国株C H /H N Y F /14虽分别被划为G 1a 亚群和G 2a 亚群,但其S 基因在遗传上实际处于G 1和G 2群之间㊂2株P E D V S 基因更接近G 1和G 2群共同的始祖株㊂H B /H E B E U /2020S基因与市售的经典疫苗株a t t e n u a t e dC V 777株和C V 777遗传距离(亲缘关系)较远,而与变异疫苗株A J 1102和L W /L 遗传距离较近㊂2.6 H B /H E B E U /2020基因组重组分析为了解H B /H E B E U /2020潜在基因重组情况,基于图4P E D V 遗传系谱,选择11株具有代表性的P E D V 毒株(S N J -P ㊁A J 1102㊁L W /L ㊁C H/H N Y F /14㊁B J -2011-1㊁S D 2014㊁P E D V -Y Z ㊁G D -X L -2019㊁H B 2018㊁a t t e n u a t e dC V 777和S C 1402)进行基因组重组分析㊂由表3可知,H B /H E B E U /2020基因组存在3个潜在重组事件,根据重组事件发生的概率,事件1㊁2均至少有5种重组检测方法呈阳性,发生概率较高㊂重组事件1主要亲本可基于64138期翟新国等:猪流行性腹泻病毒H B/H E B E U/2020株全基因组遗传变异与重组分析G2a群的S D2014进行推导,次要亲本为G2a群的S N J-P,重组断点为15918 22119b p(O R F1b-S基因部分区域);重组事件2主要亲本为G2a群的H B2018,次要亲本为G1b群的S C1402,重组断点为100 734b p(5'-U T R-O R F1a基因部分区域),发生在G1群和G2群之间;重组事件3,发生概率较低(所有7种重组检测方法中2种呈阳性,重组断点为2214 2729b p(5'-U T R基因部分区域),主要亲本为G2a群的S N J-P,次要亲本可基于G1b群的经典疫苗株a t t e n u a t e dC V777进行推导㊂图5H B/H E B E U/2020S基因遗传进化分析F i g.5P h y l o g e n e t i c a n a l y s i s o f S g e n e o fH B/H E B E U/2020表3H B/H E B E U/2020株基因组的重组分析T a b l e3R e c o m b i n a t i o na n a l y s i s o fw h o l e g e n o m e o fH B/H E B E U/2020重组事件编号N u m b e r o fr e c o m b i n a n te v e n t s重组序列R e c o m b i n a n ts e q u e n c e s主要亲本M a j o rp a r e n t s次要亲本M i n o rp a r e n t s断点(相对MW413556)B r e a k p o i n t p o s i t i o n(c o m p a r e d t oMW413556)/b p检测方法D e t e c t i o nm e t h o d sR G B M C S3 1H B/H E B E U/2020S D2014*S N J-P15918 22119+++++++ 2H B/H E B E U/2020H B2018S C1402100 734+-+++-+ 3H B/H E B E U/2020S N J-P a t t e n u a t e dC V777*2214 2729--+---+①*,用于推断可能存在的缺失亲本序列㊂②R,R D P;G,G E N E C O N V;B,B o o t s c a n;M,M a x c h i;C,C h i m a e r a;S,S i S s c a n;3,3S e q①*,P a r e n t a ls e q u e n c eu s e dt oi n f e rt h ee x i s t a n c eo fa m i s s i n g.②R,R D P;G,G E N E C O N V;B,B o o t s c a n;M,M a x c h i;C,C h i m a e r a;S,S i S s c a n;3,3S e q3讨论P E D V是威胁全球养猪业的一种重要冠状病毒,2010年后,中国的流行毒株以变异型P E D V毒株为主,许多已免疫的猪场纷纷发病,仔猪死亡率极高,损失巨大[26]㊂目前,P E D V仍在不断流行和进化,然而近2年具有代表性的P E D V毒株全基因组遗传信息却十分匮乏㊂2020年底,河北某大型猪场暴发P E D,本研究采集病料成功扩增并获得1株突变型P E D V H B/H E B E U/2020全基因组序列,并进行了遗传变异和重组分析㊂H B/H E B E U/2020属于P E D V突变株,在相似性和遗传进化上与当前主要的商业化传统疫苗株存在差异㊂虽然基于P E D V基因组和S基因相似性分析结果略有不同,H B/H E B E U/2020与以B J-2011-1㊁U S A/C o l o r a d o/2013㊁H B2018㊁S14和S D2014等为代表的分离于2010年后的P E D V毒株相似性均较高㊂本研究选择了M L法进行遗传进化分析㊂利用M L法进行遗传进化分析虽然耗时长,但却往往能反应真实的情况,结果更为准确[27-28]㊂本研究发现,不论是全基因组遗传进化分析还是S基因遗传进化分析,P E D V均呈现两种不同的进化路径,形成了G1群和G2群,G1群可进一步分化为G1a亚群和G1b亚群,G2群可进一步分化为G2a亚群和G2b亚群,该结果与W a n g等[26]的研究结果一致㊂德国株G E R/L03208/2019在全基因组遗传进化分析和S基因遗传进化分析中呈7413中国畜牧兽医49卷现不同的结果,但在遗传上都处于G1a群和G2a群之间,亲缘关系更接近于G1群和G2群共同的始祖株㊂与G E R/L03208/2019类似,C H/HN Y F/14在遗传上也更接近于G1群和G2群共同的始祖株㊂相比其他甲型冠状病毒,如猪传染性胃肠炎病毒(T r a n s m i s s i b l e g a s t r o e n t e r i t i sv i r u s,T G E V)和人冠状病毒229E株等,一种高头蝠冠状病毒512/ 2005株(S c o t o p h i l u s b a tc o r o n a v i r u s,B t C o V)与P E D V原型株C V777的亲缘性更高,说明C V777与B t C o V存在共同祖先,冠状病毒可能在猪和蝙蝠之间存在跨物种传播[29]㊂H B/H E B E U/2020属于G2a群,与P E D V原型株C V777(G1a亚群)的遗传距离较远,因此H B/H E B E U/2020的进化方向与高头蝠来源的B t C o V关系不大㊂目前,以C V777为基础的经典株P E D V疫苗在中国市场占有较大份额,本研究提示这些经典疫苗可能很难对H B/ H E B E U/2020提供有效的保护㊂在所有市售疫苗株中,A J1102(G2b亚群)与H B/H E B E U/2020遗传距离最近,同属于G2群,这提示A J1102与H B/ H E B E U/2020可能进化于同一个祖先毒株㊂由于A J1102与H B/H E B E U/2020相似性达98.3%,并且目前尚无以G2b亚群毒株开发的商品化P E D V 疫苗,本研究结果表明现阶段选择以A J1102毒株为基础的P E D V疫苗并制定免疫计划将有助于对H B/H E B E U/2020的防控㊂近年来,中国频频发生G2b亚群P E D V变异毒株感染疫情[22,26],损失惨重,建议中国及早布局G2b亚群P E D V毒株疫苗研发计划以应对P E D V疫情㊂P E D V仍在不断进化,H B/H E B E U/2020已经进化为跨G1和G2群的重组变异株㊂以冠状病毒为代表的R N A病毒通过点突变积累和同源重组进化有利于基因损伤修复㊁组织嗜性和宿主范围改变,进而增强环境适应性[22,30-33]㊂本研究发现,H B/ H E B E U/2020存在3个潜在的重组事件,其中重组事件1和2均至少有5种检测方法验证呈阳性,说明发生概率较大㊂翟新国等[22]研究发现P E D V G1群和G2群毒株S1基因可能存在重组事件㊂在本研究中,重组事件2和3证明P E D V G1b亚群和G2a亚群基因组之间存在重组事件,说明不同P E D V毒株之间普遍存在基因重组现象[19,34]㊂由于P E D V重组发生会带来潜在不可控风险,因此建议种猪场在制定免疫程序时,应基于本场病原日常监测结果,选择亲缘关系较近的疫苗毒株(避免不同疫苗株混用),从而有效防止强毒重组突变株的产生㊂B o n i o t t i等[32]报道了1株2009 2012年间流行于意大利的猪重组肠道冠状病毒(S w i n ee n t e r i c c o r o n a v i r u s,S e C o V),S e C o V由P E D V的S基因和T G E V的骨架重组产生,随后,德国和西班牙等国也暴发了这种重组型S e C o V引起的疫情[35];V a l kó等[36]发现在匈牙利S e C o V S基因和P E D V又有了新的重组现象,这些都说明冠状病毒属的成员之间也可发生基因重组,这些广泛发生的猪冠状病毒重组将持续对生猪养殖业带来严重威胁㊂因此,应继续加强对P E D V病原学监测,揭示P E D V潜在起源㊁演化和重组的分子机制㊂4结论本研究成功克隆了河北省暴发于2020年底的1株变异型P E D V(H B/H E B E U/2020)全基因组,发现其与S N J-P株相似性最高,属于P E D V G2b亚群,并且存在跨P E D V亚群重组现象㊂研究结果有助于揭示P E D V在中国的自然选择与进化规律,为制定合理的防控策略提供理论和依据㊂参考文献(R e f e r e n c e s):[1] HU Y,X I EX H,Y A N GLC,e t a l.Ac o m p r e h e n s i v ev i e wo n t h eh o s t f a c t o r sa n dv i r a l p r o t e i n sa s s o c i a t e dw i t h P o r c i n e e p i d e m i c d i a r r h e a v i r u si n f e c t i o n[J].F r o n t i e r s i n M i c r o b i o l o g y,2021,12:762358.[2] S A L AMA T S E A,C O L L A N T E S T M A,L UM B E R A W M L,e ta l.S e q u e n c ea n a l y s i so fn e wv a r i a n t s o fP o r c i n e e p i d e m i cd i a r r h e av i r u s i nL u z o n,P h i l i p p i n e s,i n2017[J].A r c h i v e s o f V i r o l o g y,2021,166(7):1859-1867.[3] HU A N C C,P A N H C,F U S Y,e t a l.C h a r a c t e r i z a t i o n a n d e v o l u t i o n o f t h e 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分类号：R181.2 学校代码：10392学科专业代码：100103 学号：1110302053厦门地区HBV感染、环境因素及基因多态性与原发性肝癌的关联研究The association study between HBV infection, environmental factors, polymorphisms and the risk of hepatocellularcarcinoma in Xiamen学位类型：医学博士所在学院：公共卫生学院申请人姓名：苏成豪学科、专业：病原生物学导师：蔡琳教授研究起止日期：2011年9月至2015年2月答辩委员会主席：吴小南教授答辩日期：2015年6月2日二○一五年六月目录英文缩略语表．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．1 中文摘要．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．3 Abstract．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．6 第一部厦门地区原发性肝癌流行特征与趋势分析．．．．．．．．．．．．．．．．．．．．．．．．101 前言．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．102 材料与方法．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．113 结果．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．124 讨论．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．20 第二部厦门地区自然人群乙肝病毒基因型分布与肝癌队列随访分析．．．．．．．．221 前言．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．222 材料与方法．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．233 结果．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．254 讨论．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．29 第三部分HBV感染、环境危险因素与原发性肝癌的关联研究．．．．．．．．．．．．．．341 前言．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．342 材料与方法．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．353 结果．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．404 讨论．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．53 第四部分原癌基因、抑癌基因单核苷酸多态性与原发性肝癌的易感性研究．．．601 前言．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．602材料与方法．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．613 结果．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．674 讨论．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．80 第五部分TP53、MDM2、KIF1B基因多态性与原发性肝癌的Meta分析．．．．．．．871 前言．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．872对象与方法．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．883 结果．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．894 讨论．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．102 全文小结．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．106 存在问题与展望．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．108 参考文献．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．109 综述一．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．121 综述二．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．136 致谢．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．160英文缩略语表英文缩写英文全称中文全称AFB1 Aflatoxin B1 黄曲霉毒素AP Attributable proportion due to interaction 交互作用归因比APLL Average potential years of life lost 平均减寿年数CCR5 C-C chemokine receptor type 5 C-C类趋化因子受体5 CI Confidence interval 可信限区间DNA Deoxyribonucleic acid 脱氧核糖核酸ECM Extracellular matrix 细胞外基质GWAS Genome-wide association studies 全基因组关联研究HBcAb Hepatitis B virus c antibody 乙型肝炎病毒核心抗体HBeAb Hepatitis B virus e antibody 乙型肝炎病毒e抗体HbcAg Hepatitis B virus e antigen 乙型肝炎病毒e抗原HBsAb Hepatitis B virus s antibody 乙型肝炎病毒表面抗体HBsAg Hepatitis B virus s surface antigen 乙型肝炎病毒表面抗原HBV Hepatitis B virus 乙型肝炎病毒HBx Hepatitis B virus X protein 乙型肝炎病毒X蛋白HCC Hepatocellular carcinoma 肝细胞癌HCV Hepatitis C virus 丙型肝炎病毒HLA Human leukocyte antigen 人类白细胞抗原ID Incidence density发病密度KIF1B Kinesin family member 1B 驱动蛋白家族成员1BMALDI-TOF Matrix assisted laser desorption-ionization time offlight mass spectrometry基质辅助激光解吸电离飞行时间质谱MDM2 Murine double minute2 双微体2基因MMP-2 Matrix metalloproteinases-2 基质金属蛋白酶-2 MMP-9 Matrix metalloproteinases-9 基质金属蛋白酶-9MMPs Matrix metalloproteinases 基质金属蛋白酶NF-kappa-B Nuclear factor kappa-light-chain-enhancer of activatedB cells核转录因子NOS Newcastle-ottawa scale NOS文献质量评价表OR Odds ration 比值比PCR Polymerase chain reaction 聚合酶链反应PY Person year 人年PLC Primary liver cancer 原发性肝癌Pooled OR Pooled odds ratio 合并OR值PYLLR Potential years of life lost rate 寿命损失率PYLL potential years of life lost 潜在减寿人数RECK Reversion inducing cysteine rich protein with kazalmotifs基序逆向诱导半胱氨酸丰富蛋白RERI Relative excess risk due to interaction 交互作用超额相对危险度RR Relative risk 相对危险度SNP Single nucleotide polymorphisms 单核苷酸多态性TNF-αTumor Necrosis Factor-α肿瘤坏死因子-αTP53 Tumor protein 53 肿瘤抑制蛋白53VIF Variance inflation factor 方差膨胀因子χ2Pearson χ2皮尔森卡方值厦门地区HBV感染、环境因素及基因多态性与原发性肝癌的关联研究中文摘要[目的]探讨厦门地区原发性肝癌的流行特征与趋势；研究厦门自然人群乙型肝炎病毒基因型分布及其与肝癌的关系；研究HBV感染、环境因素与厦门地区肝癌的关联；探索原癌基因、抑癌基因的单核苷酸多态性与肝癌易感性的关系。

木仓医学考研复试SCI长难句肝胆胃肠外科第一章-肝细胞恶性肿瘤Hepatocellular carcinoma(HCC)is the third leading cause of cancer--related deaths worldwide,and hepatitis B virus(HBV)infection is one of its leading causes.During the past several years,next-generation sequencing studies using bulk tumor samples have revealed considerable intratumor molecular and genetic heterogeneity in HCC.Such intratumor heterogeneity poses a great challenge for tumor characterization and therapeutic management of HCC patients.As is well known,tumor initiation and evolution are mediated by sequential genetic alterations in single cells.Single-cell sequencing has the potential to provide new insights into cancer bio-logical diversity that were difficult to resolve in genomic data from bulk tumor samples.在全球范围内导致癌症相关死亡的原因中，肝细胞癌(HCC)位列第三，而乙型肝炎病毒感染是其重要病因之一。

乙型肝炎病毒感染者血清HBV DNA与IL-21及IFN-γ水平的相关性陶鹏辉【摘要】目的探讨乙型肝炎病毒(Hepatitis B virus,HBV)感染者血清乙肝病毒脱氧核糖核酸(HBV-DNA)与白细胞介素21(IL-21)及γ-干扰素(IFN-γ)水平的相关性.方法选取我院收治的119例慢性乙肝患者,其中乙型肝炎(CHB)46例,乙肝后肝硬化(LC)41例,原发性肝癌(PHC)32例,同期选取35例肝功能检查表面抗体为阳性的健康正常者作为对照,采用荧光定量聚合酶链式反应(Fluorescence quantitative PCR,FQ-PCR)方法检测HBV感染者血清HBV DNA,利用酶联免疫吸附实验(En-zymes linked immunosorbent assay,ELISA)检测受试者血清IL-21及IFN-γ水平.结果 CHB、LC、PHC三组患者之间血清IL-21、IFN-γ水平比较无显著差异(P>0.05),与健康对照组比较差异显著,且三组HBV感染者血清IL-21、IFN-γ水平明显高于健康对照组(P<0.05);阴性组血清IL-21水平明显高于对照组(P<0.05),且随着HBV DNA载量增加呈现明显升高趋势;阴性组血清IFN-γ水平明显低于对照组(P<0.05),且随着HBV DNA载量增加呈现明显降低趋势;相关性分析结果显示,IL-21水平与HBV DNA载量呈较强正相关(rs=0.641,P<0.05),IFN-γ水平与HBV DNA载量呈现负性相关(rs=-0.372,P<0.05).结论IL-21、IFN-γ与HBV感染者血清HBV DNA持续感染密切相关,推测IL-21、IFN-γ有望成为HBV感染者免疫治疗新靶点.【期刊名称】《实验与检验医学》【年(卷),期】2017(035)003【总页数】4页(P347-350)【关键词】HBV;HBVDNA;IL-21;IFN-γ;相关性【作者】陶鹏辉【作者单位】信阳市中心医院检验科,河南信阳 464000【正文语种】中文【中图分类】R512.6+2;R446.62乙型肝炎病毒(Hepatitis B virus，HBV)感染是导致乙型肝炎(CHB)、肝硬化(LC)以及原发性肝癌(PHC)的主要原因[1]。

November 23,2020Mosquitos in Asia and the Americas More Susceptible to Zika Virus 亚洲和美洲的蚊子更容易感染寨卡病毒A study explains how Zika was present among mosquitoes in Africa for decades without causing the harm to human health seen outside the continent in recent years.一项研究解释了为什么寨卡病毒在非洲的蚊子中存在了几十年，近年来却没有对非洲大陆以外的地方那样对人类健康造成危害。

According to the World Health Organization, Zika virus was first isolated from an infected monkey in Uganda in 1947, but there have only been sporadic cases across the African continent since. In contrast, thousands of people in the Americas and Asia were infected in multiple outbreaks since 2007. During the worst, in 2016, the virus spread to more than 60 countries and was implicated in some 5,000 cases of microcephaly—a severe birth defect in which a baby’s head is much smaller than expected.据世界卫生组织称，寨卡病毒最早于1947年在乌干达从一只感染的猴子身上分离出来，但此后整个非洲大陆只出现过零星病例。

关注ＨＢＶ感染特殊人群：早诊、早治、早获益李艳伟，窦晓光中国医科大学附属盛京医院，沈阳１１００００摘要：ＨＢＶ感染是世界范围内的主要公共卫生问题。

随着新生儿乙型肝炎疫苗的广泛预防接种以及越来越多的患者接受抗病毒治疗，我国儿童ＨＢＶ感染方面取得了显著成就。

但我国成年人ＨＢｓＡｇ阳性率仍然很高，特殊人群也越来越多，应做到对特殊人群及时筛查和诊断，以尽早抗病毒治疗，阻滞或者延缓ＨＢＶ感染后进展至肝硬化、肝衰竭和肝细胞癌。

治疗时机及药物的选择除了关注ＨＢＶ病毒学、肝功能，还要考虑合并慢性基础疾病、母婴传播、接受化学治疗及免疫抑制剂的患者ＨＢＶ再激活等问题。

从而实现ＨＢＶ高危和特殊人群应筛尽筛、及时诊断和治疗，以使更多患者获益。

关键词：乙型肝炎病毒；特殊人群；筛查；诊断；治疗学ＦｏｃｕｓｏｎｔｈｅｓｐｅｃｉａｌｐｏｐｕｌａｔｉｏｎｗｉｔｈｈｅｐａｔｉｔｉｓＢｖｉｒｕｓｉｎｆｅｃｔｉｏｎ：Ｅａｒｌｙｄｉａｇｎｏｓｉｓ，ｅａｒｌｙｔｒｅａｔｍｅｎｔ，ａｎｄｅａｒｌｙｂｅｎｅｆｉｔｓＬＩＹａｎｗｅｉ，ＤＯＵＸｉａｏｇｕａｎｇ．（ＳｈｅｎｇｊｉｎｇＨｏｓｐｉｔａｌ，ＣｈｉｎａＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｓｈｅｎｙａｎｇ１１００００，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＤＯＵＸｉａｏｇｕａｎｇ，ｇｕａｎｇ４０＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００３－０６９４－７１２６）Ａｂｓｔｒａｃｔ：ＨｅｐａｔｉｔｉｓＢｖｉｒｕｓ（ＨＢＶ）ｉｎｆｅｃｔｉｏｎｉｓａｍａｊｏｒｐｕｂｌｉｃｈｅａｌｔｈｐｒｏｂｌｅｍｗｏｒｌｄｗｉｄｅ．ＷｉｔｈｔｈｅｅｘｔｅｎｓｉｖｅｐｒｏｐｈｙｌａｃｔｉｃｖａｃｃｉｎａｔｉｏｎｗｉｔｈｈｅｐａｔｉｔｉｓＢｖａｃｃｉｎｅｆｏｒｎｅｏｎａｔｅｓａｎｄａｎｉｎｃｒｅａｓｉｎｇｎｕｍｂｅｒｏｆｐａｔｉｅｎｔｓｒｅｃｅｉｖｉｎｇａｎｔｉｖｉｒａｌｔｒｅａｔｍｅｎｔ，ｒｅｍａｒｋａｂｌｅａｃｈｉｅｖｅｍｅｎｔｓｈａｖｅｂｅｅｎｍａｄｅｉｎＨＢＶｉｎｆｅｃｔｉｏｎａｍｏｎｇＣｈｉｎｅｓｅｃｈｉｌｄｒｅｎ．Ｈｏｗｅｖｅｒａｔｐｒｅｓｅｎｔ，ｔｈｅｒｅｉｓｓｔｉｌｌａｈｉｇｈＨＢｓＡｇ－ｐｏｓｉｔｉｖｅｒａｔｅａｍｏｎｇｔｈｅａｄｕｌｔｓｉｎＣｈｉｎａ，ａｎｄｗｉｔｈｍｏｒｅａｎｄｍｏｒｅｓｐｅｃｉａｌｐｏｐｕｌａｔｉｏｎｓ，ｉｔｉｓｎｅｃｅｓｓａｒｙｔｏｐｅｒｆｏｒｍｔｉｍｅｌｙｓｃｒｅｅｎｉｎｇａｎｄｄｉａｇｎｏｓｉｓａｎｄｇｉｖｅａｎｔｉｖｉｒａｌｔｈｅｒａｐｙａｓｅａｒｌｙａｓｐｏｓｓｉ ｂｌｅ，ｓｏａｓｔｏｐｒｅｖｅｎｔｏｒｄｅｌａｙｔｈｅｐｒｏｇｒｅｓｓｉｏｎｔｏｌｉｖｅｒｃｉｒｒｈｏｓｉｓ，ｌｉｖｅｒｆａｉｌｕｒｅ，ａｎｄｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒｃｉｎｏｍａａｆｔｅｒＨＢＶｉｎｆｅｃｔｉｏｎ．ＴｈｅｓｅｌｅｃｔｉｏｎｏｆｔｒｅａｔｍｅｎｔｔｉｍｉｎｇａｎｄｄｒｕｇｓｓｈｏｕｌｄｎｏｔｏｎｌｙｆｏｃｕｓｏｎＨＢＶｖｉｒｏｌｏｇｙａｎｄｌｉｖｅｒｆｕｎｃｔｉｏｎ，ｂｕｔａｌｓｏｃｏｎｓｉｄｅｒｃｈｒｏｎｉｃｕｎｄｅｒｌｙｉｎｇｄｉｓｅａｓｅｓ，ｍｏｔｈ ｅｒ－ｔｏ－ｃｈｉｌｄｔｒａｎｓｍｉｓｓｉｏｎ，ａｎｄｒｅａｃｔｉｖａｔｉｏｎｏｆＨＢＶｉｎｐａｔｉｅｎｔｓｒｅｃｅｉｖｉｎｇｃｈｅｍｏｔｈｅｒａｐｙａｎｄｉｍｍｕｎｏｓｕｐｐｒｅｓｓａｎｔｓ，ｔｈｅｒｅｂｙｒｅａｌｉｚｉｎｇｔｈｅｔｉｍｅｌｙｓｃｒｅｅｎｉｎｇ，ｄｉａｇｎｏｓｉｓ，ａｎｄｔｒｅａｔｍｅｎｔｏｆｔｈｅｐｏｐｕｌａｔｉｏｎａｔａｈｉｇｈｒｉｓｋｏｆＨＢＶａｎｄｓｐｅｃｉａｌｐｏｐｕｌａｔｉｏｎｓａｎｄｂｒｉｎｇｉｎｇｍｏｒｅｂｅｎｅｆｉｔｓｔｏｐａｔｉｅｎｔｓ．Ｋｅｙｗｏｒｄｓ：ＨｅｐａｔｉｔｉｓＢＶｉｒｕｓ；ＳｐｅｃｉａｌＰｏｐｕｌａｔｉｏｎ；Ｓｃｒｅｅｎｉｎｇ；Ｄｉａｇｎｏｓｉｓ；ＴｈｅｒａｐｅｕｔｉｃｓＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－５２５６．２０２２．１１．００１收稿日期：２０２２－０９－１９；录用日期：２０２２－１０－１２通信作者：窦晓光，ｇｕａｎｇ４０＠１６３．ｃｏｍ ＨＢＶ感染是世界范围内的主要公共卫生问题。

分泌及吞噬功能的影响·肝纤维化及肝硬化·DOI： 10.3969/j.issn.1001-5256.2023.09.013干扰素基因刺激蛋白（STING）通路对乙型肝炎肝硬化患者外周血单核细胞炎症因子分泌及吞噬功能的影响杨逸帆，杜万威，王霞，杨广德，李丽，傅涓涓，潘修成徐州医科大学附属医院感染性疾病科，江苏徐州 221002通信作者：潘修成，***************（ORCID： 0000－0001－9706－9458）摘要：目的　研究干扰素基因刺激蛋白（STING）通路对乙型肝炎肝硬化患者外周血单核细胞生成炎症细胞因子及吞噬功能的影响。

方法　收集2020年5月—10月于徐州医科大学附属医院感染性疾病科住院的35例乙型肝炎肝硬化患者和10例体检中心健康体检者的外周静脉全血，分离并提取外周血单核细胞；另将乙型肝炎肝硬化患者分为环鸟苷酸-腺苷酸（cGAMP）（n=12）、cGAMP+CCCP（n=12）和Control（n=11）三组。

体外以STING激活剂cGAMP和/或STING抑制剂CCCP加入单核细胞培养液中，ELISA法检测上清液IFN－α、IFN－β、IL－6和TNF－α水平。

此外，将含cGAMP和/或CCCP的单核细胞培养体系与荧光标记的E.coli共同孵育后，采用流式细胞仪检测单核细胞吞噬功能的改变。

计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD－t检验。

结果　与加入抑制剂CCCP组相比，乙型肝炎肝硬化患者外周血单核细胞经cGAMP刺激后分泌IFN－α、IFN－β、IL－6和TNF－α水平显著升高（P值均<0.05）。

乙型肝炎肝硬化患者外周血单核细胞在有或无cGAMP/cGAMP+CCCP处理下，吞噬E.coli能力均较健康组明显降低（t值分别为4.647、2.790、2.504，P值均<0.05），且有或无cGAMP与cGAMP+CCCP刺激，对乙型肝炎肝硬化患者单核细胞吞噬E.coli的能力没有显著影响（P>0.05）。

4种解释系统对HIV-1基因型耐药解释相关性评价赫天一;关琪【摘要】目的：比较HIVDB （7.0.1）、ANRS （25_09/2015）、REGA（9.1.0）和HIV GRADE （12/2015）4种解释系统对HIV-1基因型耐药结果解释的一致性。

方法：选取我国3种主要HIV-1流行亚型和流行重组模式（B’、CRF01_AE和CRF07_BC），每种选取200条序列，共600条序列。

分别用4种解释系统进行耐药结果分析，分为耐药（R）、中度耐药（I）和敏感（S）3个水平。

对4种结果进行统计学分析。

结果：4种系统基因型耐药解释结果比较相关性较高（r均＞0.57，P均＜0.01）。

针对亚型来说，CRF07_BC的解释结果相关性最高，其次为B’亚型，CRF01_AE略低。

结论：对于我国主要流行的HIV来说，4种解释系统对11种常用的抗病毒治疗药物耐药结果的解释具有较好的一致性。

%Objective:To compare the correlation of four drug-resistance interpretation system including HIVDB, ANRS, REGA and HIV GRADE for testing HIV-1 genotype drug resistance. Methods:Trendy subtypes and restructuring model of HIV-1 including B’,CRF01_AE and CRF07_BC were selected,each genotype was 200 series,600 samples were analyzed by four drug⁃resistance interpretation system, the results were divided into three levels including resistance (R), possible drug⁃resistance (I) and susceptible (S). Results:Four drug⁃resistance interpretation system had a high correlation in genotypic drug⁃resistance consequence (rs>0.57, P<0.01), CRF07_BC had the highest correlation, followed by subtype B, CRF01_AE was slightly lower.Conclusion:Four drug⁃resistance interpretation systemhas a high correlation in analyzing antiviral drug resistance for major epidemic of HIV in China.【期刊名称】《沈阳医学院学报》【年(卷),期】2016(018)004【总页数】5页(P237-241)【关键词】HIV-1;基因型耐药解释系统;耐药突变【作者】赫天一;关琪【作者单位】沈阳医学院附属中心医院检验科，辽宁沈阳 110024;沈阳医学院附属中心医院检验科，辽宁沈阳 110024【正文语种】中文【中图分类】R512.91doi：10.16753/ki.1008-2344.2016.04.004［Abstract］Objective：To compare the correlation of four drug-resistance interpretation system including HIVDB，ANRS，REGA and HIV GRADE for testing HIV-1 genotype drug resistance.Methods：Trendy subtypes and restructuring model of HIV-1 including B'，CRF01＿AE and CRF07_BC were selected，each genotype was 200 series，600 samples were analyzed by four drug⁃resistance interpretation system，the results were divided into three levels including resistance（R），possible drug⁃resistance （I）and susceptible（S）.Results：Four drug⁃resistance interpretation system had a high correlation in genotypic drug⁃resistance consequence（rs＞0.57，P＜0.01），CRF07_BC had the highest correlation，followed by subtype B，CRF01_AE was slightly lower.Conclusion：Four drug⁃resistance interpretation system has a high correlation in analyzing antiviral drug resistance for major epidemic of HIV in China.［Key words］HIV-1；genotype resistant interpretation system；drug⁃resistant mutation基于生物信息学方法建立的基因型耐药分析技术，能够在进行抗病毒治疗之前，对患者基因型进行检测，并结合分析结果制定最优的抗病毒治疗方案，它不仅能够降低由耐药性导致的治疗失败，甚至有可能决定抗病毒治疗的成败，因而在HIV-1抗病毒治疗中起到非常重要的作用，本文比较了4种最为常用的基因型耐药解释系统HIV DB、ANRS、REGA和HIV GRADE，针对我国主要流行的3种HIV-1病毒亚型解释结果上的异同，以期筛选出最符合我国国情的基因型耐药解释系统。

急性甲型肝炎病毒感染儿童总 IgE 抗体及 Th17相关细胞因子变化研究张晓俞;冯涛;席日升【摘要】Objective To explore the effect of HAV infection in allergic disease and Th17 cells effect in the process of HAV infection .Methods From June 2014 to June 2015 ,41 cases of acute HAV infection children and 41 cases of healthy controls in Maternal and Child Health Hospital of Zhangjiakou were chose as research object ,clinical data were retrospectively analyzed in detail ,and serum total IgE and Th17 cells related cytokines level were detected by ELISA .Results Serum total IgE in children with acute HAV infection was lower than healthy controls (P<0 .05) ,and IgE content further reduced with the degree increase of liver injury ;Th17 related cytokines in children with acute HAV infection were higher than healthy controls(P<0 .05);There was a negative correlation between IgE antibody and IL‐17 levels(r= -0 .533 ,P< 0 .05) .Conclusion Th17 related cytokines in children with acute HAV infection are increased significantly ,which may play a protective role against allergic diseases by reducing the IgE levels .%目的：探讨甲型肝炎病毒（HAV）感染对儿童过敏性疾病的影响及HAV感染过程中Th17所起作用。

免疫学英文名词解释（一）1. Immunity: “a condition of being able to resist a particular disease especially through preventing development of a pathogenic microorganism or by counteracting the effects of its products”2. Immunology: Immunology is the study of our protection from foreign macromolecules or invading organisms and our responses to them.3. Innate immunity: evolves with the germline and involves receptors, enzymes and cells that detect conserved aspects of microbes and parasites. It is the 1st line of defense. No specificity, no memory.4. Adaptive immunity is provided by T & B lymphocytes. It is the 2nd line of defense. It has two important characteristics: Immune response is highly specific for the antigen that triggered it. Exposure to antigen creates an immunologic “memory.”5. Primary lymphoid organs: Lymphoid organs include primary (bone marrow & thymus) and secondary lymphoid organs and tissues (lymphoid nodes、spleen、MALT). Primary lymphoid organs are the place where lymphocytes develop and mature. Lymphocytes includes B cell and T cell, respectively originating from bone marrow and thymus and mediates humoral and cellular immunity.6. Secondary lymphoid organs: Secondary lymphoid organs are the place that immune responses happen, which include lymph nodes, spleen, tonsil and MALT. Lymph nodes drain the connective tissues of the body. The spleen drains the blood. MALT are responsible for local infection.7. MALT: The majority (50%) of lymphoid tissue in the human body is located within the lining of the major tracts, including respiratory, digestive and genitourinary tracts. This is because these are the main sites of entry for microbes into the body. These are collectively called the mucosa-associated lymphoid tissues (MALT).8. Antigens are molecules which are recognized by receptors on lymphocytes. B lymphocytes usually recognize intact antigen molecules, while T lymphocytes recognize antigen fragments on the surface of antigen presenting cells.9. Epitope:Antigen molecules each have a set of antigenic determinants, also called epitopes. Epitopes are molecular shapes recognized by antibodies and T cells of the adaptive immune system.10. Clonal selection: Each lymphocyte is genetically programmed to be capable of recognizing essentially only one particular antigen. When an antigen binds to the cell that can recognize it, it is induced to proliferate rapidly. Within a few days there are a sufficient number to mount an adequate immune response. In another words, the antigen selects for and generates the specific clones of its own antigen-binding cells, a process called clone selection. In brief: clone selection involves recognition of antigen by a particular lymphocyte, this lead to clonal expansion and differentiation to effector and memory cells11. Opsonization: This s is a process of making a microbe easier to phagocyte. A number of molecules called “opsonins” do this by coating the microbes and aid attachment of the microbe to the phagocyte and also trigger activation of phagocytosis. Opsonins include the complement component C3b and antibody which acting as a bridge between antigen and phagocytes.12. NK cells: belong to lymphocyte family. But in contrast to all T and B lymphocytes, NK cell do not express antigen-specific receptors and do not possess the adaptive property of memory cell development: they are there for considered to form part of the innate immune system. However, like Tc, their main function is to kill infected cell and tumor cells using similar mechanism to those of Tc cells to induce apoptosis of their targets. NK cell are also able to kill targets coated with IgG via their receptor for IgG. This property is referred to as ADCC.13. DCs are required by T cell to enable them to respond to antigens. DCs are most important antigen presenting cells known so far and are the interface of innate and adoptive immunity. Functions: Antigen up-taking in peripheral sites & antigen presentation in lymph nodes.14. Complement: The complement system is an important component of innate immunity It can be activated by the classical and alternative pathways, both pathways will eventually lead to the lytic pathway which featured by the formation of MAC.Function of complement: anaphylaxis (C3a,C5a), chemotaxis (C5a)，opsonization (C3b,C4b), lysis (C56789)15. Interferons: Interferons are proteins involved in protection against viral infections. The two kinds of interferon, type I and type II, have different cellular origins and mediate a range of different activities. They interfere with viral replication but also are signaling molecules between cells.16. lymphocyte traffic and recirculation Lymphocytes produced in the primary lymphoid organs (thymus-T, bone marrow-B) migrate via the bloodstream to the secondary lymphoid organs/tissues where they carry out the function. They do not stay in one site but continually recirculate through the body in search of antigens.17. Affinity is the tightness of binding of an antibody binding site to an antigenic determinant (epitope)----the tighter the binding, the less likely the antibody is to dissociate from antigen.18. Valence: Valence is the maximum number of epitopes with which the antibody can react.19. Avidity: antibody binds a multivalent antigen is termed avidity, to differentiate it from the affinity of a single antigenic determinant for an individual combining site. Antibody avidity indicates the overall strength of interaction between antibody and antigen.20. Isotype: These are genetic markers on immunoglobulins shared by all the individual of same species. The genes for isotypic variants are present in all healthy members of a species. For example, the genes for γ1, γ2,γ3, γ4, μ, α1, α2, δ, ε, κ and λ chains are all present in the human genome, and are therefore isotypes.21. Allotype:These are genetic markers on immunoglobulins that segregate within the species. This refers to genetic variation between individuals within a species. For example, the variant of IgG3 called G3m(b0) is characterized by a phenylalanine at position 436 of the γ3 heavy chain. It is not found in all people and is therefore an allotype. Allotypes occur mostly as variants of heavy chain constant regions.22. Idiotype: These are unique antigenic determinant associated with antigen binding sites of antibodies and are the results of the different amino acid sequences which determine their specificities. Variation in the variable domain, particularly the hypervariable regions, produces idiotypes. These determine the binding specificity of the antigen-binding site.23. CDRs: At the amino acid level, the variable region of antibody is comprised of three regions of extreme variability (hyervarible region). They are called complementarity-determining regions, or CDRs.24. FRs: Interspersed among the CDRs are framework regions (FRs) which are less variable and more evolutionarily conserved. At the three-dimensional level, the three CDRs of each chain converge to form a combining site which recognize the antigenic determinant (epitope).25. Monoclonal antibody: In 1975, Kohler and Milstein developed a procedure to create cell lines producing predetermined, monospecific and monoclonal antibodies. The basic technology involves fusion of an immortal cell (a myeloma tumor cell) with a specific predetermined antibody-producing B cell from immunized animals or humans. The resulting hybridoma cell is immortal and synthesizes homogeneous, specific, mAb which can be made in large quantities.26. ADCC：ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY –The linking of antibody bound to target cells (virus infected cells, or some tumor cells) with FcR of natural killer cells (NK cells), neutrophils, macrophages,or eosinophils can result in killing of the target cell.27. cytokines: Definition: Cytokines are small molecules, secreted by cells in response to a stimulus. Function: As a group, cytokines induce growth, differentiation, chemotaxis, activation and/or enhanced cytotoxity. They are used for strengthening communications between cells.28. Toll like receptors (TLR)Toll like receptors are a family of proteins of which there are at least 5 known members.Using TLRs, innate immune cells can detect and respond to infection by recognizing conserved motifs of microbes. TLRs transmit signals about microbial constituents to the nucleus, thus regulating the type of genes expressed, and the subsequent response.29. Positive selection of T cells:T cells that express a TCR that can bind weakly to self MHC are spared from death and are positively selected to survive.30. Negative selection of T cells: T cells that react strongly to self-antigens on MHC are eliminated. Only those T cells that can react to MHC, but do not bind strongly to self-antigens emerge as mature T cells from the thymus.。