1995年大连麻疹病毒流行株的部分基因分析

目的 本实验以研究麻疹病毒（ＭＶ）的目的,采用反转录聚合酶链式反应（ＲＴＰＣＲ）、早期、快速、准确、灵敏地检出ＭＶ,通过对扩增产物的基因分析,判断传染源和传播途径,并研究ＭＶ基因型。方法 对１９９５年６月大连地区发生的一起５人的麻疹流行进行ＭＶ检测,用反转录套式ＰＣＲ法分别扩增ＭＶ的核蛋白基因（Ｎ）和基质蛋白基因（Ｍ）,阳性对照用长春冻干麻疹活疫苗。对疫苗株长－４７９９４０５）和任意选取的两个标本     

吉林省2009-2012年肠道病毒71型VP1编码区氨基酸位点的变异分析

目的分析2009--2012年引起吉林省手足口病（handfootandmouthdisease,HFMD）流行的肠道病毒71型（EV—A71）的VP1区氨基酸位点的特征性变异位点。方法对2009--2012年分离于吉林省HFMD病例的70株EV—A71分离株的VPl编码区进行逆转录．聚合酶链反应,然后对扩增产物进行核苷酸序列测定,使用Bioedit软件和MEGA4．0软件分析VPl区氨基酸位点的变异特征以及基因亲缘关系。结果2009--2012年在吉林省分离到的70株EV—A71均属于C4a基因亚型,其中69株与2008年安徽阜阳的分离株亲缘关系最近,并且这69株病毒在VPl编码区第22位氨基酸位点均由谷氨酰胺转变为组氨酸（Q—H）,与2008年安徽阜阳株一致。结论引起2009-2012年吉林省HFMD流行的EV-A71可能：来源于安徽阜阳流行株延续传播。     

甲型流感病毒核蛋白基因的克隆表达及纯化

目的　将甲型流感病毒核蛋白(NP)基因克隆到原核表达载体进行可溶性融合表达,制备纯化的病毒核蛋白,为制备甲型流感病毒单克隆抗体提供材料。方法　提取甲型流感病毒RNA ,设计引物,RT PCR扩增NP基因,利用基因工程的手段,将甲型流感病毒的NP基因在大肠埃希菌中进行融合表达,并将表达产物进行亲和层析。结果　成功构建了甲型流感病毒NP基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。结论　通过合理控制发酵时间、生长温度和诱导物浓度,制备了较为理想的可溶性甲型流感病毒核蛋白。     

基于核蛋白的麻疹病毒IgG抗体间接ELISA检测方法的建立及应用

目的 以原核系统表达的麻疹病毒核蛋白作为包被抗原,初步建立检测麻疹病毒抗体的间接ELISA方法,并将其应用于人群抗体水平的评估。方法 对各项实验条件进行筛选和优化,确定间接ELISA法的操作流程,并检测了157份健康儿童和新生儿母亲血清中的抗麻疹病毒IgG抗体,与商品化麻疹病毒ELISA IgG抗体检测试剂盒进行比较。结果 本研究建立的间接ELISA方法批内和批间的重复性试验变异系数均小于10%,与试剂盒检测结果相比,血清标本的总符合率为95.5%,灵敏度和特异度分别可达94.8%和98.3%。其中,两种方法检测的85份0～15岁健康儿童血清麻疹病毒IgG抗体阳性率,无论是<8月龄组或8月龄～15岁组,结果差异均无统计学意义(χ2＝0.313,P>0.05;χ2＝0.000,P>0.05),且通过本研究所建立的ELISA方法检测的麻疹病毒抗体水平表现出与试剂盒检测结果在不同年龄组一致的增减趋势,两种方法定量结果的相关系数r为0.893(P<0.001),显示出该方法具备的定量潜力。结论 本研究建立的ELISA方法具有良好的稳定性,且灵敏度高、特异度强,与商品化试...     

人类精子核的研究──100例不育病人精子碱性核蛋白的分析

对１００例随机选择的不育病人精子核碱性蛋白质进行分析，结果表明有５４％病人的碱性蛋白异常，并可将其分为３类：１．组蛋白－精核蛋白（ｈｉｓｔｏｎｅ－ｐｒｏｔａｍｉｎｅ）取代反应受阻，占总病历２３％；２．３种精核蛋白（ＨＰ１，ＨＰ２，ＨＰ３）比例异常，占总病历３０％；３．精核蛋白完全缺失，占总病历１％。文中还对精核蛋白与生育力的关系以及精子碱性核蛋白分析在不育病人诊断中的重要性进行了讨论。     

狂犬病毒核蛋白基因的克隆及核蛋白主要抗原表位分析

目的:克隆狂犬病毒ERA株核蛋白基因并对核蛋白主要抗原表位进行分析.方法:根据GenBank中已发表的RV N基因序列,设计合成了1对特异性引物,对RV ERA株N基因进行了RT-PCR扩增.将PCR产物纯化后与pMD18-T连接得到重组质粒pMD-N,并进行核苷酸序列测定.结果:该基因全长1 353 bp,编码450个氨基酸.RV ERA株与Gen-Bank中RV不同固定毒株和分离毒株N基因相比,核苷酸的同源性为98.0%～99.6%,推导的氨基酸序列的同源性为98.3%～99.60A,.核蛋白主要抗原表位分析表明,ERA株与其他固定毒株和分离毒株相比,仅在BC抗原表位(369～383位氨基酸)和Th抗原表位(394～408位氨基酸)处有1～3个氨基酸的不同,其他表位无差异.但与Iagos bat、Mokola和Duvenhage毒株相比,抗原位点氨基酸组成差异明显.结论:RV ERA株N基因可用于基因工程疫苗研制和核酸检测的靶基因;核蛋白可作为抗原用于狂犬病的检测.     

引起乌鲁木齐成人麻疹暴发的麻疹病毒基因特征分析

目的 分析引起新疆乌鲁木齐市2008年成人麻疹暴发流行的麻疹野病毒的基因特征.方法 用Vero/Slam细胞从成人麻疹暴发患者的标本中分离出麻疹野病毒,应用逆转录-聚合酶链反应（RT-PCR）对麻疹野病毒分离株扩增核蛋白N基因羧基末端676个核苷酸片段,再对扩增产物进行核苷酸序列测定和分析.以N基因羧基末端450个核苷酸片段构建基因亲缘性关系树,进行核苷酸变异分析.结果 共分离到11株麻疹野病毒,这些病毒N基因450个核苷酸之间的差异为0～0.2%,均为H1基因型中的H1a基因亚型.11株麻疹野病毒与H1基因型代表株（Chin9372）的核苷酸和氨基酸差异分别为：2.2%～2.4%之间和4.0%～4.6%之间.结论 引起新疆乌鲁木齐市2008年成人麻疹暴发流行的麻疹野病毒为H1a基因亚型.     

福建省2014—2019年麻疹病毒基因型监测情况分析

目的:分析福建省2014—2019年不同基因型麻疹野病毒流行情况,为制定后续麻疹防控策略,完成消除麻疹目标提供参考。方法:收集福建省2014—2019年麻疹发病监测数据及各地市上送的麻疹疑似病例咽拭子样本,采用逆转录-聚合酶链反应扩增麻疹病毒N蛋白羧基端450个核苷酸序列,对扩增产物进行序列鉴定和分析。结果:2014—...     

Smith-Fineman-Myers综合征与X连锁核蛋白基因的连锁分析

目的 明确Smith-Fineman-Myers综合征（Smith-Fineman-Myers syndrome,SFMS)与X连锁核蛋白(X-linked unclear protein,XNP）基因之间的连锁关系。方法 采用聚合酶链反应结合变性聚丙烯酰胺凝胶电泳方法，分析SFMS家系中各成员XNP基因内多态位点基因型。结果 两个多态位点中，XNPSTR1有多态，家系分析结果表明XNP基因与SFMS致病基因之间存在重组。结论 XNP基因不是导致中国山东SFMS家系的致病基因，SFMS存在位点异质性。     

RT-PCR-RFLP用于麻疹病毒疫苗株和H1基因型的分析

目的 建立RT-PCR-RFLP方法 用于天津地区2002-2008年流行的麻疹野病毒基因型研究.方法 采集疑似麻疹患者的尿标本和咽拭子,传代细胞分离病毒.提取病毒液中的RNA,用一步RT-PCR法扩增麻疹病毒核蛋白(nucleoprotein,N)基因C端594个核苷酸片段,扩增产物经Bcn I酶切后琼脂糖凝胶电泳进行限制性片段多态性分析(RFLP),同时与序列分析进行对比验证.根据结果 构建基因系统发生树进行亲缘关系和遗传距离分析.结果 2002-2008年189份标本,分离获得69株麻疹病毒,N基因C端RT-PCR检测全部阳性,RFLP分析H1a是优势流行亚型,占98.55%(68/69),仅1株(1.45%)为H1b基因亚型,分型结果 与序列测定完全一致.系统发生树分析显示H1a亚型毒株分为2个亚枝,变异范围为0.2%～3.8%,存在不同病毒株引起的传播链共循环.结论 基于Bcn I限制性内切酶的RT-PCR-RFLP方法 能够特异性区分A基因型、H1a、H1b基因亚型,具有快速、简便、准确和经济的优点,更适用于国内大规模麻疹病毒的监测.     

Effect of gene location on the evolutionary rate of amino acid substitutions in herpes simplex virus proteins

In an effort to understand the organization of genes in the herpes simplex virus (HSV-1) genome, I tested the idea that the location of a gene may be related to the evolutionary rate of amino acid sequence variation in the encoded protein. A measure of protein sequence divergence was calculated for homologous proteins in the UL region of six alphaherpesviruses including HSV-1, and this parameter was plotted against position in the HSV-1 genome. The results revealed a cluster of highly conserved proteins (UL27-UL33) encoded near the middle of UL. A similar analysis was restricted to HSV-1 and HSV-2 permitting an examination of U(S) proteins and proteins encoded in repeated regions at the segment ends. This analysis showed that U(S) proteins as a group are more highly divergent than those encoded in UL. A high degree of divergence was also observed in proteins coded at the segment ends including RL1 (gamma(1)34.5), RL2 (alpha0), UL1 (glycoprotein L), UL56, U(S)1, and U(S)12. It is suggested that conserved proteins UL27-UL33 are encoded near the middle of UL to take advantage of a low local mutation rate. Highly divergent proteins are suggested to be encoded selectively in U(S) because of a comparatively rapid evolutionary rate with which genes can be introduced and removed from S in response to environmental variation.     

吉林省麻疹野病毒的基因型别和基因特征

目的 阐明吉林省流行麻疹野病毒的基因型别和基因特征,为制定麻疹防控策略措施提供科学依据.方法 用逆转录-聚合酶链反应.限制性片段长度多态性分析(RT-PCR-RFLP)方法对2001-2006年分离的38株麻疹病毒进行基因分型;选择麻疹病毒代表株RT-PCR扩增N基因C-末端450个核苷酸,对比野毒和疫苗株核苷酸和氨基酸同源性,并构建N基因亲缘关系树.结果 经RT-PCR-RFLP基因定型,38株麻疹病毒分离珠均为H1基因型;对其中的29株进一步进行N基因C-末端450个核苷酸的序列测定和分析证实均为H1基因型的H1a亚型.吉林分离株与我国麻疹疫苗S191株450个核苷酸同源性为88.0%～89.4%,所提示氨基酸的同源性为91.8%～92.7%;吉林省内分离株核苷酸平均变异小于1.4%.结论 H1a基因亚型为吉林省近年来流行的麻疹野病毒绝对优势基因亚型.不同年份存在相同麻疹病毒的持续循环传播,同一年份也存有H1a亚型内不同病毒的共循环;与其他省之间存在相同麻疹病毒引起的传播链.     

以新型杆状病毒载体在昆虫、哺乳动物细胞中表达克里米亚-刚果出血热病毒核蛋白基因

目的 构建可以在哺乳动物细胞中高效表达的新型杆状病毒载体，并利用其将克里米亚—刚果出血热病毒(CCHFY)中国分离株(新疆出血热病毒，xHFY)BA88166的核蛋白(NP)基因在昆虫和哺乳动物细胞中进行表达。方法 将人巨细胞病毒(CMv)立即早期(IE)启动子连接至杆状病毒载体PFastBacl多角体启动子下游形成新载体PCB1，然后将xHFY NP基因克隆至该载体，通过重组质粒转染和病毒感染，检测其在哺乳动物细胞(COS—7和vero)及昆虫细胞中的表达。结果 连接至PCB1的xHFV NP基因均能在相应的细胞中获得良好表达；以重组杆状病毒感染的vero细胞可以作为抗原检测xHF血清，与ELISA的检测结果完全一致，并与临床诊断有很好的平行性。结论 新型杆状病毒载体能够驱动外源基因在昆虫和哺乳动物细胞中高效表达，不仅能方便快速地制备诊断抗原，还具有发展重组病毒疫苗和基因治疗的潜力。     

Sequence analysis of the hemagglutinin gene of measles virus isolates in Denmark 1997-1998: no evidence of persistent circulation of measles virus in Denmark

The hemagglutinin-coding region of 17 virus samples from 12 measles cases in Denmark during 1997-1998 was analysed by partial nucleotide sequencing. The cases appeared as three sporadic cases and two epidemics, both with a limited time course and geographical distribution. The measles strains identified from the three sporadic cases and two epidemics could be allocated to five different previously well-defined sequence groups consistent with the assumption that cases of measles in Denmark are due to repeated introduction from abroad rather than persistent circulation of strains in the population.     

The nucleocapsid protein of measles virus blocks host interferon response

Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-α/β and γ-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.     

2009-2013年菏泽市曹县麻疹病毒感染特征分析

目的 分析曹县麻疹病例实验室检测结果、流行病学和临床特点,为当地预防控制麻疹提供指导.方法 从国家疾病监测信息报告管理系统收集2009-2013年曹县麻疹疫情数据,以及麻疹疑似病例,开展麻疹IgM、风疹IgM检测和分析麻疹病毒分离情况、病例特点.结果 2009-2013年度共报告227例麻疹疑似病例,血清标本采集率72.69％,麻疹IgM阳性率为18.71％,12例咽拭子标本中分离出5株麻疹病毒,为H1a基因型.确诊168例麻疹病例,＜4岁年龄组的构成比为80.95％,幼托儿童、散居儿童是麻疹发病的主要人群,占89.29％;主要临床表现包括发热,超过38.5℃者占86.63％,皮疹首发部位为面部,占72.95％,有口腔黏膜斑者占72.80％,麻疹并发症前三位分别为肺炎、心肌炎、喉炎.结论 经血清学和病毒学证实,曹县当地存在麻疹病毒传播,病例以散发为主,麻疹野病毒为H1基因型,麻疹发病与年龄、性别、职业、季节有关.     

An internal element of the measles virus antigenome promoter modulates replication efficiency

The cis-acting sequence elements that direct measles virus (MV) genome synthesis reside in the 109 base non-coding region at the 5′ trailer (3′ antigenomic) end of MV genome that makes up the antigenomic promoter (AGP). The MV-AGP nucleotides 79–96, corresponding to nucleotide hexamers 14, 15 and 16 (the C′ element), show sequence similarity with the equivalent region of many paramyxoviruses and are analogous to the three nucleotide hexamers that form the second replication control element in the Sendai virus AGP. In this study, results of two independent procedures demonstrate that the MV C′ element also is a replication control sequence. Results of in vivo nucleotide selection experiments show that selection pressure for retaining the wild type nucleotides at the first position of each of the three hexamers, and for the fifth position of the 14th hexamer was relatively high. However, with continued replication, preference for the conservation of wild type nucleotides across the entire C′ element was clearly evident. Results of mutational analysis of individual nucleotides in one or more hexamers in a measles-helper-virus driven reporter gene rescue system agreed with these results. Substitutions at the first position of the 14th, the 15th or the 16th hexamers reduced minireplicon activity dramatically. In contrast, changes at the other five positions of any one hexamer had little or no effect on minireplicon activity, even when all the five bases were changed at the same time. However, when minireplicons were analyzed which contained point mutations at equivalent positions in all three hexamers, it was evident that the nucleotides, particularly those at the 5th position, were also important components of the C′ element. This pattern of sequence requirement in the C′ element based on mutational analysis could be described as a distinct motif, 5′-(GNNNAN)₂GNNNCN-3′, that is important for MV replication.