新疆乌鲁木齐HIV—1流行毒株膜蛋白基因C2—V3区序列测定和亚型分析

应用ＰＣＲ方法对７份１９９６年３－５月采集于新疆乌鲁木齐ＨＩＶ－１阳性静脉吸毒者的外周血单核细胞样品进行扩增,获得了ＨＩＶ－１膜蛋白基因的核酸片段,并对其Ｃ２－Ｖ３区及邻区３５０－４５０个核苷酸序列进行了测定和分析。     

猪瘟病毒石门株NS2—3基因片段的序列测定及比较

根据猪瘟病毒Ｃ株的序列,以计算机辅助设计,化学合成１对引物（ＰＦ５６４８／ＰＲ６６０４）,应用ＲＴＰＣＲ技术从感染猪血中成功地扩增了我国猪瘟病毒强毒石门株ＮＳ２３基因片段,大小为９５７ｂｐ,位于ＮＳ３基因的中部ＮＴＰａｓｅ和Ｈｅｌｉｃａｓｅ活性区。克隆后测序,结果表明该段基因产物具有解旋酶超家族全部七个特征性保守序列,包括共同的ＮＴＰ结合基序Ａ位点（ＧＸＧＫＴ／Ｓ）和Ｂ位点（３ｈｙ,２ｘ）Ｄ。序列同源性比较表明,石门株与日本的ＡＬＤ和ＧＰＥ－株同源性最高,与其它３株猪瘟病毒（Ｃ株、Ｂｒｅｓｃｉａ株和Ａｌｆｏｒｔ株）的同源性也很高,并与２株牛病毒性腹泻病毒（ＢＶＤＶ）（ＮＡＤＬ株和ＳＤ１株）也有较高的同源性,尤其是由核苷酸序列推导的氨基酸序列,同源性均大于９０％,是瘟病毒属基因组中最保守的区段,这与该基因产物在病毒复制及聚蛋白前体加工过程中所具有的重要功能是一致的     

我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .     

HIV—1长末端重复序列对重组痘苗病毒表达Gag蛋白的影响

将系列缺失的ＨＩＶ１长末端重复序列（ＬＴＲ）和全长的ｇａｇＯＲＦ置于痘苗病毒载体中,经同源重组和血球吸附试验,成功地构建了６株重组痘苗病毒。免疫印迹和免疫酶试验检测均表明,６株重组病毒的Ｇａｇ蛋白表达量因ＬＴＲ不同而有明显差异,表明ＨＩＶ１的ＬＴＲ及其下游基因置于痘病毒启动子控制下,在痘苗病毒中表达时有下述特点：（１）不同的痘苗病毒启动子与全长ＬＴＲ相互作用,对ｇａｇ基因表达有显著不同的调控效果;（２）ＮＲ序列对Ｇａｇ蛋白表达没有明显影响;（３）ＥＮ序列不能被重组痘苗病毒表达系统识别;（４）ＴＡＲ序列可提高Ｇａｇ蛋白的表达量;（５）Ｕ５区及下游非翻译序列不影响Ｇａｇ蛋白的表达。     

我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

 为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .     

江苏省2013—2014年3株柯萨奇病毒B组5型流行株全基因组序列分析

目的分析引起江苏省疱疹性咽峡炎的柯萨奇病毒B组5型(coxsackievirus B5, CV-B5)毒株基因组特征。方法对江苏省2013—2014年3例疱疹性咽峡炎患儿临床标本进行病原分离,获得3株CV-B5毒株,进行全基因组序列测定,与GenBank中世界各地不同年份CV-B5流行株序列进行比对分析,使用Mega 5.2软件构建系统进化树,并应用Simplot软件进行重组分析。结果获得3株CV-B5毒株,分别命名为417/JS/CHN/2013、492/JS/CHN/2013和759/JS/CHN/2014,均属于GI.D3亚型,且在位点5 224～5 696 bp(以Faulkner为参考株)与2008年北京市的CV-B3流行株(GQ141875)发生重组,在位点5 696 bp之后与2009年昆明市的CV-B3流行株(JX843810)发生重组。结论 CV-B5在婴幼儿人群中的感染可能存在严重危害,应加强对CV-B5的分子流行病学研究,监测可能发生的重组。     

鸡传染性支气管炎病毒(IBV)广西流行株3'端非编码区序列分析

本研究应用RT-PCR方法扩增出分离自广西的26株鸡传染性支气管炎病毒(IBV)以及参考株M41和疫苗株H120、Ma5和4/91的3'端非编码区(3'UTR)的cDNA,并对其进行了克隆、序列测定、比较及其系统进化分析。序列分析结果表明,与Beaudette株的3'UTR序列相比较,26株分离的IBV中有1株和3株的分离株3'UTR序列分别插入了23个和2个碱基,另外有12株、6株、1株、2株和1株的分离株3'UTR序列分别缺失了1个、2个、22个、29个和84个碱基,不同毒株之间碱基数量最大相差达107个碱基。系统进化分析结果表明,26株分离株可分为4群,其中21株属于第Ⅰ群,与经典疫苗株H120的核苷酸同源性均较低(54.4%～75.5%);3株与4/91同属于第Ⅱ群;1株与H120同属于第Ⅲ群;另外1株单独属于Ⅴ群。综上所述,广西流行IBV的绝大部分毒株在3'UTR序列上与目前常用的疫苗株相比都发生了很大的变异,而且存在多种形式的碱基缺失和插入现象。由此,我们认为流行株的变异可能是疫苗不能提供有效保护的主要原因。     

河南HIV感染者的HIV-1B型株tat基因的克隆及序列分析

克隆河南人免疫缺陷病毒(HIV)感染HIV-1B型株tat基因完整编码框序列,并分析比较其编码产物的序列结构特点。使用重叠PCR技术,从河南省1名HIV-1感染外周血标本中扩增出to／基因第一和第二外显子并重组为完整的tat基因序列。获得的HIV-1B病毒株tat基因,第一外显子为263bp,第二外显子为214bp。将该基因编码产物与其他HW-1株Tat蛋白经DNA软件编辑并翻译成蛋白质,使用Clustal X1．81进行多序列对比分析发现,第一外显子编码产物的3个保守区域的氨基酸组成大致相同,只有少数氨基酸存在差异。由于Tat蛋白不同病毒株间有高度保守的Cys富集区、核心区和碱性氨基酸富集区,tat基因的克隆为研究其功能并以其为靶点设计和筛选抗艾滋病的药物奠定了基础。     

汉滩病毒A9株强毒和弱毒克隆M基因片段G1糖蛋白编码区核苷酸序列的分析比较

采用逆转录－ＰＣＲ方法,用３对引物分３个片段扩增强毒克隆（Ａ９ｖ）及弱毒克隆（Ａ３％）的Ｍ基因片段ｃＤＮＡ,并克隆入ｐＧＥＭ－Ｔ载体中。采用Ｓａｎｇｅｒ双脱氧法测定了Ａ３９ｓ、Ａ９ｖ病毒Ｇ１糖蛋白编码区的全序列,发现二者均由１９７８个核苷酸组成,比７６／１１８株少６个核苷酸,并发现Ａ３９ｓ在Ｇ１编码区有３３个碱基及８个氨基酸与Ａ９ｖ不同。进一步与７６／１１８、ＨＶ１１４的相关序列比较,发现Ｇ１编码区的第６０６、６１４位氨基酸的变异同Ａ３９ｓ病毒的减毒有一定相关。从Ａ３９ｓ、Ａ９ｖ、７６／１１８及ＨＶ１１４４株病毒Ｇ１糖蛋白编码区推导的氨基酸序列亲疏水性资料显示,在位于Ｇ１糖蛋白Ｃ末端的最大亲水区域的６００～６２０位氨基酸区域,弱毒株Ａ３９ｓ同强毒株Ａ９ｖ、７６／１１８、ＨＶ１１４的亲水性有明显不同,而这一差异又主要由第６１４位氨基酸的变异所引起。因此推测,Ｇ１糖蛋白Ｃ端亲水区域同汉滩病毒毒力相关,而第６１４位的精氨酸被谷氨酰胺取代,同Ａ３９ｓ病毒毒力的减弱可能有关。     

Relationship of HIV-1 Envelope V2 and V3 Sequences of the Primary Isolates to the Viral Phenotype

We examined the relationship between the amino acid sequences of the V2 and V3 regions of the envelope protein and the biological properties of ten human immunodeficiency virus type 1 (HIV-1) primary isolates. The infectivity, cytopathic effect (CPE), and syncytium forming activity of these primary isolates were tested against three T cell lines (CEM, MT2, and MOLT4/CL.8 cells), CD8-depleted peripheral blood mononuclear cells (PBMC), and primary monocyte-derived macrophages (MDM) from seronegative donors. In addition to the viral groups which had the syncytium inducing/T-cell line tropic (SI/TT) phenotype or non-syncytium inducing/non-T cell line tropic (NSI/NT) phenotype (including the NSI/macrophage tropic (NSI/MT) phenotype), there was a group of viruses that infected one or two T cell lines and PBMC but could not mediate syncytium formation. We therefore classified this group of viruses as a non-syncytium inducing/partial T-cell line tropic (NSI/pTT) virus. To investigate the relationship between these viral phenotypes and the sequence variability of the V2 and V3 regions of the envelope, we cloned the viral gene segment and sequenced the individual isolates. The sequence data suggested that the SI/TT type changes in the V3 sequence alone mediate a partial T cell line tropism and mild cytopathic effect and that an isolate became more virulent (SI/TT phenotype) if there were additional changes in the V2 or other regions. On the other hand, sequence changes in the V2 region alone could not mediate phenotypic changes but some additional changes in the other variable regions (for example, V3) might be required for the phenotypic changes in combination with changes in V2. These findings also suggested that amino acid changes in both the V2 and V3 region are required for the development of virulent variants of HIV-1 that outgrow during advanced stages of the disease.     

Study on conformational homology of the HIV-1 gp120 protein V3 loop. Structural analysis of the HIV-RF and HIV-thailand viral strains

Based on NMR spectroscopy data, conformation of the HIV-RF gp120 protein V3 loop giving rise to the virus principal neutralizing determinant and also determinants of cell tropism and syncytium formation was calculated by computer modeling approaches. Elements of the HIV-RF V3 loop secondary structure and conformational states of its irregular stretches were determined. The calculated structure was compared with the conformation of the homologous stretch of the HIV-Thailand protein gp120 V3 loop, and structural elements preserved in the two viral strains were identified. Conservative elements of the HIV-1 V3 loop structure are considered to be promising targets for deriving chemically modified forms of this loop with the enhanced immunogenicity and cross-reactivity of neutralizing antibodies and also for creation of effective antiviral drugs on this base.     

Determination of structurally conservative amino acids of the HIV-1 protein gp120 V3 loop as promising targets for drug design by protein engineering approaches

Based on the published NMR spectroscopy data, three-dimensional structures of the HIV-1 gp120 protein V3 loop were obtained by computer modeling in the viral strains HIV-Haiti and HIV-MN. In both cases, the secondary structure elements and conformations of irregular stretches were determined for the fragment representing the principal antigenic determinant of the virus, as well as determinants of the cellular tropism and syncytium formation. Notwithstanding the high variability of the amino acid sequence of gp120 protein, more than 50% of the V3 loop residues retained their conformations in the different HIV-1 virions. The combined analysis of the findings and the literature data on the biological activity of the individual residues of the HIV-1 V3 loop resulted in identification of its structurally conservative amino acids, which seem to be promising targets for antiviral drug design by protein engineering approaches.     

EB病毒LMP1-CTAR3对NP69细胞增殖和蛋白质表达的影响

为了探讨EB病毒潜伏性膜蛋白 1(LMP1)第三个功能活性区域(CTAR₃)在鼻咽上皮细胞NP69中的转化作用机制,采用逆病毒感染的方法,将浓缩的逆病毒RV-LMP1和RV-LMP1^Δ232～351分别感染鼻咽上皮细胞NP69,建立NP69-LMP1与NP69-LMP1^Δ232～351稳定表达细胞系．通过绘制生长曲线、平皿克隆形成试验和软琼脂集落形成试验比较野生型和突变型LMP1对NP69细胞增殖的影响,运用蛋白质组学方法鉴定NP69-LMP1与NP69-LMP1^Δ232～351细胞间的差异表达蛋白,选用实时荧光定量RT-PCR与Western blot对其中部分蛋白质点差异表达进行验证．结果发现：a．突变型LMP1^Δ232～351促NP69细胞增殖的能力较野生型LMP1明显降低(n=3,P < 0.05);b．鉴定了LMP1-CTAR₃在NP69细胞中参与调节的16个蛋白质(表达上调的蛋白质8个,下调的8个)．c．实时荧光定量RT-PCR和Western blot证实了部分上述蛋白质的差异表达．以上结果说明,LMP1-CTAR₃是其发挥促细胞增殖的重要活性部位,可能通过参与调节G蛋白和异柠檬酸脱氢酶等蛋白质的表达而起作用．     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain Comparison of predicted amino acid sequences from G-protein α-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human G_i2α gene and rat G_i2α cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat G_i1α and G_oα cDNAs, respectively.     

Applications of biotinylated V3 loop peptides of human immunodeficiency virus type 1 to flow cytometric analyses and affinity chromatographic techniques

A principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) lies within the V3 loop of gp120, the external major envelope glycoprotein. V3 loop peptides derived from two HIV-1 strains, HTLV-III BH-10 (V3-BH10) and LAV_ELI (V3-ELI), were synthesized and biotinylated. The binding of both biotinylated V3-BH10 and V3-ELI to the surfaces of MOLT-4 clone 8 cells was demonstrated by flow cytometric analyses. Both the peptides (more than 2 μM) bound to the cells (2 · 10⁵) in a dose-dependent manner. The binding of biotinylated V3-BH10 was specifically inhibited by a neutralizing monoclonal antibody (0.5β). The binding of both of the biotinylated V3 loop peptides was enhanced by the addition of unlabeled V3-BH10. In addition, the peptides were employed as ligands on affinity columns. A major V3 loop binding protein (V3BP) was purified from the membrane soluble fraction of MOLT-4 cells by successive application to two different V3 loop columns. V3BP consisted of two major polypeptides (32 and 33 kDa). The SDS-PAGE profile of V3BP did not change under non-reducing conditions, but only a single band was observed after analysis on native PAGE. The major peak of the eluate as determined by size exclusion chromatography was abroad and the estimated relative molecular mass was much larger than 33 kDa, suggesting that V3BP comprises several subunits. Taken together, we confirmed that the V3 loop peptides are useful in the characterization of V3BP(s) of which they are conformational ligands.     

Amino acid sequence of the Homo sapiens brain 21-23-kDa protein (Neuropolypeptide h3), comparison with its counterparts from Rattus norvegicus and Bos taurus species,and expression of its mRNA in different tissues

The amino acid sequence of neuropolypeptide h3 from Homo sapiens brain has been determined. It revealed that h3 is the exact counterpart of the 21-kDa protein from Bos taurus brain and the 23-kDa protein from Rattus norvegicus brain: The three proteins belong to the same 21-23-kDa protein family. Multiple tissue Northern blots showed that the mRNA encoding the 21-23-kDa protein is expressed in different amounts according to tissues and species; it is particularly abundant in Rattus norvegicus testis.Correspondence to: F. Schoentgen