鹅副粘病毒WF00 G分离株HN蛋白基因的测序与序列分析

用鹅副粘病毒凤阳分离株WF00G做9～10日龄SPF鸡胚的尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF00G病毒的HN基因,获得了1条约1．8kb的特异性条带。PCR产物回收纯化后测序。测序结果表明,扩增片段大小为1844bp,含有1个1716bp的开放性阅渎框,编码571个氨基酸。核苷酸同源性分析表明：WF00G株与其他12株鹅副牯病毒的同源性为89．9％-99．2％,与国内外其他NDVHN基因的同源性为81．7％～95．7％,其中与国内标准强毒株F48E9的同源性为84．7％,说明WF00G与国内外的传统毒株有较大变异。与Taiwan95株和NL／96株的同源性为94．3％和95．7％,说明WF00G与Taiwan95株和NL／96株亲缘关系较近,具有较高的相似性。     

鹅副粘病毒WF（01）G株F基因的测序与蛋白特性分析

用鹅副粘病毒WF01G分离株进行9-10日龄SPF鸡胚尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF01G病毒的F基因,获得了1条长约1.7 kb的特异性条带。对PCR扩增产物测序。结果表明,扩增片段大小为1782 bp,含有1个1662 bp的开放性阅读框,编码554个氨基酸。核苷酸同源性分析表明：WF01G株与其他7株鹅副粘病毒的同源性为84.8%-98.8%,与国内外其他NDV F基因的同源性为84.7%-93.8%,其中与国内标准强毒株F48E9的同源性为86.8%,说明WF01G与国内外的传统毒株有较大变异。与Tai-wan95株的同源性为93.8%,说明WF01G与Taiwan95株亲缘关系较近,具有较高的相似性。F蛋白裂解位点的氨基酸顺序为112Arg-Arg-G ln-Lys-Arg-Phe117,表明为副粘病毒强毒株。蛋白疏水性和抗原性分析表明与标准强毒F48E9株相比没有太大的变异。     

大熊猫犬瘟热病毒附着或血凝蛋白基因的序列分析

首次对犬瘟热病毒(CDV)大熊猫(GP)毒株附着或血凝蛋白(H)基因进行了序列测定并与疫苗株Onderstepoort进行了比较.我们设计合成了4对引物,对GP株进行了RT-PCR扩增与测序.H蛋白基因全长为1 946 bp,开放阅读框架(ORF)始于21-23位的ATG,终止于1 842-1 844位的TGA,编码607个氨基酸,该基因序列已被GenBank收录.将GP毒株与GenBank中疫苗弱毒株Onderstepoort进行比较,二者核苷酸序列的同源性为91.4%,推导的氨基酸序列的同源性为90.2%,GP和Onderstepoort株H蛋白的半胱氨酸残基数目均为12个且相对位置不变;疏水性有一定的变化,但推测的穿膜区位置(约35-55位氨基酸)是一致的;Onderstepoort株的H蛋白潜在的N-联糖基化位点为4个,GP株H蛋白为9个,糖基化位点的不同可能对GP株H蛋白的抗原性产生影响.     

鸡传染性支气管炎病毒中国分离株LX4纤突蛋白基因分子特征

应用RT-PCR方法分别扩增了中国地方分离株IBV LX4 S1和S2基因并进行了基因的克隆和序列测定.结果发现,LX4 S基因由3495个核苷酸组成,编码一条1164个氨基酸残基组成的多肽.S基因编码产物裂解后形成的S1和S2亚单位分别由539和625个氨基酸残基组成.LX4 S基因推导氨基酸切割识别位点序列为HRRRR,与A2、SD/97/02和Z株相同,而与其它国内外参考毒株不同.与国内外10株已报道的具有全S基因序列的IBV参考毒株比较,LX4与国内分离毒株SD/97/02的核苷酸和氨基酸同源性最高.与32株国内外参考毒株的S1基因进化树分析比较表明,LX4与A2、SD/97/02、Z、TJ/96/02、JX/99/01和SAIBWJ等7个国内分离株在同一亚群内.在该亚群内,LX4与A2和SD/97/02亲缘关系更近,且三者在高变区和抗原表位均具有高度的同源性,而与本亚群内其它参考毒株对应的高变区和抗原表位同源性差异较大.LX4与H120 S1基因编码氨基酸的同源性虽然高于其它国外参考毒株,但同源性仍然较低,为75%.与参考毒株比较,LX4 S2基因的点突变造成其推导的氨基酸序列有11个位点发生改变,这些突变可能影响S2与S1蛋白之间的相互作用,从而影响S蛋白与特异性抗体的结合.     

口蹄疫病毒O/NY00株基因组L片段的克隆及其基因特征

采用RT—PCR方法对口蹄疫病毒O／NY00株基因组L片段进行了分子克隆和序列测定。结果表明：获得的O／NY00株基因组L片段长7805nt,其中包括715nt的5’非编码区和6999nt的多聚蛋白编码区。将O／NY00株与其它参考毒株进行序列比较,结果显示：O／NY00株与O／SKR／2000、O／1JKG／3／2001遗传关系最近,而与其它毒株遗传关系较远,属于0型口蹄疫ME-SA拓扑型Pan／ksia株的成员。与Cathay拓扑型的代表毒株O／TAW／97比较,两者在主要抗原位点1和位点3呈现出明显的以拓扑型为特征的氨基酸差别,提示两拓扑型毒株之间有较高的抗原差异。     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCVDXMV)纤突蛋白基因进行了克隆和序列测定。该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白。与GenBank中已发表的11个CCV毒株s基因相比,s基因核苷酸序列同源性在40．2％-99．5％之间;推导的氨基酸序列同源性在15．9％-99．O％之间。DXMV株s基因变异区主要集中在该基因前1／2处,其中350．370、439．478、1718．1818三个区域碱基变异较大,而1060．1700区却十分保守。基于s全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和USpatent株亲源关系最近。推导的DXMV株s蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc．1弱毒多一个;其中第566．568位糖基化位点为多数强毒拥有而弱毒没有的。另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究。     

我国四株狂犬病毒糖蛋白基因序列分析和位点比较

测定我国人用狂犬疫苗株(aG)、减毒株(CTN-181)及两株街毒 糖蛋白(GP)基因cDNA的核苷酸序列及推导氨基酸序列。结果两株街毒仅相差两个碱基和一个氨基酸残基;两株街毒与CTN的同源性(85.9%)高于aG株(81.9%);聚类分析将街毒和固定毒分为两支。比较GP嗜神经位点的氨基酸序列与蛇神经毒素同AChR结合部位高度同源。被认为决定毒力的333位Arg,CTN发生了Q替换,其它毒株均为Arg333。所比较的毒株均存在319位糖基化位点,此外37位糖基化位点也相对保守;GP两个主要抗原表位,GⅡ的氨基酸构成完全一致,GⅢ只在一些减毒株中发生与毒力密切相关碱基的替换。     

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。     

我国四株狂犬病毒糖蛋白基因序列分析和位点比较

测定我国人用狂犬疫苗株 (aG)、减毒株 (CTN 181)及两株街毒糖蛋白 (GP)基因cDNA的核苷酸序列及推导氨基酸序列。结果两株街毒仅相差两个碱基和一个氨基酸残基 ;两株街毒与CTN的同源性 (85.9% )高于aG株 (81.9% ) ;聚类分析将街毒和固定毒分为两支。比较GP嗜神经位点的氨基酸序列与蛇神经毒素同AChR结合部位高度同源。被认为决定毒力的 333位Arg ,CTN发生了Q替换 ,其它毒株均为Arg333。所比较的毒株均存在 319位糖基化位点 ,此外 37位糖基化位点也相对保守 ;GP两个主要抗原表位 ,GⅡ的氨基酸构成完全一致 ,GⅢ只在一些减毒株中发生与毒力密切相关碱基的替换。     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCV DXMV)纤突蛋白基因进行了克隆和序列测定.该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白.与GenBank中已发表的11个CCV毒株S基因相比,S基因核苷酸序列同源性在40.2%～99.5%之间;推导的氨基酸序列同源性在15.9%～99.0%之间.DXMV株S基因变异区主要集中在该基因前1/2处,其中350-370、439-478、1718-1818三个区域碱基变异较大,而1060-1700区却十分保守.基于S全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和US patent株亲源关系最近.推导的DXMV株S蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc-1弱毒多一个;其中第566-568位糖基化位点为多数强毒拥有而弱毒没有的.另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究.     

鸭副粘病毒WF01 D株F基因的测序与蛋白特性分析

用鸭副粘病毒凤阳分离株WF01 D作9～10日龄SPF鸡胚的尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF01 D病毒的F基因,获得了1条长约1．7kb的特异性条带。用PCR产物直接测序。测序结果表明,扩增片段大小为1782bp,含有1个1662bp的开放性阅读框,编码554个氨基酸。核苷酸同源性分析表明：WF01 D株与国内外其他NDVF基因的同源性为84．5％～97．0％,其中与国内标准强毒株F48E9的同源性为86．6％,说明WF01 D与国内外的传统毒株有较大变异。与Taiwan95株和J株的同源性为93．6％和97．0％,说明WF01 D与Taiwan95株和J株亲缘关系较近,具有较高的相似性。F蛋白裂解位点的氨基酸顺序为112Arg-Arg-Gln-Lys-Arg-Phe117,表明为NDV的强毒株。蛋白疏水性和抗原性分析表明与标准强毒F48E9株相比没有太大的变异。     

Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors

The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage.     

Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.     

Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor.

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.     

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.     

诺如病毒CHN02/LZ35666株RdRp和VP1基因序列分析

诺如病毒（Noroviruses,NVs）为杯状病毒科的一个属,是引起人类病毒性胃肠炎暴发的重要病原。在美国、欧洲和日本,病毒性胃肠炎暴发中由NV引起的占93％。NV的基因组为单股正链RNA,全长约7．7kb,由3个开放阅读框（open reading frames,ORFs）组成,ORF1编码非结构蛋白,其中包括RNA聚合酶（RNA dependent RNA polymerase,RdRp）,0RF2和0RF3分别编码主要（VP1）和次要（VP2）衣壳蛋白。VP1蛋白折叠成两个区域,壳区（Shell,S）和突出区（Protruding,P）,S区形成内壳,P区形成拱样结构突出于内壳外。P区进一步分为P1和P2亚区,后者位于衣壳的最外面,P2区相对于S区和P1区序列高度变异,被认为是免疫识别和受体结合的关键部位。     

Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist

Newcastle disease virus (NDV) edits its P gene by inserting one or two G residues at the conserved editing site (UUUUUCCC, genome sense) and transcribes the P mRNA (unedited), the V mRNA (with a +1 frameshift), and the W mRNA (with a +2 frameshift). All three proteins are amino coterminal but vary at their carboxyl terminus in length and amino acid composition. Little is known about the role of the V and W proteins in NDV replication and pathogenesis. We have constructed and recovered two recombinant viruses in which the expression of the V or both the V and W proteins has been abolished. Compared to the parental virus, the mutant viruses showed impaired growth in cell cultures, except in Vero cells. However, transient expression of the carboxyl-terminal portion of the V protein enhanced the growth of the mutant viruses. In embryonated chicken eggs, the parental virus grew to high titers in embryos of different gestational ages, whereas the mutant viruses showed an age-dependent phenomenon, growing to lower titer in more-developed embryos. An interferon (IFN) sensitivity assay showed that the parental virus was more resistant to the antiviral effect of IFN than the mutant viruses. Moreover, infection with the parental virus resulted in STAT1 protein degradation, but not with the mutant viruses. These findings indicate that the V protein of NDV possesses the ability to inhibit alpha IFN and that the IFN inhibitory function lies in the carboxyl-terminal domain. Pathogenicity studies showed that the V protein of NDV significantly contributes to the virus virulence.