黄瓜绿斑驳花叶病毒湖南邵阳株系基因组MP片段的测定及生物信息学分析

本文运用RT-PCR方法克隆了黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)湖南邵阳株系的基因组MP(CGMMV-HuNSY movement protein)片段,测序并进行了分析。结果显示,片段全长共有803bp(KC684977),编码由264个氨基酸组成的蛋白质,推测分子量约28.87kD,理论等电点pI为9.06,ProtParam预测显示为不稳定蛋白,与已报道的辽宁分离物病毒的MP作比较,核苷酸相似性为99.6%,氨基酸相似性为98.9%;湖南邵阳株系CGMMVMP蛋白无高度卷曲螺旋部位,有跨膜结构区域,该部位表现为疏水性,可能为蛋白互作位点;磷酸化位点均匀分布于整个多肽链中,存在5个主要的B细胞抗原表位预测位点;对烟草花叶病毒属病毒MP氨基酸序列进行了motif查找,发现了该属病毒氨基酸序列的3个保守区段,还进行了密码子偏向性分析。此外,发现了1个酰胺化位点和1个依赖于cAMP和cGMP的蛋白激酶磷酸化位点,可能与病毒的侵染机制有关。     

蓝舌病毒血清5型毒株S7基因编码区的分子克隆与序列分析

目的:对蓝舌病毒(BTV)血清5型毒株(BTV-5)的S7基因编码区(ORF)进行克隆和序列分析。方法:用TRIzol LS试剂提取病毒总RNA,经反转录-聚合酶链反应(RT-PCR)扩增BTV-5型毒株S7基因的编码区,将扩增片段克隆到pGEM-T Easy载体上,对阳性克隆进行核苷酸序列测定;采用DNAStar和DNASIS v2.5软件对环状病毒属不同种群的S7基因ORF序列及其推导的氨基酸序列进行同源性及系统进化树分析。结果:克隆的基因片段长1050bp,为S7基因开放性读码框的全长序列,编码349个氨基酸残基;与环状病毒属不同血清型毒株比较,核酸序列同源性范围为42.2%～96.6%,推导的氨基酸序列同源性范围为40.4%～99.7%。结论:蓝舌病毒与非洲马瘟病毒、鹿流行性出血热病毒分属于不同种群,群内不同血清型的S7基因ORF序列及其推导的氨基酸序列显示出很高的同源性,而不同种群之间的同源性很低。     

江苏水稻黑条矮缩病毒S10的cDNA克隆序列分析

从来自江苏连云港并在本实验室保存的水稻黑条矮缩病毒接种的病株玉米中提取dsRNA,采用改进的单引物扩增技术获得了病毒基因组片段S10的cDNA克隆并测定了其全序列.结果表明S10全长1 801bp,含有一个ORF,组织结构与日本报道的RBSDV基本一致,核苷酸和推导的氨基酸序列与MRDV的相似性分别为87.5%和92.6%,与RBSDV的相似性分别为93.3%和96.4%.该研究也为病毒dsRNA克隆和序列分析奠定了基础.     

水稻黑条矮缩病毒基因组片段S7的cDNA克隆及全序列分析

应用RTPCR技术克隆了2个水稻黑条矮缩病毒 (rice blackstreaked dwarf virus,RBSDV)中国分离物,即浙江分离物和河北分离物的基因组片段S7,并测定了他们的全序列。结果表明：RBSDV浙江分离物(RBSDVZj)基因组片段S7全长2193nts(EMBL登录号为AJ297427),RBSDV河北分离物基因组片段S7全长2190nts(EMBL登录号为AJ297428),二者均含有两个开放阅读框(open reading frame,ORF),分别编码约41kD和36kD多肽,2个中国分离物核苷酸同源性高达99%,相应的ORF编码的多肽同源性分别为100%和94.4%,与日本RBSDV基因组片段S7核苷酸同源性为93.4%和93.8%,相应ORF编码的多肽同源性分别为98.1%(ORF1)、96.5%和97.8%(ORF2),与意大利MRDV S6核苷酸同源性为85.1%和85.3%,相应多肽同源性分别为92.3%(ORF1)、85.5%和86.8%(ORF2)。     

中国西藏小反刍兽疫病毒China/Tib/Gei/07-30磷蛋白基因的分子特征及其编码蛋白特性分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行磷蛋白基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出病毒磷蛋白基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。小反刍兽疫病毒China/Tib/Gej/07-30磷蛋白基因由1 655个核苷酸组成,编码2个相互交叠的开放阅读框(ORF)。第一个ORF长度为1 530个核苷酸,编码的P蛋白长度为509个氨基酸。第二个ORF长度为534个核苷酸,编码的C蛋白长度为177个氨基酸。第一个ORF通过基因编辑在751位插入1个G核苷酸,转录生成第二个mRNA,长度为897个核苷酸,编码的V蛋白长度为298个氨基酸。小反刍兽疫病毒China/Tib/Gej/07-30的P蛋白与其他分离株氨基酸序列同源性为86.1%～97.3%,C蛋白氨基酸序列相似性为84.3%～94.9%,V蛋白为82.9%～96.3%。China/Tib/Gej/07-30的P蛋白第315～387位氨基酸是一段高度保守的七肽重复序列。     

江苏水稻黑条矮缩病毒S10的cDNA克隆序列分析

从来自江苏连云港并在本实验室保存的水稻黑条矮缩病毒接种的病株玉米中提取dsRNA ,采用改进的单引物扩增技术获得了病毒基因组片段S10的cDNA克隆并测定了其全序列。结果表明S10全长 180 1bp ,含有一个ORF ,组织结构与日本报道的RBSDV基本一致 ,核苷酸和推导的氨基酸序列与MRDV的相似性分别为 87.5 %和92 .6 % ,与RBSDV的相似性分别为 93.3%和 96 .4%。该研究也为病毒dsRNA克隆和序列分析奠定了基础     

鲤春血症病毒中国分离株糖蛋白基因和氨基酸序列的初步解析

2002～2004年间从国内养殖鲤科鱼类和观赏鱼类中分离出8株鲤春血症病毒(SVCV).根据SVCV参考株全序列,设计引物,用逆转录聚合酶链式反应扩增出8株SVCV糖蛋白编码基因片段,并对扩增产物进行了克隆和序列测定.用生物信息学方法对测得的序列进行分析,结果8个国内分离株的糖蛋白基因序列与参考株的基因序列相似性均在92%以上,8个国内分离株之间基因序列相似性均在97.7%以上;8个国内分离株之间糖蛋白推导出的氨基酸序列相似性均在94.5%以上,与参考株氨基酸序列相似性在92.9%-94.9%之间.系统发育树分析结果表明,SVCV国内分离株与USA株、980451株、980528株和970469株的进化方向一致,与其它SVCV毒株在进化方向上不同.8个毒株有19个共同的酶切位点,推导出的氨基酸序列中有10个亲水区、10个可能的抗原位点和10个跨膜蛋白区域,其峰值基本一致.对SVCV国内分离株的糖蛋白6个功能位点(天冬酰胺糖基化位点、精氨酸-甘氨酸-天冬氨酸序列、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、酪氨酸磷酸化位点和肉豆蔻酰基化位点)进行了初步分析.     

苏云金芽胞杆菌拟步行甲亚种YBT-1765中稳定质粒pBMB175的克隆和功能分析

从苏云金芽胞杆菌拟步行甲亚种YBT_1765中克隆得到一个大小约15.2kb的质粒pBMB175,构建了该质粒的限制性图谱,通过功能验证,将其最小的复制区定位在一个1151bp的片段上。分析了包含有这个复制区的一个大小为4152bp的核苷酸序列,该片段包含有3个编码框(ORF1、OFR2和ORF3)。氨基酸序列同源性比较发现,ORF1(767AA)与UvrD_旋促酶、重组酶RecD和RecB家族具有20%～30%的相似性;ORF2(149AA)没有发现与任何已知序列具有同源性;ORF3(83AA)与pGI3中一个未知功能的蛋白(ORF7)具有34%的相似性。通过缺失及序列比较分析推测ORF2可能编码一种新的复制蛋白。因此pBMB175的复制类型可能属于一类新的复制家族。利用最小复制区构建的重组质粒在无抗生素选择压力下可稳定遗传40多代,具备构建稳定遗传质粒载体的潜力。     

马尾松毛虫质型多角体病毒S4的cDNA克隆及序列分析

从马尾松毛虫质型多角体病毒湖南株中提取病毒核酸,回收、纯化第四片段s4,经RT-PCR扩增得到了S4的cDNA克隆并测定了其全序列.结果表明,S4全长由3 262个碱基组成,包含一个编码1058个氨基酸的完整开放阅读框架.Blast同源分析显示,DpCPV S4与LdCPV S4、BmCPV S4和RRSV S2编码蛋白氨基酸的同源性分别为94%、92%和22%.另外,氨基酸序列部分区域与Methanosarcina mazei Goel的甲基化转移酶有同源性,且序列中含有鸟苷酸转移酶活性位点.     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析。结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%。RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定。利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp prote-ase proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性。     

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.     

玉米粗缩病毒基因组第七组份的cDNA克隆及序列分析

从采自中国河北滦城感病的玉米材料中提取玉米粗缩病毒(MRDV)的双链RNA。根据已知MRDV的部分序列设计引物,反转录、PCR扩增,克隆并测序分析了MRDV的第七片段(S7)cDNA序列。结果表明,S7 cDNA序列全长为1936bp,与国外所测的MRDV S7的序列长度相等,而且S7包含的两个阅读框(ORF1和ORF2)位置无变化。它们的核苷酸和最大开放阅读框(ORF1)同源性分别为877%和91.6%,然而,MRDV S7的片段与水稻黑条矮缩病毒(RBSDV)S8片段的核苷酸和最大开放阅读框(ORF1)有更高的同源性,分别为95.5%和93.5%。     

Sequence analysis of segment 8 of five Chinese isolates of Rice gall dwarf virus and expression of a main outer capsid protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8 (S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%–98.8% and 97.3%–99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%–95.6% and 95.0%–96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta (DE3) II cells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression. Foundation item: National natural science foundation of China (30370929) and Guangdong province natural science foundation (C036845)     

汉坦病毒HTN261株S基因片段的核苷酸序列测定及分析

黑龙江省是肾综合征出血热(HFRS)的重疫区。近年来HFRS年的发病人数曾超过万人。流行病学和血清学研究表明黑龙江省HFRS疫区主要是姬鼠型,但目前尚缺乏病毒的分子生物学资料。我们对从疫区捕获的宿主动物-黑线姬鼠肺中分离的汉坦病毒HTN261株的S基因片段的全基因序列进行了测定和初步分析。结果如下,HTN261株的S基因片段的全序列长为1697nt。只有一个主要的编码N蛋白的ORF,起始位置为第37nt,终止于1326nt,编码的蛋白长为429aa。没有发现存在ORF2。HTN261株的S基因片段核苷酸序列与HTN型中的病毒株的同源性很高,而与汉坦病毒其他型的同源性较差。从种系发生树分析来看,HTN261株归结于汉坦病毒的HTN型。在HTN型之内,HTN261株和HTN76-118株在一个分枝内。就其核苷酸和蛋白的同源性来说,HTN261株和HTN76-118株的同源性分别是89%(全S基因)和98%(蛋白)。而与中国境内发现的其他汉坦病毒株Z10,HU,Chen4,NC167等基因和蛋白的同源性相对较差。汉坦病毒除具有其宿主的依赖性外,还具有其地理的簇集性。HTN261株和HTN76-118株之间S基因和N蛋白序列的变异性的差异分别为11%和2%,表明HTN261株和HTN76-118株还有不同,可能是不同的亚型。不过,尚有待于进一步研究证明。     

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.     

汉坦病毒HTN261株S基因片段的核苷酸序列测定及分析

黑龙江省是肾综合征因热（HFRS）的重疫区。近年来HFRS年的发病人数曾超过万人。流行病学和血清学研究表明黑龙江省HFRS疫区主要是姬鼠型,但目前尚缺乏病毒的分子生物学资料。我们对从疫区捕获的宿主动物－黑线姬鼠肺中分离的汉坦病毒HTN261株的S基因片段的全基因序列进测定和初步分析。结果如下,HTN261株的S基因片段的全序列长为1697nt。只有一个主要的编码N蛋白的ORF,起始位置为第37nt,终止于1326nt,编码的蛋白长为429aa。没有发现存在ORF2。HTN261株的S基因片段核苷酸序列与HGTN型中的病毒株的同源性很高,而与汉坦病毒其他型的同源民生较差,从种系发生树分析来看,HNT261株归结于汉坦病毒的HTN型。在HTN型之内,HTN261株和HTN76－118株在一个分枝内,就其核苷酸和蛋白的同源性说,HT N261株和HTN76－118株的同源性分别是89％（全S基因）和98％（蛋白）。而与中国境内发现的其他汉坦病毒株Z10,HU,Chen4,NC167等基因和蛋白的同源性相对较差,汉坦病毒除具有其宿主的依赖性外,还具有其地理的簇集性。HTN261株和HTN76－118株之间S基因和N蛋白序列的变异性的差异分别为11％和2％,表明HTN261株和HTN76－118株还有不同,可能是不同的亚型。不过,尚有待于进一步研究证明。     

Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA.

Aquatic birnaviruses, such as infectious pancreatic necrosis virus (IPNV), cause serious diseases in a variety of fish species used worldwide in aquaculture and have also been isolated from a variety of healthy fish and shellfish species. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. To better understand genetic and biological diversity among these viruses, the nucleotide and deduced amino acid sequences were determined from cDNA of the large open reading frame (ORF) of genome segment A of the 9 type strains of Serogroup A and 4 other representative strains of Serotype A1, the predominant serotype in the United States. In addition, nucleotide and deduced amino acid sequences were determined for the VP2 coding region of a variety of isolates representing 5 of the 9 serotypes. VP2 is the major outer capsid protein of aquatic birnaviruses. RT-PCR was used to amplify a 2904 bp cDNA fragment including all but a few bp of the large ORF of genome segment A or a 1611 bp fragment representing the entire VP2 coding region. Nucleotide and deduced amino acid sequences were determined from the PCR products. Pairwise comparisons were made among our data and 2 other aquatic birnavirus sequences previously published. Several hypervariable regions were identified within the large ORF. The most divergent pair of viruses exhibited a similarity of 80.1% in the deduced amino acid sequence encoded by the large ORF. Genomic relationships revealed in a phylogenetic tree constructed from comparison of the deduced amino acid sequences of the large ORF demonstrated that these viruses were clustered into several genogroups. Phylogenetic comparison of the deduced amino acid sequences of the VP2 coding region of 28 aquatic birnavirus isolates, including the type strains of all 9 serotypes, demonstrated 6 genogroups, some of which were comprised of several genotypes. The most divergent pair of viruses exhibited a similarity of 81.2% in the deduced amino acid sequence from the VP2 coding region. In contrast to previous studies of much shorter genomic sequences within the C-terminus-pVP2/NS junction coding region, these genogroups based on the entire large ORF or the VP2 coding region generally correlated with geographical origin and serological classification. Isolates from the major Canadian serotypes were more closely related to the European isolates than to isolates from the United States.