中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关.淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原.在己完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF 011L的TS基因结构、及其推定蛋白结构进行了分析.该基因全长858bp,GC含量为28.2%,编码一个长为286aa、分子量为32.7kD、等电点为7.1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS).该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性.二级结构预测结果显示LCDV-C TS含8个α螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征.这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS.对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-CTS被单独分为一枝.进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨.     

淋巴囊肿病毒中国株TNFR类似物的原核表达与结构分析

肿瘤坏死因子受体(TNFR)是细胞因子受体家族中的一员,在大DNA病毒的免疫逃避中起着重要的作用。 淋巴囊肿病毒中国株(LCDV-C)是一种大DNA病毒,属于虹彩病毒科。参照已知虹彩病毒TNFR基因设计引物: P1,5′GGATCCAAAACTATGATTAAAATAAAGA 3′;P2:5′ATTACTCGAGAATGTTAAAAATTAAGCTT 3′。以LCDV-C基因组DNA为模板,PCR扩增得到一个834bp的DNA片段,并对该片段进行测序。构建原核表 达重组质粒后,在大肠杆菌DE3中诱导表达,其产物经SDS-PAGE电泳后,显示为45kDa的融合蛋白带。对测序 结果进行计算机辅助分析的结果显示,LCDV-C TNFR类似物是一个含278个氨基酸的多肽,具有典型的半胱氨 酸富集区功能结构域,与宿主牙鲆TNFRII氨基酸同源性为34％。     

淋巴囊肿病毒中国株TNFR类似物的原核表达与结构分析

肿瘤坏死因子受体(TNFR)是细胞因子受体家族中的一员,在大DNA病毒的免疫逃避中起着重要的作用.淋巴囊肿病毒中国株(LCDV-C)是一种大DNA病毒,属于虹彩病毒科.参照已知虹彩病毒TNFR基因设计引物P1,5'GGATCCAAAACTATGATTAAAATAAAGA 3';P25'ATTACTCGAGAATGTTAAAAATTAAGCTT3'.以LCDV-C基因组DNA为模板,PCR扩增得到一个834bp的DNA片段,并对该片段进行测序.构建原核表达重组质粒后,在大肠杆菌DE3中诱导表达,其产物经SDS-PAGE电泳后,显示为45kDa的融合蛋白带.对测序结果进行计算机辅助分析的结果显示,LCDV-C TNFR类似物是一个含278个氨基酸的多肽,具有典型的半胱氨酸富集区功能结构域,与宿主牙鲆TNERII氨基酸同源性为34%.     

牙鲆淋巴囊肿病毒中国株(LCDV-C)羟类固醇脱氢酶基因的特征分析

属于虹彩病毒科的淋巴囊肿病毒(Lymphocystis disease virus,LCDV)是一类能引起全球各地上百种淡、海水鱼产生囊肿的病原。在新分离到淋巴囊肿病毒中国株(Lymphocystis disease virus isolate from China,LCDV—C)并完成序列测定的基础上,用计算机辅助分析了LCDV-C羟类固醇脱氢酶(hydroxysteroid dehydrogenase,HSD)基因结构特征,LCDV—CHSD读码框为1023bp,推导其编码含340个氨基酸、分子量约为39．3kD的蛋白质。对来自10个不同病毒株或物种的HSD蛋白序列进行的比较分析显示,LCDV-C与淋巴囊肿病毒代表株(LCDV-1)的HSD氨基酸同源性最高,为60．5％。对LCDV-CHSD的二级结构进行了预测,有亲水区5个,抗原区、α螺旋区和β折叠区各有6个。经密度排列界面(dense alignment surface,DAS)的方法预测,表明LCDV-CHSD有一个跨膜结构域。     

鳜传染性脾肾坏死病毒主衣壳蛋白基因结构及序列分析

分析了鳜传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)的主衣壳蛋白(MCP)基因结构及其序列。对ISKNV DNA Kpn I L酶切片段的序列分析结果发现,该序列中含有完整的MCP基因。ISKNV MCP基因完整读码框为1362bp,比含量为56．24％,编码一个长为453aa、分子量为49．61kD、等电点为6．25的推定蛋白。结构分析发现,该基因具有启动子元件TATA框和CAAT基序。根据对虹彩病毒MCP系统进化树和脊椎动物虹彩病毒的生物学特性的分析比较发现,ISKNV、RSIV、SBIV、GIV和ALIV等在养殖海、淡水鱼类中引起其脾、肾、固有层和表皮细胞肿大的虹彩病毒,是独立于蛙病毒属和淋巴囊肿病毒属的又一新脊椎动物虹彩病毒类群。     

胸苷酸合成酶表达调控的分子机制

胸苷酸合成酶(thymidylate synthase,TS)是生物体内催化胸苷酸合成所必需的酶．多年来一直作为肿瘤化疗的重要靶酶。对TS基因调控机制的研究表明：基因扩增、转录、翻译和翻译后过程都参与了TS表达的调控。先前的研究表明：TS可与自身的mRNA结合形成TS-mRNA复合物,使mRNA翻译受阻,5-氟尿嘧啶(5-fluorouracil,5-FU)等抗代谢药物可与TS蛋白结合,结合后的复合物不能与TS mRNA作用,导致体内TS的表达升高,是肿瘤细胞产生抗药性的重要分子机制之一。现对TS基因表达调控研究进展、翻译调控与抗药性产生的分子机制进行综述。     

蛙病毒核糖核酸酶Ⅲ基因结构及其序列分析

对蛙病毒(TFV)核糖核酸酶Ⅲ基因序列进行分析.TFV基因组中 含有完整的核糖核酸酶Ⅲ基因序列,全长为1 113bp,GC含量为56.63%.其推定蛋白质的分子 量为40.47kD,等电点为\{7.99\}.序列结构分析发现在编码区的下游有可形成茎环的反向重复序 列和形成发夹结构的回文序列.与其它物种相比,TFV与虹彩病毒的LCDV-1和CIV的核糖核 酸酶Ⅲ基因的氨基酸序列同源性较高,与酵母、线虫等物种的相应基因的同源性较低.     

T4病毒研究进展

T4病毒科由一类单股正链RNA病毒组成,分为松天蛾β样病毒属和松天蛾ω样病毒属。这2个属的病毒具有不同的基因组结构,β样病毒含单组分基因组,其结构蛋白由一亚基因组RNA表达; 而ω样病毒含双组分基因组,2个RNA分子分别编码复制酶蛋白和结构蛋白。在T4病毒基因组RNA 3′端有类似tRNA的二级结构。ω样病毒壳蛋白的氨基酸序列一致性高达66%～86%, 而β样病毒壳蛋白的氨基酸同源性则要低得多。在昆虫细胞中表达壳蛋白基因时都能形成病毒类似粒子。该文还介绍了T4病毒复制机理以及T4病毒与其他病毒的进化关系。     

棉铃虫核型多角体病毒HindⅢ—K片段的核苷酸序列及其分析

测定了棉铃虫核型多角体病毒基因组ＤＮＡ的Ｈｉｎｄ Ⅲ－Ｋ片段苷酸序列。该片段全长３２５５ｂｐ,含可编码大于４０个氨基酸残基的多肽的开放阅读框１５个,包括多体蛋白基因编码区３’端４８９ｂｐ和蛋白激酶ＨａｖＰＫ基因编码区８０１ｂｐ。     

蛙病毒核糖核酸酶Ⅲ基因结构及其序列分析

对蛙病毒 (TFV)核糖核酸酶Ⅲ基因序列进行分析。TFV基因组中含有完整的核糖核酸酶Ⅲ基因序列 ,全长为 1113bp ,GC含量为 5 6 .6 3%。其推定蛋白质的分子量为 4 0 .4 7kD ,等电点为7.99。序列结构分析发现在编码区的下游有可形成茎环的反向重复序列和形成发夹结构的回文序列。与其它物种相比 ,TFV与虹彩病毒的LCDV 1和CIV的核糖核酸酶Ⅲ基因的氨基酸序列同源性较高 ,与酵母、线虫等物种的相应基因的同源性较低     

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.     

军曹鱼淋巴囊肿病毒主衣壳蛋白基因全序列分析

军曹鱼(Rachycentron canadum)亦称海鲡,是我国南方沿海一带的重要海水网箱养殖对象。2005年8月,广东省海水网箱养殖的军曹鱼首次暴发类似的淋巴囊肿病,病鱼的口唇、鳃、鳍、尾及体表等处,可看到大小不一的单个或成群的肿瘤,个别网箱的感染率在80%以上,死亡率近30%。病鱼形象丑陋,严重影响其市场价值,造成了较大的经济损失。淋巴囊肿病(Lymphocystis disease)发现于1874年,1965年正式确认该病病原为淋巴囊肿病毒[1],现已知可感染9目34科140种以上鱼类。我国在20世纪90年代陆续在养殖石斑鱼、鲈鱼、牙鲆中发现淋巴囊肿病[2-5],随后对其病原…     

鳜鱼传染性脾肾坏死病毒的胞嘧啶5-甲基转移酶基因结构及其序列分析

对鳜鱼传染性脾肾坏死病毒（infectious spleen and kidney necrosis virus,ISKNV）的胞嘧啶5－甲基转移酶（MTase）基因的结构及序列进行了分析。序列比较分析表明,ISKNV MTase编码区全长684bp,编码长227个氨基酸的蛋白质,推测分子量为25855D。与一些细菌的MTase比较,ISKNV MTase也含有负责转移甲基的4个保守区,但缺乏识别靶序列的保守区。比较ISKNV与其它6种脊椎动物虹彩病毒的MTase序列并建立系统树,ISKNV显著不同于蛙病毒属和淋巴囊肿病毒属。7种脊椎动物虹彩病毒MTase具有高度保守区,可以此设计引物用PCR方法鉴定脊椎动物虹彩病毒。     

Comparative studies of vertebrate lipoprotein lipase: a key enzyme of very low density lipoprotein metabolism

Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58-99% identity as compared with 33-49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5'-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2-3 times) than for LIPC and LIPG genes and proteins.     

Ascovirus and its evolution

Ascoviruses, iridoviruses, asfarviruses and poxviruses are all cytoplasmic DNA viruses. The evolutionary origins of cytoplasmic DNA viruses have never been fully addressed. Morphological, genetic and molecular data were used to test if all four cytoplasmic virus families (Ascoviridae, Iridoviridae, Asfarviridae, and Poxvirirdae) evolved from nuclear replicating baculoviruses and how the four virus groups are related. Molecular phylogenetic analyses using DNA polymerase predicted that cytoplasmic DNA viruses might have evolved from nuclear replicating baculoviruses, and that poxviruses and asfarviruses share a common ancestor with iridoviruses. These three cytoplasmic viruses again shared a common ancestor with ascoviruses. Morphological and genetic data predicted the same evolutionary trend as molecular data predicted. A genome sequence comparison showed that ascoviruses have more baculovirus protein homologues than do iridoviruses, which suggested that ascoviruses have evolved from baculoviruses and iridoviruses evolved from ascoviruses. Poxviruses showed genetic and morphological similarity to other cytoplamic viruses, such as ascoviruses, suggesting it has undergone reticulate evolution via hybridization, recombination and lateral gene transfer with other viruses. Within the ascovirus family, we tested if molecular phylogenetic analyses agree with biological inference; that is, ascovirus had an evolutionary trend of increasing genome size, expanding host range and widening tissue tropism for these viruses. Both molecular and biological data predicted this evolutionary trend. The phylogenetic relationship among the four species of ascovirus was predicted to be that TnAV-2 and HvAV-3 shared a common ancestor with SfAV-1 and the three virus species again shared a common ancestor with DpAV-4.      

Ascovirus and its Evolution

Ascoviruses, iridoviruses, asfarviruses and poxviruses are all cytoplasmic DNA viruses. The evolutionary origins of cytoplasmic DNA viruses have never been fully addressed. Morphological, genetic and molecular data were used to test if all four cytoplasmic virus families (Ascoviridae, Iridoviridae, Asfarviridae, and Poxvirirdae) evolved from nuclear replicating baculoviruses and how the four virus groups are related. Molecular phylogenetic analyses using DNA polymerase predicted that cytoplasmic DNA viruses might have evolved from nuclear replicating baculoviruses, and that poxviruses and asfarviruses share a common ancestor with iridoviruses. These three cytoplasmic viruses again shared a common ancestor with ascoviruses. Morphological and genetic data predicted the same evolutionary trend as molecular data predicted. A genome sequence comparison showed that ascoviruses have more baculovirus protein homologues than do iridoviruses, which suggested that ascoviruses have evolved from baculoviruses and iridoviruses evolved from ascoviruses. Poxviruses showed genetic and morphological similarity to other cytoplamic viruses, such as ascoviruses, suggesting it has undergone reticulate evolution via hybridization, recombination and lateral gene transfer with other viruses. Within the ascovirus family, we tested if molecular phylogenetic analyses agree with biological inference; that is, ascovirus had an evolutionary trend of increasing genome size, expanding host range and widening tissue tropism for these viruses. Both molecular and biological data predicted this evolutionary trend. The phylogenetic relationship among the four species of ascovirus was predicted to be that TnAV-2 and HvAV-3 shared a common ancestor with SfAV-1 and the three virus species again shared a common ancestor with DpAV-4.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Complete Genomic Sequence of Transmissible Gastroenteritis Virus TS and 3＇ End Sequence Characterization Following Cell Culture

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains.Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster.Continued culture in different cell types indicated that TGEV TS virulence could be attenuated alter fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.