猪圆环病毒ORF2基因和细小病毒VP2基因真核表达载体的构建及鉴定

猪圆环病毒2型的ORF2基因编码主要的结构蛋白(cap蛋白).有研究证明ORF2基因与病毒的毒力密切相关.猪细小病毒的VP2基因编码主要衣壳蛋白,VP2蛋白在细小病毒感染过程起着极为重要的作用.     

犬细小病毒新型疫苗的研究进展

正犬细小病毒(CPV)属细小病毒科细小病毒属成员。CPV为无囊膜的线性单股负链DNA病毒,其基因组全长约为5 300 nt,由4个开放阅读框(ORF)组成,为编码结构蛋白的VP1和VP2,非结构蛋白NS1和NS2组成。衣壳蛋白VP1和VP2蛋白构成互相叠加,其中VP2蛋白占总衣壳的90%。VP2蛋白是构成病毒抗原决定簇的主要组成部分,该蛋白可使机体产生中和抗体[1]。将近40年的流行过程,CPV发生了多次抗原变异,出现了CPV-2a、CP     

水貂肠炎细小病毒的分离鉴定及其VP2基因的序列分析

本试验从疑似细小病毒感染的病死水貂中分离到1株病毒。经PCR鉴定、细胞培养、蛋白质电泳鉴定最终确定为水貂肠炎细小病毒(MEV),命名为MEV-WFD。对该分离病毒的衣壳蛋白VP2基因进行克隆测序分析,结果表明此病毒VP2基因3处碱基发生点突变,其中一处的突变导致第328处氨基酸残基由疏水性丙氨酸(Ala)变为亲水性苏氨酸(Thr)。将此株病毒VP2基因与GenBank上公布的所有MEV的VP2基因碱基序列进行同源性比较及进化树分析,结果表明该病毒与ZYL-1、MEV/LN/-10和Manzhouli的VP2基因同源率最高,为99.8%;进化树构建结果表明,该病毒与6株已公布的病毒属于同一进化分支。     

关于水貂肠炎病毒结构蛋白基因5'-非翻译区调控其基因表达的评述

正水貂肠炎病毒(MEV)是细小病毒科细小病毒属的一种病毒,感染水貂引起水貂病毒性肠炎。作为感染真核细胞的最小DNA病毒之一,细小病毒基因组DNA仅有5 kb左右。为了利用有限的基因序列来完成病毒的复制周期,病毒利用了一些特殊的表达调控方式。比如:该属病毒成员(如:MVM,CPV)利用非结构蛋白NS1基因编码区的基因内启动子来调控结构蛋白VP1和VP2的表达;腺依赖病毒和浓核病毒利用leaky-scanning机制来表达衣壳蛋     

口蹄疫病毒蛋白对先天免疫的影响

正口蹄疫病毒(FMDV)抑制宿主的翻译机制,阻断蛋白质分泌,并裂解与信号转导和先天免疫应答有关的细胞蛋白。FMDV是一种单链阳性RNA病毒,由衣壳和核酸组成,其基因组长度约为8500个碱基,该基因组被一个二十面体衣壳包裹,衣壳由VP1、VP2、VP3和VP4四种结构蛋白各60个拷贝组成(图1)。VP1、VP2、VP3折叠成一个八链的楔形β-折叠形成外层     

我国猪博卡病毒的流行现状及检测技术

<正>猪博卡病毒(PBo V)属于细小病毒科(Parvovirus)细小病毒亚科(Parvovirus)博卡病毒属(bocavirus),为无囊膜的单股DNA病毒,病毒粒子大小为25~30 nm,呈正二十面体结构。病毒基因组全长5.2 kb,编码3个主要的开放阅读框(ORF1~3),其中ORF1编码非结构蛋白NS1,ORF2编码衣壳蛋白VP1和VP2,     

表达PCV-2 Cap和PPV-1 VP2蛋白的重组PRV的构建及鉴定

为了构建表达猪圆环病毒2型(PCV-2)Cap蛋白和猪细小病毒1型(PPV-1)VP2蛋白的重组伪狂犬病病毒(PRV)及鉴定其免疫原性,试验采用经典同源重组技术构建重组病毒,利用密码子优化的VP2基因(VP2_(opti))和含PRV gG蛋白信号肽序列的VP2_(opti)基因分别构建重组质粒pMD18T-LR(TK)-VP2_(opti)和pMD18T-LR(TK)-VP2_(opti)(SP)。用已构建的重组病毒rPRV-TK~-/gE~-/gG~-/3Cap~+为骨架,以EGFP为标签经同源重组获得重组病毒。采用IPMA法鉴定Cap蛋白和VP2蛋白抗原在重组病毒感染的PK-15细胞中的表达,采用IFA法鉴定VP2蛋白在重组病毒感染的PK-15细胞中的表达与定位。用这2株重组病毒免疫小鼠,通过检测血清中PRV、PCV-2和PPV-1抗体水平对构建的重组病毒在小鼠体内的免疫原性进行评价。结果表明:经同源重组获得重组病毒rPRV-TK~-/VP2~+/gE~-/gG~-/2Cap~+和rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2Cap~+;在重组病毒感染的PK-15细胞中均能检测到阳性Cap蛋白和VP2蛋白抗原;重组病毒rPRV-TK~-/VP2~+/gE~-/gG~-/2Cap~+表达的VP2蛋白定位于细胞浆和细胞核,而重组病毒rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2Cap~+表达的VP2蛋白定位于细胞浆;用这2株重组病毒免疫小鼠能够检测到PRV中和抗体;用灭活的重组病毒免疫小鼠后可产生低水平的Cap和VP2抗体,而重组病毒活毒免疫的小鼠未检测到相应抗体。说明这2株重组病毒在体外能够表达Cap蛋白和VP2蛋白,PRV gG蛋白信号肽序列的加入促进了VP2蛋白从细胞核的释放。     

犬细小病毒病防治研究

犬细小病毒（Canine parvovirus;CPV）是细小病毒科、细小病毒属成员,具有细小病毒属病毒典型形态和结构。病毒粒子细小,直径20～22nm,呈20面体对称,无囊膜,在氯化铯中的浮密度1．438／cm3。基因组为单股DNA,大小5233bp病毒粒子有VP1、VP2和VP3三种多肽,其中VP2为衣壳蛋白主要成分,有血凝活性。     

犬细小病毒病防治研究

犬细小病毒（Canine parvovirus；CPV）是细小病毒科、细小病毒属成员，具有细小病毒属病毒典型形态和结构。病毒粒子细小，直径20～22nm，呈20面体对称，无囊膜，在氯化铯中的浮密度1．438／cm3。基因组为单股DNA，大小5233bp病毒粒子有VP1、VP2和VP3三种多肽，其中VP2为衣壳蛋白主要成分，有血凝活性。     

蓝舌病病毒衣壳蛋白VP2、VP5、VP7酵母双杂交诱饵质粒的构建及鉴定

蓝舌病病毒(bluetongue virus,BTV)的结构主要由3层衣壳蛋白组成,其中VP2、VP5蛋白构成了BTV的外层衣壳,VP7蛋白构成了BTV的中间衣壳,最内层衣壳则由VP3蛋白构成。VP2、VP5及VP7蛋白在BTV侵染宿主细胞的过程中起着非常重要的作用。为了研究BTV与宿主细胞相互作用的分子机制,本研究将BTV的VP 2、VP 5、VP 7基因分别克隆到pGBKT7载体中,成功构建了pGBKT7-VP2、pGBKT7-VP5与pGBKT7-VP73个诱饵质粒,且通过自激活和毒性验证,证明所构建的3个质粒均无自激活作用,对酵母细胞无毒性作用。本研究为今后利用酵母双杂交筛选VP2、VP5、VP7蛋白中与宿主细胞相互作用的蛋白做好了铺垫,为深入研究BTV与宿主细胞的相互作用奠定了基础。     

Effects of proteolytic enzymes on the infectivity, haemagglutinating activity and protein composition of bluetongue virus type 20

The effects on virus infectivity, haemagglutinating (HA) activity and polypeptide composition of bluetongue virus type 20 (BTV 20) were determined after digestion with the proteolytic enzymes, chymotrypsin, thermolysin and trypsin. Virus infectivity increased eight to 50-fold after exposure periods which reflected the activity of the proteases. Identical maximum increases in HA activity (i.e. 4096, 1024 and 128 HAU per 0.05 ml with sheep, bovine and human erythrocytes, respectively) occurred with each of the three proteases. Peak increases in virus infectivities and HA activities occurred after similar exposure periods. Outer capsid protein VP2 was the most sensitive virus protein to proteolytic digestion, being cleaved into a number of smaller polypeptides that remained attached to the virus particle. Digestion with chymotrypsin and thermolysin yielded four common cleavage products, designated P93, P76, P54 and P25 according to their estimated molecular weight, which suggested that they shared at least three cleavage sites. VP2 cleavage products resulting from digestion with trypsin differed somewhat from those of chymotrypsin and thermolysin, although the generation of polypeptides P93, P54 and P25.5 suggested the existence of common cleavage sites for the three proteases. Possible mechanisms whereby proteolytic cleavage of VP2 may enhance the infectivity and HA activity of BTV 20 are discussed.     

Chicken anemia virus induced apoptosis: underlying molecular mechanisms

In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis.     

Expression or subcellular targeting of virus capsid proteins with cloning genome of a canine parvovirus from China

A strain of canine parvovirus (CPV), designated B2004, was isolated from the stool of a sick dog in Beijing. The partial genome (4623 bp) was cloned, sequenced with sequence showing B2004 to be a member of the widely distributed CPV-2a subclade. A completed VP2 or 11-residue N-terminal peptide (MAPPAKRARRG) of VP1 from B2004 was also tested for its ability to mediate nuclear transport of a heterologous protein, in this case enhanced green fluorescence protein (EGFP). EGFP was detected in the nucleus when it fused with the VP1 peptide; it was distributed primarily in the nucleus and also in the cytoplasm either when it fused with VP2, or in the cytoplasm when expressed on its own. In common with other parvoviruses the CPV VP1 N-terminal peptide contributes to the nuclear localization of the gene product.     

雏鹅新型病毒性肠炎病毒的提纯和核酸链型及结构蛋白分析

本文报道雏鹅新型病毒性肠炎病毒（NGVEV）的提纯方法、核酸类型及结构蛋白分析的研究结果。采用氯仿处理－饱和硫酸铵沉淀－透析清除NH4^ 、SO4^2-柱层析分离的技术程序可获得纯将的NGVEV病毒粒子。于电镜下可观察到NGVEV具有典型的腺病毒特征。经吖啶橙染色和S1核酸酶消化鉴定NEVEV的核酸类型为双链DNA。经SDS－PAGE分析，病毒结构蛋白由15种多肽组成，即VP1（116KD）、VP2（104KD）、VP3（88KD）、VP4（68KD）、VP5（60.7KD）、VP6（54KD）、VP7（40.1KD）、VP8（36KD）、VP9（34.5KD）、VP10（24.2KD）、VP11（22KD）、VP12（20KD）、VP13（18KD）、VP14（14.2KD）和VP15（13.4KD)。其中VP4、VP7、VP8、VP9、VP14为主要结构多肽，占蛋白总量的90.2622%。     

兔病毒性出血症病毒次要结构蛋白（VP2）的表达和细胞内定位分析

病毒蛋白VP2是兔出血症病毒（Rabbit Hemorrhagic Disease Virus,RHDV）的一种次要结构蛋白。本文旨在构建VP2与绿色荧光蛋白融合的真核表达载体,进而利用它研究VP2在细胞内的定位情况。首先在大肠杆菌中表达GST-VP2融合蛋白,然后用纯化的重组蛋白免疫小鼠制备抗VP2的抗血清。对制备的抗VP2抗血清进行特异性分析,再利用该抗体对转染pEGFP-VP2的BHK-21细胞进行检测,以研究VP2蛋白在细胞内的定位情况。结果表明,pEGFP-VP2转染BHK-21细胞后,不仅VP2蛋白得到了成功表达,而且通过检测偶联的绿色荧光蛋白可判断出VP2蛋白主要分布于BHK-21细胞的细胞质。本研究成功构建了VP2与绿色荧光蛋白融合的表达载体,并在真核细胞内实现了表达,通过检测荧光蛋白,发现VP2主要在细胞质内表达并分布,为深入探讨VP2的生物学功能奠定了基础。     

AsiaⅠ型口蹄疫病毒VP1基因结构预测及亚细胞定位

利用PCR方法扩增到VP1基因，序列测定后利用Mega4．0、swiss—model、GOR4和RasMol软件预测了VP1基因的二、三级结构。将VP1基因融合EGFP基因后定向克隆入PcDNA3．1（＋）真核表达载体，构建正确的重组质粒命名为PVP1E，在脂质体介导下将PVP1E质粒转染BHK-21细胞，WesternBlotting试验证实VP1基因成功表达，经DAPI细胞核染色后在共聚焦显微镜下观察VP1亚细胞定位。结果表明本研究预测了VP1基因的二、三级结构，其在BHK-21细胞中呈现以细胞核为主的弥散性分布，VP1基因亚细胞定位及结构预测为进一步深入探究AsiaI型FMDVVP1结构和功能提供丰富的资料。     

The ovine cytotoxic T lymphocyte responses to bluetongue virus

This study uses recombinant vaccinia viruses expressing truncated or entire bluetongue virus (BTV) proteins to map the location of epitopes recognized by cytotoxic T lymphocytes (CTL) from Australian merino sheep. The non-structural protein, NS1, was recognised by CTL from all sheep, while VP2, VP3, VP5 and VP7 were recognised by CTL from only some sheep. The remaining proteins (except for VP1, which was not tested) did not contain CTL epitopes. When truncated genes were used to map the location of CTL epitopes, it was found that sheep often have CTL that recognise more than one epitope in NS1 or VP2. Overall there was considerable diversity in the CTL recognition patterns in the sheep tested.     

水貂阿留申病病毒分子生物学研究进展

水貂阿留申病病毒是一种在水貂中广泛存在的重要病原体.该病毒属阿留申病毒属,主要编码4种蛋白(结构蛋白VP1、VP2和非结构蛋白NS1、NS2).VP1蛋白在协助病毒产生感染性方面起着重要作用;VP2蛋白是该病毒的主要免疫原性抗原,能体外中和病毒;NS1和NS2对病毒在宿主细胞中的复制起重要的调节作用.该病毒的分子生物学诊断技术主要有核酸杂交技术、PCR和基因芯片检测技术.     

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.     

水貂阿留申病毒结构蛋白与非结构蛋白的研究进展

水貂阿留申病毒(Aleutian mink disease virus,ADV)是一种主要侵染水貂的自主复制型细小病毒,是一种在水貂中广泛存在的重要病原体。病毒粒子的蛋白分为结构蛋白(VP1、VP2)和非结构蛋白(NS1、NS2)两类。VP1蛋白对病毒粒子产生感染性有重要作用;VP2蛋白是主要免疫功能区,能刺激机体产生中和抗体;NS1和NS2主要参与病毒的复制和基因的表达调节。文中对近年来国内外学者关于水貂阿留申病毒结构蛋白和非结构蛋白的研究情况进行归纳和总结。