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2001年16卷4期

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Review

干扰素诱导的鱼类Mx蛋白

张义兵, 桂建芳

2001, 16(4): 291

Mx蛋白是干扰紊诱导表达的蛋白家族中的成员,当机体和细胞受病毒感染或诱生荆处理时产生。 蛋白和 其它干扰素诱导蛋白一起构成宿主细胞的抗病毒状态,以达到抗病毒的目的。研究表明, 蛋白具有抗病毒活 性,还可能与其它基本生命活动如发育或分化,蛋白质分送和生长有关。在鱼类也发现多种Mx蛋白,具有Mi蛋白 家族的共有特征:在肽链末端有一个三联ATP/GTP结合区和发动蛋白家族的结构特征序列;在蛋白C端存在使Mx 蛋白形成三聚体的ku拉链结构以及定位信号。但是迄今没有发现龟类Mx蛋白的抗病毒活性。文章最后对目前 鱼类病毒病的防治及利用抗病毒基目进行鱼类基臣工程抗病毒育种进行了探讨。Mx proteins are members of a family of interferon inducible genes expressed in organisms or cells which are treated with all sorts of inducers or virus infection.These proteins together with other interferon inducible proteins form the antiviral state in host cells and the first line of the body’s defense against virus infection.Apart from the inhibtion of a specific virus,Mx proteins may be related with other basic cellular functions such as development/differentiation,protein sorting and growth.Mx proteins in several fishes are found and a comparsion of their sequences with that of avian and mammalian species reveals striking conservation of domains.They all maintain a tripartite ATP/GTP binding motif and a signature of the dynamin family in the amino terminal of the protein.In addition,the C terminal region of the Mx proteins contains the localization signals and the leucine zipper motif which account for the trimerization of Mx in the cell.So far,the antiviral function of the fish Mx proteins has not b

汉坦病毒疫苗研制进展

肖春惠, 杨占秋

2001, 16(4): 299

本文从脑组织灭活疫苗、培养细胞单双价灭活疫苗、基因重组疫苗、DNA疫苗、减毒活疫苗、佐剂、灭活方法、 免疫策略等方面概述了汉坦病毒疫苗研制近况We outlinethe development ofthe hantaanvims vaccine in ree~t years∞ brain dsaneinavtivated vaccine,cultural cell bivalent and univalent inactivated vaccine,recu~ inadou vaccine,DNA vaccine,attenuat— ed live vaccine,adjuvarIt,inacfivatima methods and lmnlulle strategy.
Research Article

猴免疫缺陷病毒引起多形核嗜中性白细胞凋亡的可能机理

李刚, 杨非易, 刘新华, 王丹, 李平风

2001, 16(4): 304

本实验目的是研究猴免疫缺陷病毒(srv)~l起多形核嗜中性白细胞(r~tNs)凋亡的机理。实验用PCR技术扩 增gag基因,用Western blot法测定p53和bel-2基因的表达。结果显示P唧自在被SIV感染后随着保温时间的延长 存话卑下降,在感染后24h可以从PMNs中扩增出gag基因。PMNB中p53基因的的表达在感染后24h增加。同时 一2基因的表达在对照组和SIV感染组都增加,但在SIV感染组 .2蛋白的表达明显低于对Jf}i组。结果揭示SIV 能够感染PMNs,p53和6 2基因表达的改变可能是SIV感染P咖 引起细胞凋亡的机理。The aim ofthis stud),istoinvestigatethemechanis~s of apoptosisin polymorphonudear neutrophils (PMNs)induced by simian immunodeficiency v~ (siv).In this experiment.gag gene was amplified by the polymerase chaill reactio~andthe expression ofp53 and bc/一2 gC~12e were determined byWestern blot assay. The results showedtllattl1e viability ofPMNs aftertreated with SIV was declined with the cLlk time pro 1~ged. gene could be amplified from PMNs at 24 h postinfection. expression of p53 gene in PMNs W83 enhaneed at 24 h postinfee6on.At tlle s8me time。the expression of bcI一2 gene were elevated in both of control and SIV infected group.but the production of 一2 protein in SIV infected group was obviously lower than contro1.0tilt"dataindicatethat PMN$could beinfected witll SIV andthe ch~ged expression ofp53 and bcI一2 gene~,66t be one oftlle meehanlsms of apoptosis occurred in PMN infected witll sⅣ.

汉坦病毒K24株M和S片段基因的结构特征

姚智慧, 董关木, 俞永新

2001, 16(4): 309

为研究汉坦病毒1(24株M和S片段基因的结构特征,从病毒感染细胞提取细胞总RNA,经 个循环的一次 性PCR得到了与理论值相符合产物,将RT—PCR产物直接克隆T载体,经K24毒株M片段的全基因序列共3 653 个核苷酸,四种核苷酸的酶切和PCR鉴定正确的克隆通过柱纯化后进行序列测定,结果比例分别为A 30.44% ,T 30.14%,G 20.72%,C18.70% ,GC含量为39.42% .AT含量为6o 58%,编码1 133个氪基酸。S片段的全基因序列 共1 772个核苷酸,四种核苷酸的比例分别为A.31 30%,G26.05%,"1’22.79% ,C19.77%,GC含量为45.81% ,AT含量 为54 19%,共编码429个氨基酸。1(24株M片段桉苷酸全基因和氪基酸与HTN型毒株同源率分别为70.7% 一 71.2%和75.9% 一76.9% ,与SEO型为95 2% ~99.2%和95 9% 一99.4%。S片段与I/rlq 76 118和SEO SR-II病 毒的比较,核苷酸同源率分别为74.0%和95 6%,氨基酸同源率为83.3%和97.9% ,与M片段的结果相类似。M 片段氪基酸与SEO型1O个毒株存在4个氪基酸替代。应用DNASTAR软件的系统发生分析方法分别绘出了基于M 片段核苷酸厦推导氪基酸的系统发生树。

肾综合征出血热疫苗候选毒株A16株的分子特性

姚智慧 董关木 俞永新 贾克丽 严玉辰

2001, 16(4): 315

为研究肾综合征出血热疫苗候选毒株A16株的分子基础 ,应用RT -PCR方法扩增并测定了A16株的M和S片段的序列 ,结果A16株M片段的全基因序列共 36 15个核苷酸 ,编码 1133个氨基酸。S片段的全基因序列共1770个核苷酸 ,编码 4 2 9个氨基酸。A16株M片段核苷酸全基因序列及其推导的氨基酸与HTN型毒株同源率为75 .5 %~ 90 .3%和 85 .9%~ 97.1% ,即A16与HTN同型毒株间的差异高达 9.7%~ 2 4 .3%和 2 .9%~ 14 .1% ,而与SEO型的同源率比较 ,核苷酸和氨基酸分别仅为 70 .2 %~ 70 .8%和 73.4 %~ 76 .7%。S片段的序列同源率与M片段的结果相类似 ,即核苷酸和氨基酸的同源率与HTN型毒株分别为 76 .0 %~ 90 %和 92 .1%~ 97.7% ,与SEO型为6 7.0 %~ 6 7.7%和 81.7%~ 82 .6 %。结果显示 ,A16株虽然属于HTN型 ,但该毒株为HTN型病毒的新亚型病毒株

乙肝病毒表面抗原和抗体双阳性者中病毒S区基因序列分析

辜文洁, 林京香, 王佑春一, 张华远, 蓝海云, 李河民

2001, 16(4): 321

为了解乙肝病毒 (HBV)表面抗原和抗体双阳性患者中病毒的基因型及其HVBS区是否有变异。用放射免疫试剂检测HBsAg阳性样品中的抗 HBs抗体 ,用聚合酶链反应法检测双阳性样品中的HBVDNA ,然后对阳性样品进行克隆和基因序列分析 ,并将所得序列与HBV不同基因型的代表株进行比较分析。结果显示 389例HBsAg阳性样品中有 10例为抗HBs抗体阳性 ;该 10例双阳性样品中有 5例为HBVDNA阳性 ;序列分析显示该 5株HBV均为B基因型 ,其中 4株为adw亚型 ,1株为adr亚型 ;其中有 2株在S区的“a”决定簇的氨基酸发生了变异In order to investigate HBV genotype and variants from the blood donors positive for both HbsAg and anti HBs antibody,389 sera from HBsAg positive blood donors were tested for anti HBs antibody with RIA assay.The sera positive for both HbsAg and anti HBs were tested for HBV DNA with polymerase chain reaction(PCR).The positive PCR products were cloned and sequenced.The sequences were compared with the representative sequences of different HBV genotypes.The results showed that 10/389 sera positive for HBsAg were positive for anti HBs.Five of these ten sera were positive for HBV DNA.The sequence analysis showed that all five sequences belonged to HBV genotype B,four sequences were adw serotype and one was adr serotype.Amino acids of antigen determinant "a" of two sequences have been changed.

肝癌中HBV和HCV基因和抗原的分布及意义

王文亮 王春杰 师建国 王冰枫

2001, 16(4): 325

采用原位分子杂交方法检测HCVRNA及HBVX基因 ;采用免疫组织化学方法研究HCV核心抗原 ,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布。结果表明 (1)HCVRNA、HBVX基因在肝细胞肝癌组织检出率分别为 4 0 % (5 5 / 136 )和 82 % (112 / 136 )。HCVRNA定位于癌细胞的胞浆内 ,阳性细胞呈散在、灶状及弥漫分布三种形式 ;HBVX基因在肝癌细胞中的分布呈胞浆型、核型及核浆型 ,阳性细胞也呈上述三种分布形式 ;(2 )HCVC33c抗原、核心抗原在肝细胞肝癌中的阳性率为 81% (133/ 16 4 )及 86 % (14 1/ 16 4 )。C33c抗原定位于癌细胞及肝细胞的胞浆内 ;核心抗原既定位于癌细胞核中 ,又可定位于胞浆中。C33c抗原阳性细胞以灶状分布为主 ;而核心抗原阳性细胞在癌组织以弥漫核阳性常见 ,在癌旁肝组织以胞浆阳性为主 ;(3)HBxAg在肝细胞肝癌中的检出率为 75 % (12 3/16 4 ) ,C33c和HBxAg二者同时阳性占 6 3% (10 3/ 16 4 )。HCV感染在我国肝细胞肝癌中比较普遍 ,HCV和HBV重叠感染占相当比例 ,可能在肝细胞肝癌的发生中起着重要作用 HCV RNA and HBV X gene were detected by in situ hybridization and HCV core?C33c antigens and HBxAg were localized by immunohistochemical method.Results showed(1) The positive rate of HCV RNA and HBV X gene were 40%(55/136) and 82% (112/136) in HCC respectively,both of them were positive simultaneously,34%(46/136).HCV RNA was localized in the cytoplasm of cancerous cells and hepatocytes,the positive cells distributed in scatter,local or diffusion pattens.The distribution of HBV X gene in HCC cancerous cells showed cytoplasm,nuclear and nuclear cytoplasm,the positive cells distributed just as that of HCV RNA.(2) The positive rates of HCV C33c and core antigens in HCC were 81%(133/164) and 86%(141/164) respectively,and C33c antigen was localized in the cancerous cells intracytoplasmically,either in the form of focal or diffuse in distribution,some were access to the nuclear membrane.Not only was HCV core antigen positive stain distributed in the nuclei of the cancerous cell but it was also found in the cytoplas

棉铃虫单核衣壳核多角体病毒囊膜蛋白odv.e66基因 及其邻近区域的序列分析

吴东, 胡志红, Just M.Vlak, 陈新文

2001, 16(4): 330

杆状病毒ODV.E66蛋白是包涵体来源病毒(ceclusion-de~ved viIll8,0DV)囊膜的结构蛋白,ODV囊膜对0DV的 稳定性和感染性具有重要作用。本文报道了H且sNPv。 66基因及其邻近区域共4 2376v的核苷酸序列及其分析 结果。HaSNPV的odv-e66基因编码区全长2 019bp,推测编码一个由672个氨基酸残基组成的,分子量为74.51~D的蛋白质。在起始密码子m 上游具有杆状病毒晚期转录起始信号ATAAG。与其他杆状病毒ODV-E66的氪基酸序列比较分显示HaSNPVODV-E66蛋白具有多个保守区域,包括N末端的强疏水功能区、序列中部的一个可能的棱定位信号RⅪ ,两个1.eu—zipper以及5个跨膜区。~ -e66基因上游的两个0RF分别与AddNPV的o,Do~和0r九09具有同源性.下游的ORF与LsNPV的p13基因具有同源性。 ODV E66 is the major protein of occlusion derived virions envelope of baculovirus.An odv e66 gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus was identified and sequenced.The HaSNPV odv e66 gene had a size of 2 109 bp and potentially coding a protein of 672 amino acids,with a molecular weight of 74.5 kD.A late transcriptional motif,ATAAG,was found at 20~ 24nt uppstream of the translational start codon ATG.There was a TATA box,a GATA box and a CAGT motif at 13nt, 92nt, 141nt upstream of ATG,respectively.Amino acid sequences alignment with so far identified baculovirus’ODV E66 had revealed a conserved hydrophobic region at the N terminal of ODV E66 proteins.A potential nuclear locating signal RR(K)IW was found conserved in all ODV E66 proteins.Two leu zipper motifs were found in the middle of ODV E66 proteins.Six potential transmembrane regions were found in HaSNPV ODV E66 protein.Two of them were conserved in the other baculoviruses’.Analysis with computer shown

棉铃虫核多角体病毒iap2基因的克隆和序列分析

张小霞 张忠信 丁清泉

2001, 16(4): 338

棉铃虫核多角体病毒 (Helicoverpaarmigerasinle nucleocapsidnucleopolyhedrovirus,HaSNPV)基因组中发现了一个抗细胞凋亡基因iap2。该基因位于病毒基因组的BamHI F片段 ,全长 75 3个核苷酸 ,编码 2 5 0个氨基酸 ,预计蛋白质分子量 2 9.2kD。在iap2基因上游 - 30~ - 33的位置有一个TATA框 ,在 5 0~ 5 3的位置有一个早期转录信号CAAT ,在终止密码子下游有一个poly(A)信号AATAAA ,HaSNPViap2基因编码一个锌指结构和两个BIR结构。与LdMNPV、AcMNPV、SeMNPV和OpMNPV的IAPs及果蝇DIAP2和人类XIAP等九种IAPs相比较 ,HaSNPVIAP2与其它的BIR(baculovirusIAPrepeater)和RING结构在Cys/His基元 (motif)位点上高度保守 An iap 2 gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus was identified.It was localized Bam H I F fragment of the genome.Nucleotide sequencing showed the open reading frame was 753nt,encoded 250 amino acids with a predicted size of 29.2kD.A"TATA"box was found-30~-33nt upstream of the translational start codon ATG.There is an early consensus translational motif CAAT,-50~-53nt upstream of the translational start codon ATG.A typical poly(A)signal was identified 46~51nt downstream of the translational stop codon TAA.HaSNPV iap 2 gene encode one typical RING and two BIRs.The alignment of AcMNPV?OpMNPV?LdMNPV?LdMNPV?SeMNPV?DIAP and XIAP indicated that they had consensus BIR motifs and RING zinc finger motif.It was suggested HaNPV IAP2 had a potential anti apoptotic function.

棉铃虫单核衣壳核多角体病毒解螺旋酶基因的序列分析

范凌云, 胡志红, 陈新文

2001, 16(4): 344

对棉铃虫单核衣壳核多角体病毒 (Helicoverpaarmigerasingle -nucleocapsidnucleopolyhedrovirus,HaSNPV)基因组中EcoRI N片段进行序列分析 ,获得了完整的解螺旋酶基因 (hel) ,其开放阅读框大小为 376 2bp ,编码一个分子量为 14 6kD的蛋白质。在hel起始密码子ATG上游 5 0位有强晚期启动子转录起始信号ATAAG ,在 112位和 189位存在两个TATAbox ,但未发现早期转录信号CAGT。其在终止密码子下游第 12位有一PolyA终止信号AATAAA。在其它真核或原核解螺旋酶中存在的 7个保守基元 (I、Ia、II、III、Ⅳ、Ⅴ、Ⅵ) ,只有 5个 (I、Ia、II、III、Ⅳ)在杆状病毒中保守同源性比较发现 ,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒 (SpodopteraexigueMNPV ,SeMNPV)的解螺旋酶具有最高的同源性 (6 6 % ) ,与Xestiac nigrum颗粒体病毒 (XcGV)解螺旋酶的同源性最低 (43% )。HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因 In order to investigate the genomic organization of the single nucleocapid nucleopolyhedrovirus of Helicoverpa armigera ,the Eco R I N fragment located at 54.8~59.3 kbp of the viral genome was sequenced.The fragment contained 3 762 bp helicase gene potentially encoding a protein with a molecular mass of 146kDa.A late transcriptional motif,ATAAG,was found at 50nt upstream of the translational start codon ATG,while two TATA box was located at 112nt and 189nt upstream of ATG.A typical poly(A) signal was found at 12nt downstream of the translational stop codon.Compared HaSNPV helicase with the helicases of Autographa colifornica MNPV(AcMNPV),( Bombyx bori NPV (BmNPV), Lymantria dispar MNPV(LdMNPV) Spodoptera exigue MNPV (SeMNPV), Orgyia pseudotsugata MNPV (OpMNPV),and Xestia c nigrum granuloviurs(XcGV),only 5motifs(I,Ia,II,III,IV)were found conserved in baculovirus.The homology of the other two notifs (V and VI),which are also quite conserved in other helicases,were lowe

马尾松毛虫质型多角体病毒多角体蛋白基因的 eDNA克隆及序列分析

杜建字, 张珈敏, 郭海涛, 张晓东, 胡远扬

2001, 16(4): 350

通过对马尾橙毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经 SDS一酚抽提,琼脂糖凝胶电泳分离基因组dsPdgDA,回收纯化第十片段SIO。SIO经DMSO变性,逆转录合成cDNA 第一链,PCR扩增后,克隆在DGE T载体上。对重组子进行限制性内切酶分析及序列测定,结果表明,克隆片段全 长763b口,起始密码AUG位于3—5残基,终止密码UGA位于747—749残基。推测Dt,C2V多角体蛋白基因编码248 个氨基酸的多肽,分子量28kD。和家蚕质型多角体病毒(BmCPV)多角体蛋白基因相比较,核苷酸和编码氨基酸序 列同源性分别为89,3%和97.6%。CPVs belong to the genus Cypovirus in the family Reoviridae and infect midgut epithelial cells of the wide range of insects.For the synthesis of cDNA of Dendrolimus punctatus CPV (DpCPV) polyhedrin gene,the primers were constructed on the basis of the terminal RNA sequence of BmCPV segment 10,After RT PCR,the amplified cDNA was cloned into the pGEM T.The S10 cDNA of DpCPV was found to be 763 nucleotides in length with a single open reading frame in one strand capable of coding a predicted protein of 248 residues (Mr of 28411).Comparison of the nucleotide sequence of the polyhedrin gene of DpCPV with that of BmCPV showed that nucleotides homology is 89.3%,but there are only 6 amino acid differences between the two CPVs.

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind III.L片段的序列分析

方明刚, 陈新文, 王汉中, 胡志红

2001, 16(4): 355

本文报道了棉铃虫单核衣壳核多角体病毒 (Helicoverpaarmigerasingle nucleocapsidnucleopolyhedrovirus,HaSNPV)基因组的HindIII L片段的全序列。该片段全长 2 6 35bp ,包括 5个有意义的开放阅读框 :HaSNPVORF2 2 7,晚期表达因子 10基因 (lef10 ) ,vp10 5 4基因 ,Ac5 5 (AcMNPVORF5 5的同源基因 ) ,Ac5 6 (AcMNPVORF5 6的同源基因 )。与其它 6种杆状病毒的氨基酸序列比较表明 ,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒 (SeMNPV)的同源性最高 ,为6 4 % ,与冷杉毒蛾核型多角体病毒 (OpMNPV)的同源性最低 ,为 4 3% ;HaSNPV的vp10 5 4基因与SeMNPV的同源性最高 ,为 6 5 % ,与OpMNPV的同源性最低 ,为 4 9%。序列比较表明 ,HaSNPV的LEF10与VP10 5 4蛋白与其它 6种杆状病毒具有相同的保守区和亮氨酸拉链 (leucinezipper)In order to investigate the genetic organization of the single nucleocapsid nucleopolyhedrovirus (SNPV) of Helicoverpa armigera (HaSNPV),the Hin d III L fragment of the viral genome was sequenced.The fragment contained five complete open reading frames(ORFs) representing ORF227 (a unique gene),a late expression factor 10 gene ( lef 10),a baculovirus structural protein gene( vp 1 054), Ac 55(a homologue of the AcMNPV ORF55) and Ac 56 (a homologue of the AcMNPV ORF56),respectively.These five genes were identified for the first time in HaSNPV.Alignment of baculovirus LEF10 s indicates that they have conserved leucine zipper regions,and aligament of baculovirus VP1054s indicates that they are highly conserved.

甜菜夜蛾核型多角体病毒中国分离株的分离鉴定及毒力测定

张俊杰 李茂俭 未荣杰 张友清

2001, 16(4): 361

本文对分离自中国的甜菜夜蛾病毒进行提纯、鉴定。生物测定的结果表明其对二龄、三龄甜菜夜蛾的Ⅱ 分别为6.6x1 ,2.6x105PIB/mL.A strain of Spodoptera exigua Nuclear Polyhedrosis Virus(SeNPV)isolated from China was purified and identified.The results of bioassay indicated that the LC 50 of 2nd and 3rd instar of Spodoptera exigua was 6.6 ×10 4 and 2.6×10 5 PIB/ml

棉铃虫颗粒体病毒增效蛋白基因2.6kb片段的表达

胡蓉, 孟小林一, 徐进平, 王健, 鲁伟

2001, 16(4): 364

以棉铃虫颗粒体病毒 (Helicoverpaarmigeragranulosisvirus ,简称HaGV)基因组的DNA为模板设计引物 ,PCR扩增病毒增效蛋白 (Enhanicn)基因 ,然后经BamHI/PstI双酶切消化 ,得到近乎全长的约 2 .6kb的增效蛋白基因片段 ,再与pQE32质粒连接 ,构建了重组表达载体pQE32 /En ,转化大肠杆菌M15 (pREP4 ) ,在IPTG诱导下表达出分子量约为 10 2kD的融合蛋白并命名为P10 2 ,纯化的P10 2 包涵体显示了明显的增效活性 ,在感染后 16 8h时统计可提高HaNPV对棉铃虫幼虫的感染死亡率 6 .2 5 %~ 2 7.0 9% ,缩短LT50 12 .3h以上 ;在感染后 72h时统计可提高Bt对棉铃虫幼虫的感染死亡率 2 8.18% ,缩短LT50 12 .33h。The 2.6kb fragment of enhancing gene from Helicoverpa armigera granulosis virus was inserted into vector pQE32 and expressed successfully in E.coli M15.The synergy of expression product(P 102 ) on Helicoverpa armigera nuclear polyhedrosis virus(HaNPV)and Bt against the 2nd instar larvae of H.armigera was studied.The results indicated that the accumulated mortality of the larvae increased 6.25%~27.09% on 168h post infection and the median lethal time decrease at least 12.3h in HaNPV+En treated compared with those in HaNPV treated.The accumulated mortality of the larvae increase 28.18% on 72h postinfection and the median lethal time decreased 12.33h in Bt+En treated compared with that in Bt treated.

番木瓜环斑病毒畸叶株系的CP基因克隆和序列分析

贺国安, 肖火根“, 张曙光, 李华平, 范怀忠

2001, 16(4): 369

采用RT PCR方法合成了本研究室保存的番木瓜畸叶病毒 (PMaLV)的外壳蛋白 (CP)基因 ,将其CP基因克隆进Promega公司的pGEM -TandpGEM -TEasyVectorSystem(简称T -载体 ) ,并进行了序列分析。结果表明 ,PMaLVCP基因核苷酸序列全长为 86 1nt,推导其编码 2 87个氨基酸。与番木瓜环斑病毒 (PRSV)美国夏威夷HA株系和澳大利亚W株系的CP基因相比 ,在第 6 6nt处开始连续缺失 3个核苷酸。与PRSV的华南Ys、Sm和G株系以及夏威夷的HA和澳大利亚的W株系相比 ,其CP基因序列同源率分别为 96 %、98%、95 %、89%和 89%。其推导的氨基酸序列同源率分别为 98%、97%、97%、96 %和 95 %。此结果表明 ,PMaLV属于PRSV的一个株系 ,不是一种新病毒。因此 ,我们称其为番木瓜环斑病毒畸叶株系 (ML株系 )。The coat protein(CP)gene of an isolate of papaya malformed leaf virus(PMaLV),preserved in Plant Virology Laboratory of South China Agricultural University by Luo Xuehai,was synthesized by reverse transcription and polymerase chain reaction(RT PCR) and cloned into pGEM T and pGEM T Easy Vector System(T vector).The full length of the CP gene was sequenced,and it shown that the CP gene was 861 nucleotides(nt) in length,with 3nt deletion beginning from 66nt as compared with that of Hawaii strain(HA) and Australian strain W of papaya ringspot virus(PRSV).Compared with Ys(yellow ringspot strain)?Sm(severe mosaic strain)?G(Hainan isolate)?HA and W strains of PRSV,the CP gene sequence of PMaLV shared 96%?98%?95%?89% and 89% nucleotide sequence identity respectively.The coat protein encoded by CP gene of PMaLV consisted of 287 amino acids residues,and shared 98%?97%?97%?96% and 95% amino acid identity with those of the five strains respectively.This results of sequence analysis shown conclusively that PMaLV is a s

中红侧沟茧蜂多分DNA病毒基本特征研究

李馨 秦启联 丁翠 刘海虹

2001, 16(4): 373

本文首次报道中红侧沟茧蜂 (Microplitismediator)雌蜂卵巢中存在多分DNA病毒 (MicroplitismediatorPlolyd navirus,MmPDV) ,初步研究了MmPDV形态和基本生理生化特征。利用蔗糖密度梯度超速离心分离纯化了MmPDV粒子 ,电镜负染显示PDV粒子分三段 ,带有一明显的尾部结构 ,大小约为 130× 35nm ;SDS PAGE电泳条带较多 ,至少可以分辨出 2 6个电泳条带 ,表明病毒粒子衣壳蛋白复杂 ;琼脂糖凝胶电泳显示MmPDV基因组至少由大小不同、丰度不等的 14个DNA分子组成 ,用 6种内切酶 (EcoRI,HindIII,BssHII,PstI,BamHI,BglI)酶切MmPDV基因组后 ,估算出MmPDV基因组大小约为 10 8kb。用雌蜂输卵管萼液注射小地老虎幼虫 ,注射后的小地老虎体重和龄期发育动态表明 ,MmPDV具有抑制寄主生长发育的生理功能 A polydnavirus(PDV)was purified by sucrose density gradient centrifugation from the calyx region of female parasitoid wasp Microplitis mediator (Hymenoptera:Braconidae).Negative stain technique showed that the virus was tadpole like and measured 130×35nm.A diverse protein profile of the virus’capsid proteins was displayed by SDS PAGE.Its genome was composed of over 14 DNA molecules,which were different in molecular size and abundance.Molecular size of the genome was approximate 108 kbp calculated by patterns from six restrict endonucleases digestion.Micro injection experiment with calyx fluid of the female wasps showed that the virus suppressed growth and development of host of the wash.

IBV青岛腺胃分离株(SD/97/02)S1蛋白基因的序列测定和分析

潘杰彦 陈德胜 戴亚斌 陈溥言

2001, 16(4): 377

参考Genbank上发表的IBVS1纤突蛋白基因序列 ,设计了一对引物 ,对鸡传染性支气管炎病毒青岛腺胃分离株 (SD/ 97/ 0 2 )RNA进行RT PCR扩增。将PCR产物克隆入pMD18 T载体中进行序列测定和分析。序列分析表明 ,该毒株的S1基因的G +C %含量较少 ,为 37.0 % ,存在HindIII,BamHI,BgIII,SacI和SalI位点 ,无EcoRI位点 ,与其他毒株的同源性在 87.0 2 % 94 .2 1%之间 ,在第 15 4~ 4 2 9nt处为高度的变异区 ;将基因序列翻译成氨基酸后 ,假定的S1蛋白由 5 4 0个氨基酸组成 ,等电点 8.2 4 ,在蛋白质内部存在 18个Cys,在S1与S2蛋白之间的剪切位点为HRRRR ,这与大多数IBV毒株 (RRF/SRR)不一样 ,有三个区域的氨基酸序列高度保守 ;16 9~ 181aa,2 30~ 2 5 0aa ,4 85~ 5 0 6aa ;与其他毒株进行抗原性比较后发现 ,在该毒株的 32 0~ 32 6aa及 390~ 4 0 1aa处的抗原表位消失 ,而在 32 5~345aa、379~ 389aa处则出现了很强的抗原表位 ;第 4 38~ 4 4 4aa处 ,其他IBV毒株 (除ZJ971株外 )原来存在的强抗原位点在本毒株中消失 ;在 5 3~ 6 5位的氨基酸抗原性与其他毒株相比明显变弱A pair of primers were designed and synthesized according to the reported S1 spike protein gene sequence of IBV.The viral RNA of SD/97/02 strain was amplified by reverse transcription polymerase chain reaction(RT PCR).The amplified product was analyzed by agarose gel electrophoresis,all appeared a fragment of 1657 bp as expected.The RT PCR product was cloned into pMD18 T vector,and then the recombinant plasmid was sequenced.The result suggested that it was the S1 spike gene of IBV.The recombinant plasmid was designated pMDQXS1.The sequence has been published in the Genbank,and the accession number is AF193423.Sequence analysis showed that the S1 gene has low G+C content at about 37.0%.There existed several RE cleavage sites such as Hin d III, Bam H I, Bgl III, Sac I and Sal I,but have no Eco R I site,which is different from that of other strains.Its homology compared with other IBV strains was between 87.02% and 94.21%.In the site between 154 and 429 of this S1 gene is a highly variation r

鳜鱼病毒结构特征与形态发生

方勤 艾桃山 汪亚平 朱作言

2001, 16(4): 382

从患流行病的鳜鱼脾脏组织超微切片中观察到大量的病毒颗粒。该完整病毒颗粒直径约 135nm± 10 ,具包膜。成熟病毒核壳体约 90nm± 5 ,包膜厚度约 18nm± 3,核壳体与包膜间的非电子致密区约有 2 7nm± 2。通过对不同发病阶段发病鳜鱼脾脏组织切片的电镜观察 ,在感染初期的鳜鱼脾脏组织观察到病毒的吸附及典型的内吞入侵方式 ,在感染中后期的脾脏组织细胞质内观察到病毒发生基质及病毒核壳、包膜形成与病毒的释放。此外 ,在染病鳜鱼的肾、心、肝、及鳃组织亦观察到相同结构的病毒粒子。回接实验证实该病毒为引起鳜鱼暴发流行病的病原 The structure and morphogensis of Mandarin fish( Siniperca chuats ) virus were studied.The matured virons were about 135nm±10 in diameter with envelope outside.The intact virus is composed of three parts,its nucleocapsid was about 90nm±5,and the envelope was around 18nm±3,the air space between core and envelope was about 27nm±2?The morphogenesis of the virus was observed in the ultra thin section of spleen tissue cells with outbreak lethal disease,which were collected in different stages of infected mandarin fish.At early stage infected fish section,it showed typical virus entry into cell cytoplasm via viropexis.There were many viromatrix,unmatured,matured and viron released phases,which were found in middle and late phase of diseased fish.In addtion,the same virions were also detected in diseased kidney,liver,heart and gill tissue.Artificial infected fish also caused acute lethal disease with same virus found in its tissue section.

应用噬菌体展示随机肽库淘筛mAb 5H5识别的抗原表位

李光玉, 白雪帆, 潘蕾, 杨为松

2001, 16(4): 386

汉滩病毒 随机肽库 单克隆抗体 表位 Hantaan virus Random peptides library Monoclonal antibodies Epitope
Brief Reports

茶毛虫核型多角体病毒【EpNPV】多角体蛋白基因的定位及克隆

常国辉, 陈绳亮“, 罗保君, 李天宪

2001, 16(4): 390

茶毛虫核型多角体病毒 (Euproctispseudoconsper saNuclearPolyhedrosisVirus简称EpMPV) ,属于杆状病毒科核型多角体病毒属 ,能使茶毛虫染病死亡。EpNPV据报道最初由日本学者于 195 7年发现[1] ,随后在其它国... The polyhedrin gene of EpNPV was located on Hin d III E, Xba I B, Eco RI J, Kpn I A, Pst I F, Bam H I G fragments under stringent condition(68℃,in water),by using the DIG labeled Bam H I F fragment of AcMNPV DNA as the probe.In order to sequence the EpNPV polyhedrin gene,the Bam H I G fragment was cloned into pTZ19R vector.Then the fragment was digested by Xba I? Hin d III,and subcloned into pTZ19R.

RT-PCR检测蓝舌病毒技术的建立

张苜玮, 董长垣“, 涂攀, 郭淑芳, 陈晓, 严银芳

2001, 16(4): 393

Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment 7,a part of the 5′end of segment 7 was synthesized and amplified by RT PCR method.We adapted this method to test on 12 blood samples suspected to be with BTV and the blood sample of a health sheep as a negative control.The results suggested that eleven of the twelve samples were positive,one was negative.In order to compare the sensitivity and reliability of RT PCR technique,Vero cells were used to isolate BTV from these suspected samples and the results will be a control.The study proved that RT PCR is a rapid,sensitive and precise method for detection and identification of bluetongue virus in clinical samples,animal quarantine and scientific research.