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水稻黑条矮缩病毒(RBSDV)p8蛋白由其基因组片段S8编码,根据RBSDV浙江分离物S8序列(AJ297431)设计特异性引物扩增编码p8蛋白N端部分的片段,并亚克隆至原核表达载体pET-32a ,然后以大肠杆菌BL21plysS为宿主菌进行高水平地表达,利用纯化的p8蛋白免疫小鼠,制备了p8蛋白的特异性抗血清。Western blot分析表明,p8蛋白在水稻病株中含量较为丰富,易于检测,而且p8蛋白参与病毒粒子的组成,表明p8是一个病毒结构蛋白。ELISA检测显示p8蛋白的抗血清可与不同来源的水稻、小麦、玉米病汁液发生强烈的血清学反应,表明该抗血清适用于RBSDV田间病株的快速诊断。 相似文献
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灰飞虱Laodelphax striatellus的囊泡相关膜蛋白相关蛋白B(VAP-B)是内质网膜蛋白家族的蛋白,其主要参与调节囊泡运输、脂类的转运代谢以及未折叠蛋白应答等。本研究克隆了灰飞虱VAP-B基因,构建了pCold-SUMO-VAP-B原核表达载体,再将其转化大肠杆菌感受态细胞Rosetta(DE3),经过IPTG低温诱导表达得到了含有HIS和SUMO标签的VAP-B可溶性蛋白,经过Ni2+-NTA亲和层析柱纯化后的蛋白再利用rTEV酶去除SUMO标签,得到无标签的VAP-B蛋白。通过免疫新西兰雄兔制备了VAP-B的多克隆抗体。Western blot检测结果表明该抗体可以特异性检测到灰飞虱的VAP-B蛋白;同时利用该抗体进行免疫荧光标记,发现其能特异性定位灰飞虱唾液腺内的VAP-B蛋白。研究结果表明制备的VAP-B抗体能够成功用于该蛋白的体内外检测,为阐明VAP-B在灰飞虱体内的关键作用奠定了基础。 相似文献
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本实验提取被菜蛾盘绒茧蜂寄生后24h的小菜蛾幼虫体内总RNA,反转录合成cDNA,以此为模板PCR扩增出菜蛾盘绒茧蜂多分DNA病毒(Cotesia vestalis polydnavirus,CvBV)EP-1-like基因,将其分别克隆到表达载体pET30a、pET28a中,转化宿主菌BL21(DE3),获得单克隆重组质粒pET-30a-EP1L和pET-28a-EP1L。经IPTG诱导,pET-28a-EP1L成功表达了约34.8kDa的包涵体蛋白。将诱导条件优化以后,采用割胶回收的方法纯化包涵体,将纯化后的表达蛋白免疫新西兰大耳兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1:128000,Western blot检测证明抗体具有良好的免疫反应特异性。 相似文献
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本研究是利用异源表达的小麦黄花叶病毒(Wheat yellow mosaic virus, WYMV)复制酶Nib蛋白创建了病毒核酸的体外复制系统。将WYMV的Nib编码序列扩增并插入到大肠杆菌表达载体pMAL-C2X中以构建原核表达质粒pMAL-WY-Nib。0.4 mmol·L-1 IPTG诱导可特异性表达分子量约为100 kDa的MBP-Nib融合蛋白。根据温度梯度测定,20℃下诱导MBP-Nib的可溶性比例较高,约为30％,可满足MBP标签的亲和层析。通过与WYMV的其他蛋白和烟草丛顶病毒的RdRp进行比较,纯化的MBP-Nib融合蛋白可以特异性识别WYMV RNA1和RNA2的3′末端区域并体外合成其互补链,此体外转录体系可用于WYMV复制调控的研究。 相似文献
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表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染 总被引：7，自引：0，他引：7
利用RT-PCR分别克隆了CMV P3613株系的RNA2片段、MP(movement protein)基因片段及CMV AN株系的CP(coat protein)基因片段。以CP基因为中间间隔序列,分别构建了含有RNA2片段和MP基因反向重复片段的原核表达载体。体外转录试验表明:两个载体转录后都能形成预期大小的dsRNA。经过IPTG诱导,在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase和RNaseA消化处理,证实为dsRNA。将表达病毒基因dsRNA的细菌超声破碎后处理烟草,进行保护和治疗试验,结果表明:表达CMV MP基因和RNA2片段dsRNA的细菌破碎液能够诱导烟草对CMV产生抗性。接种病毒60d后,保护效果试验病株率分别为45%和60%,治疗效果试验病株率分别为75%和85%,而其他对照发病率均为100%。本研究结果证明了利用RNA沉默的原理,构建具有反向重复序列的原核表达载体,用细菌表达dsRNA的粗提取物可防治CMV对烟草的侵染。 相似文献
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采用RT-PCR方法克隆了编码禾谷镰刀菌单端孢酶烯3-O-乙酰转移酶Tri101基因的cDNA序列,并连接到原核表达载体pGEX-4T2上,将获得的重组载体pGEX-4T2/Tri101转化大肠杆菌BL21后用IPTG进行诱导表达。SDS-PAGE和Western blot分析表明,经IPTG诱导后,Tri101基因在大肠杆菌BL21中获得了高效表达,融合蛋白GST-Tri101分子量为75.45 kDa。将该融合蛋白切胶纯化后免疫家兔,制备兔抗GST-Tri101多克隆抗体。经ELISA法测定抗体效价大于1∶256 000。Western blot分析表明制备的抗体与原核细胞体外表达的Tri101蛋白可以特异性结合,表明该抗体的特异性良好。应用该抗体验证了感赤霉病小麦中Tri101基因的表达。兔抗GST-Tri101抗体的成功制备,为进一步研究Tri101的生物学功能、细胞定位以及在其它植物中的表达等奠定了基础。 相似文献
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根据同源分析,在脱水素wzy1-2基因序列中选择298bp的cDNA序列作为干扰片段,将其插入高效植物干扰表达载体pTCK303的多克隆位点,成功构建含反向重复序列的RNA干扰表达载体。采用基因枪法轰击2512个郑引1号小麦的幼胚愈伤组织,获得再生植株26株,对其T_0代再生植株进行特异引物PCR检测,获得6株阳性植株,阳性转化率为0.24%。对脱水素wzy1-2基因实时定量分析发现,小麦中目的基因表达量显著下降。为进一步了解该基因在小麦整个生长过程中表达模式,选取2个不同抗旱性小麦品种(陕合6号和郑引1号),分别在小麦4个不同生长时期(苗期、分蘖期、拔节期和开花期)进行干旱胁迫处理,分析脱水素wzy1-2基因的表达规律。结果表明,随着干旱程度增加小麦WZY1-2蛋白的表达量升高;苗期小麦WZY1-2蛋白的表达量均高于其它3个生长时期。Western blot分析显示陕合6号和郑引1号的非特异性条带单一,表明小麦脱水素wzy1-2是干旱诱导型基因。 相似文献
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抑制南方水稻黑条矮缩病毒非结构蛋白P5-1的表达阻碍病毒在昆虫体内的增殖 总被引：2，自引：2，他引：0
为明确南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)编码的非结构蛋白P5-1参于SRBSDV在介体白背飞虱体内侵染过程中的作用机制,通过原核表达蛋白制备SRBSDV编码的非结构蛋白P5-1的多克隆抗体,并应用Western blot和免疫荧光标记法检测抗体的特异性,以注射法将来源于P5-1基因的dsRNA(ds P5-1)注入获毒1 d的白背飞虱体内,5 d后通过免疫荧光标记法检测ds P5-1对SRBSDV在白背飞虱体内增殖的影响,同时以注射来源于GFP基因的dsRNA(ds GFP)为对照。结果显示,Western blot和免疫荧光标记分别检测到SRBSDV侵染水稻和白背飞虱表达的P5-1蛋白,表明所制备的P5-1抗体具有特异性。ds GFP处理的对照组白背飞虱带毒率高达81%,而ds P5-1处理的白背飞虱带毒率仅为21%,且P5-1蛋白的表达和SRBSDV在昆虫体内的增殖均受到抑制。表明P5-1蛋白是病毒在昆虫体内增殖的关键因子,可作为阻断病毒在昆虫体内增殖的理想靶标。 相似文献
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利用Bac-to-Bac杆状病毒表达系统,在Sf9昆虫细胞中表达抗对硫磷单链抗体,并评价该重组抗体scFv-4C6的分子识别活性。以分泌能特异性识别对硫磷的单克隆抗体的杂交瘤细胞株4C6为RNA来源,采用RT-PCR方法扩增抗体的重链和轻链可变区基因,经重叠延伸PCR方法串联拼接获得单链抗体基因片段(scFv-4C6)。构建包含目的片段的重组杆粒Bacmid-scFv-4C6,转染Sf9细胞表达目的蛋白,采用免疫印迹法(Western blotting)检测表达产物,间接竞争酶联免疫吸附(ic-ELISA)法评价产物的生物活性。结果表明:scFv-4C6基因片段拼装正确,成功转染Sf9细胞,并在转染后72 h表达量最高,表达的单链抗体大小为28.3 kD;表达产物能特异性识别对硫磷,IC50值为7.9 ng/mL,对甲基对硫磷和杀螟硫磷分别有12%和1.8%的交叉反应率,与亲本单克隆抗体的识别性能相似。该研究表明,具有生物活性的抗对硫磷单链抗体scFv-4C6可在昆虫细胞中成功得到表达。 相似文献
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A soil-borne wheat virus causing severe mosaic and stunting symptoms on wheat in China has been characterized. It had been considered to be soil-borne wheat mosaic virus (SBWMV) because of its rod-shaped virions and similarities to epidemiology and host range. In this study, the virions purified from infected wheat tissue were approximately 20 nm in diameter and of two lengths (140–160 nm and 280–300 nm), with a coat protein of 19 kDa and two RNA components of approximately 7 and 3.5 kb. A rabbit antiserum was produced against the virus and a serological relationship to SBWMV from the USA (Oklahoma) was demonstrated. However, the coat protein was not recognized by most monoclonal antibodies against Oklahoma SBWMV in either ELISA, ISEM or Western blot analysis, indicating epitope differences. In RT-PCR experiments the viral nucleotide sequences were significantly different from those of SBWMV, and this was confirmed by partial sequencing of the cloned PCR fragments generated from RNA1 ( c . 1100 nt) and RNA2 ( c . 1400 nt), which showed homologies of about 79 and 63%, respectively, to corresponding regions of SBWMV. Because of these significant differences in serology and nucleotide sequence it is suggested that it is a new furovirus for which the name Chinese wheat mosaic virus (CWMV) is proposed. 相似文献
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J. B. Valente F. S. Pereira L. A. Stempkowski M. Farias P. Kuhnem D. Lau T. V. M. Fajardo A. Nhani Junior R. T. Casa A. Bogo F. N. da Silva 《Plant pathology》2019,68(3):588-600
Soilborne wheat mosaic disease (SBWMD), originally attributed to infections by Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV), is one of the most frequent virus diseases and causes economic losses in wheat in southern Brazil. This study aimed to characterize molecularly the viral species associated with wheat plants showing mosaic symptoms in Brazil. Wheat leaves and stems displaying mosaic symptoms were collected from different wheat cultivars in Passo Fundo municipality, Rio Grande do Sul State, southern Brazil. Double-stranded RNA was extracted and submitted to cDNA library synthesis and next-generation sequencing. No sequences of SBWMV and WSSMV were detected but the complete genome sequence of a putative new member of the family Benyviridae was determined, for which the name wheat stripe mosaic virus (WhSMV) is proposed. WhSMV has a bipartite genome with RNA 1 and RNA 2 organization similar to that of viruses belonging to Benyviridae. WhSMV RNA 1 has a single open reading frame (ORF) encoding a polyprotein with putative viral replicase function. WhSMV RNA 2 has six ORFs encoding the coat protein, the major protein (read-through), triple gene block movement proteins (TGB 1, 2 and 3) and ORF 6 (hypothetical protein). In addition to the genomic organization and nucleotide and amino acid sequence identities, phylogenetic analyses also corroborated that WhSMV is a virus species of the Benyviridae. However, isolates of WhSMV formed a clade distinct from members of the genus Benyvirus. It was also demonstrated that the plasmodiophorid Polymyxa graminis is associated with wheat roots showing SBWMD symptoms and infected by WhSMV. 相似文献
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小麦矮缩病毒外壳蛋白基因的原核表达、抗体制备及应用 总被引：1，自引：0，他引：1
小麦矮缩病毒(Wheat dwarf virus,WDV)引起的小麦矮缩病是近年来我国小麦生产中的一种重要病毒病害,急需研发快速精准的检测技术用于预测预报和病毒-介体相互作用的研究。本研究应用Gateway重组技术构建了外壳蛋白基因(Coat protein, CP)的原核表达载体,将重组表达载体转化大肠杆菌Rosetta,经IPTG诱导获得CP基因原核表达蛋白。以重组蛋白为抗原免疫新西兰大白兔制备得到了相应的抗体,Western blot检测表明制备的抗体能与CP重组蛋白、感病小麦和带毒叶蝉特异性结合,说明获得的抗体特异性高。用获得的抗体进行免疫荧光标记,观察到病毒分布在介体叶蝉的前中肠和中中肠部位,为WDV的预测预报和介体条沙叶蝉传毒机制的研究奠定了基础。 相似文献
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在实验室中利用灰飞虱接种小麦时出现一种新的病毒病症状,鉴定表明其病原为大麦黄条点花叶病毒(Barley yellow striate mosaic virus, BYSMV)。采用生物学测定、电镜观察、RT-PCR检测和序列分析的方法,明确了该病毒的粒体特性、危害症状及田间发生情况。接种试验表明该病毒通过灰飞虱传播,接种7~10 d后小麦新生叶片出现黄色斑点、斑驳,继而发展成黄色条纹,叶片对生且细而窄,重病株新叶扭曲,叶鞘不能伸长,病株矮化。对小麦病叶超薄切片电镜观察,发现细胞质中存在大量弹状病毒粒子,病毒粒体大小为（315~353）nm×（46~57） nm。利用特异性引物从病株总RNA中扩增出 565 bp基因片段,序列同源性分析显示与大麦黄条点花叶病毒(Barley yellow striate mosaic virus, BYSMV) Zanjan-1分离物聚合酶（L）基因对应序列的一致性为97 %,与北方禾谷花叶病毒(Northern cereal mosaic virus, NCMV) L基因对应序列的一致性为78 %~79 %。对采自河北邯郸、石家庄、保定、唐山的31株样品进行RT-PCR检测,25株检测到BYSMV,7株检测到水稻黑条矮缩病毒(Rice black streaked dwarf virus,RBSDV),其中5株为2种病毒复合侵染,结果表明BYSMV的田间分布较广。系统发育分析表明BYSMV-Lab/TS/ZX/QY 4个分离物与本研究的BYSMV亲缘关系密切。BYSMV是我国小麦上新发现的一种弹状病毒,并已形成危害,暂定名为小麦黄条纹矮缩病,应加强流行动态监测。 相似文献
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采用RT-PCR法从感染齿兰环斑病毒(Odontoglossum ringspot virus, ORSV)贵州分离物的苋色藜叶片中扩增出病毒的依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase, RdRp)基因保守序列。测序结果显示,该保守片段长516 bp,编码171个氨基酸残基。构建了该片段的原核表达载体pET32a-ORSV RdRp并转化BL21(DE3)菌株,在25℃以0.4 mmol·L-1 IPTG诱导表达重组蛋白。SDS-PAGE分析表明,重组蛋白分子量约为36 kDa,与预测相符合。将该重组蛋白作为抗原免疫BABL/C小鼠,制备的多克隆抗体效价达1∶102 400。间接ELISA结果表明,该多抗具有较强的特异性,能检测到ORSV感染的病叶汁液中的RdRp,而与其它感染4种同属或不同属病毒的病叶汁液不发生血清学交叉反应,本实验为进一步研究RdRp的结构和功能以及从分子水平上探讨该病毒的致病机制奠定基础。 相似文献
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N. L. Robertson 《Plant pathology》2004,53(5):569-576
A new virus named Nootka lupine vein-clearing virus (NLVCV) was isolated from Lupinus nootkatensis plants that were confined to a relatively small area in the Talkeetna mountains of south-central Alaska. Annual surveys (2000–03) consistently found leaf symptoms of pronounced vein clearing and mosaic on 3- to 4-week-old plants in late June. Spherical particles ≈30 nm in diameter were isolated from these leaves. Virions contained a single-stranded RNA of ≈4·0–4·2 kb and one species of capsid protein estimated to be ≈40 kDa. The double-stranded RNA profile from naturally infected leaves consisted of three major bands ≈4·2, 1·9 and 1·5 kbp. Protein extractions from either sap or virions of diseased plants reacted to polyclonal antiserum made against the virions in Western blot assays. A predicted PCR product ≈500 bp was synthesized from virion RNA using primers specific to the carmovirus RNA-dependent RNA polymerase (RDRP) gene. The nucleotide sequence of the amplified DNA did not match any known virus, but contained short regions of identity to several carmoviruses. Only species belonging to the Fabaceae were susceptible to NLVCV by mechanical inoculation. Based on dsRNA profile, size of virion RNA genome and capsid protein, and similarity of the RDRP gene to that of other carmoviruses, it is suggested that NLVCV is a member of the family Tombusviridae , and tentatively of the genus Carmovirus . As the host range, RDRP gene and dsRNA profile of NLVCV are different from those of known viruses, this is a newly described plant virus. 相似文献