大豆花叶病毒CP基因原核表达与抗血清制备

根据已报道的大豆花叶病毒(SMV)序列设计引物,扩增SMV的外壳蛋白(CP)基因,序列同源性分析结果表明,与目前已报道的SMV不同分离物CP基因核苷酸序列同源性为85.0%～96.8%,相应推测的氨基酸序列同源性为87.9%～98.9%.将CP基因插入原核表达载体pSBET后在大肠杆菌BL21(DE3)Plys S中诱导表达.通过12%SDS-PAGE和5%～20%梯度SDS-PAGE两次制备电泳纯化诱导产物,免疫家兔获得抗CP血清,Western blot分析对CP具有高度特异性.硫酸铵沉淀法与Protein A-Red Sepharose 亲和层析相结合提取IgG,获得效价达1∶ 3 800的一抗,可用于田间SMV病样检测.     

侵染辣椒的黄瓜花叶病毒CP基因的序列分析、原核表达及抗血清制备

从吉林长春感病辣椒上获得一黄瓜花叶病毒(Cucumber mosaic virus,CMV)分离物(CMV-CC),根据GenBank中已登录的CMV外壳蛋白(coat protein,CP)基因核苷酸序列设计简并引物,通过RT-PCR的方法克隆到了长度为657 bp的目的片段。序列分析表明,CMV-CC与CMVI组各分离物核苷酸同源性为93.2%-97.9%。根据完整CP基因核苷酸序列构建的系统进化树显示:38个CMV分离物可分为3个组,CMV-CC属于CMV的IB亚组。将CMV-CC CP基因与原核表达载体pET-22b(+)连接,在大肠杆菌BL21(DE3)诱导表达出分子量约27 kD的融合蛋白。表达的融合蛋白经树脂纯化后免疫家兔制备了抗血清。用间接ELISA测定抗血清效价为1/4 096。Western blotting分析表明制备的抗血清对CP有高度特异性,为准确、快速地检测CMV奠定了基础。     

大蒜A病毒CP基因的原核表达及抗血清制备

根据GenBank报道的大蒜A病毒(GarV-A)序列设计引物、扩增其外壳蛋白基因并进行序列分析.结果表明,GarV-A的CP基因与目前已报道的两种GarV-A不同分离物CP基因的核苷酸序列同源性为98％-99％;氨基酸序列同源性均为98％.将GarV-A CP基因插入表达载体pSBET,在大肠杆菌BL21(DE3)Plys E菌株中诱导表达.CP经12％SDS-PAGE和5％-20％SDS-PAGE两次纯化,免疫小鼠获得抗CP血清,Western blotting分析表明确定制备的抗体对CP具有高度特异性,ELISA检测表明制备的抗体能够与天然病毒离子结合,因此可以作为该病毒的检测.     

小麦黄花叶病毒RNA228kDa蛋白基因的表达及表达产物抗血清的制备

根据小麦黄花叶病毒（ Ｗ Ｙ Ｍ Ｖ） 核苷酸序列测定结果,将 Ｗ Ｙ Ｍ Ｖ Ｒ Ｎ Ａ２ 上的２８ ｋ Ｄａ 蛋白基因克隆到ｐ Ｅ Ｔ１１ａ 上,构建了原核表达载体ｐ Ｅ２８３９ 。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 分析表明,经 Ｉ Ｐ Ｔ Ｇ 诱导,２８ ｋ Ｄａ蛋白基因在大肠杆菌 Ｂ Ｌ２１（ Ｄ Ｅ３）ｐ Ｌｙｓ Ｓ 中得到高效表达。以含表达产物的凝胶为抗原,免疫家兔,首次制备了小麦黄花叶病毒 Ｒ Ｎ Ａ２ 蛋白特异性抗血清。     

侵染节瓜的小西葫芦黄花叶病毒CP基因的克隆、表达及其抗血清的制备

通过鉴别寄主反应、病毒部分序列测定确定了采自广州白云区表现花叶、斑驳症状的节瓜上的病毒为ZYMV。采用RT PCR方法扩增和克隆了该病毒的外壳蛋白基因 ,连接到原核表达载体pET 2 2b( )上。获得的重组子pET ZCP转化大肠杆菌BL2 1(DE3)后 ,用IPTG进行诱导表达。SDS PAGE和Westernblot分析表明 ,CP基因在大肠杆菌中获得了高效表达 ,融合蛋白分子量约为 33 0kD。将融合蛋白纯化后免疫兔子 ,获得了特异性较高的抗血清。ELISA测定其效价为 1 4 0 96     

紫藤脉花叶病毒cp基因在大肠杆菌中的表达及抗血清的制备

采用RT-PCR方法自紫藤脉花叶病毒北京分离物(WVMV-BJ)的基因组中分离出其CP基因,连接到原核表达载体pET22b( )上。获得的重组子pET-WVMVCP转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE和Western blot分析表明,cp基因在大肠杆菌中获得了高效表达,融合蛋白分子量约为34．4kDa。将融合蛋白纯化后免疫兔子,获得了特异性较高的抗血清。微量免疫沉淀法测定该抗血清的效价为1／1024,酶联法(enzyme-linked immunosorbant assay,ELISA)测定的效价为1／8192。     

黄瓜花叶病毒CP基因原核表达及抗血清的制备

把黄瓜花叶病毒 (CMV)西番莲分离物的外壳蛋白 (CP)基因 ,通过BamHI SacI位点定向插入pET 2 2b( )载体 ,转化大肠杆菌BL2 1 (DE3)中 ,经IPTG诱导下 37℃培养 6h ,SDS PAGE电泳示表达蛋白分子质量为 31 8kDa ,表达量占菌体总蛋白的 2 8 9%,表明该蛋白得到了高效表达。用冰冷的氯化钾溶液显色 ,用表达的特异蛋白质条带制备抗原 ,免疫家兔制备出病毒特异抗血清。采用ID ELISA测定抗血清效价为 1 0 -6;抗血清和CMV几个分离物均有特异反应 ,和TMV、菌体蛋白不发生非特异性反应 ;检测病毒灵敏度达 30ng ml,能够从稀释 31 2 5倍的感病植物汁液中检测出病毒     

高粱花叶病毒外壳蛋白的原核表达及其抗血清制备

高粱花叶病毒(Sorghum mosaic virus,SrMV)是世界上分布最广的侵染甘蔗的病毒之一,引起甘蔗花叶病,对甘蔗产业危害很大。根据SrMV外壳蛋白(coat protein,CP)基因序列合成一对引物,以海南(SrMV-HN)染病植株总RNA为材料,采用RT-PCR方法克隆了长约987 bp的目的片段。将CP基因与质粒pET32a(+)连接,构建了含SrMV CP基因的融合蛋白原核表达载体pET32a-SrMVCP。然后用正确的重组质粒转化大肠杆菌Rosetta(DE3),经IPTG诱导后,SDS-PAGE检测出一条约36kD的特异融合蛋白表达谱带。融合蛋白主要以可溶性蛋白形式稳定表达。通过优化诱导条件,确立了CP基因表达的最佳条件:IPTG终浓度为0.1 mmol/L,诱导时间为4 h,诱导温度为30℃。用Ni2+-NTA琼脂糖亲和层析纯化融合蛋白,免疫家兔制备出抗血清。通过酶联法(ID-ELISA)测定本试验制备的SrMV CP抗血清工作浓度为1∶1 000,Western blotting检测结果表明,抗血清与SrMV-HN诱导表达的CP蛋白发生特异性反应。对田间25个甘蔗样品进行检测,结果表明该抗血清的效价高、灵敏度高、特异性强,可以应用于田间样品的检测。     

甘蔗花叶病毒E株系外壳蛋白基因原核表达载体的构建与表达

目的:用原核表达的方法获取大量带6个His标记的甘蔗花叶病毒E株系(ScMV-E)外壳蛋白(CP)。方法:用带有BamHⅠ和SalⅠ酶切位点的特异引物,以带有多个基因的重组质粒pNUSCP为模板,扩增出片段长度为942bp的ScMV-E外壳蛋白基因,亚克隆到pMD18-T载体上,转化E.coliDH5α,经双酶切检测获得阳性克隆。BamHⅠ和SalⅠ双酶切阳性克隆质粒,回收目的片段ScMV-E的CP基因。把目的片段插入表达载体pET29a( ),转化E.coliBL21(DE3),测序。结果:阳性质粒pET29a-CP在E.coliBL21(DE3)中得到大量特异表达。SDS-PAGE分析表明,该蛋白的相对分子质量约36000,与预测一致。结论:以上方法可以得到带6个His标记的目的蛋白,有利于纯化并获取高纯度的ScMV-E的外壳蛋白。     

紫藤脉花叶病毒cp基因在大肠杆菌中的表达及抗血清的制备

采用RT-PCR方法自紫藤脉花叶病毒北京分离物(WVMV-BJ)的基因组中分离出其CP基因,连接到原核表达载体pET22b(+)上.获得的重组子pET-WVMVCP转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达.SDS-PAGE和Western blot分析表明,cp基因在大肠杆菌中获得了高效表达,融合蛋白分子量约为34.4 kDa.将融合蛋白纯化后免疫兔子,获得了特异性较高的抗血清.微量免疫沉淀法测定该抗血清的效价为1/1024,酶联法(enzyme-linkedimmunosorbant assay,ELISA)测定的效价为1/8192.     

香蕉花叶病毒外壳蛋白基因克隆及表达载体的构建

从海南大田感染香蕉花叶病的香蕉叶片 ,获得香蕉花叶病毒 ,提纯其 RNA,在 AMV反转录酶作用下合成 c DNA第一链 ,经 PCR扩增 ,获得一约 70 0 bp的 DNA片段 ,测序结果显示所克隆的 DNA片段包含一完整的香蕉花叶病毒株系 ( CMV-BHI)外壳蛋白基因 ,长度为 6 5 7bp,然后将此 DNA片段 ,分别克隆到p BI1 2 1和 p KHG4质粒 ,构成两个含 Ca MV35 s启动子 ( 5 '-端 )、NOS终止子 ( 3'-端 )和分别含 NPT 标记基因和 NPT 及 HPT标记基因的植物表达载体 ( p TBB和 p TBK)。然后用 p AHC1 8中的 UBI promoter换下p BI1 2 1的 Ca MV35 s promoter,构成 p BIAH;再用 CMV-BHI外壳蛋白基因换下 p BIAH中 GUS基因 ,构成一含单子叶植物启动子 UBI和 NPT 标记基因的植物表达载体 ( p TBBU)。从而为 CMV-BHI外壳蛋白基因在香蕉中表达打下了基础     

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell-to-cell and long-distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79–128 in MP are vital for the MP–CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79–128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MP^T107) is important for the MP–CP interaction. Substitution of T107 with alanine (MP^T107A) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP–CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP-independent manner. Moreover, two movement function-associated point mutants (MP^F17A and MP^D97A) failed to cause systemic infection in plants without impacting on the MP–CP interaction. Furthermore, we have found that co-expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid-associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP–CP interaction facilitates virus systemic infection.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3′-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and Cl proteins.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3'-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and Cl proteins.     

小西葫芦黄花叶病毒外壳蛋白抗体制备

根据已报道的小西葫芦黄花叶病毒（ZYMV）序列设计特异引物,扩增ZYMV的全长外壳蛋白（CP）基因,插入原核表达载体pSBET后在大肠杆菌B121（DE3）plysS中诱导表达。通过12％SDS—PAGE和5％～20％梯度SDS—PAGE二次制备电泳纯化诱导产物,免疫小鼠,获得经过Western blot分析为特异的抗CP血清。硫酸铵沉淀法与ProteinA—Red Sepharose亲和层析相结合提取IgG,获得效价达1：4800的一抗,对西瓜和甜瓜田间样品的间接ELISA检测表明,ZYMV在田间普遍发生,研究制备的IgG可用于ZYMV检测。