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苹果坏死花叶病毒CP基因原核表达及其抗血清制备
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作者 邢飞 王红清 李世访 《西南大学学报(自然科学版)》 CAS CSCD 北大核心 2024年第4期63-69,共7页
苹果坏死花叶病毒(Apple necrotic mosaic virus,ApNMV)是近年新发现的病原物,并且是与我国苹果花叶病症状高度相关的重要病毒,进行ApNMV外壳蛋白(coat protein,CP)基因原核表达、制备多克隆抗血清,以建立快速、灵敏、准确的ApNMV常规... 苹果坏死花叶病毒(Apple necrotic mosaic virus,ApNMV)是近年新发现的病原物,并且是与我国苹果花叶病症状高度相关的重要病毒,进行ApNMV外壳蛋白(coat protein,CP)基因原核表达、制备多克隆抗血清,以建立快速、灵敏、准确的ApNMV常规检测方法尤为必要.通过设计特异性引物,以RT-PCR方法成功获得ApNMV CP基因序列,插入到原核表达载体pET-28a(+)中构建重组质粒,转化至大肠杆菌BL21(DE3),利用IPTG进行重组蛋白(含His-tag)诱导表达.SDS-PAGE和Western blot分析结果表明,CP基因在大肠杆菌中获得了高效表达,用Ni Sepharose 6 Fast Flow进行蛋白纯化,回收共得到重组蛋白2.4 mg.在屏障环境下免疫2只SPF级新西兰兔,制备出多克隆抗血清,稀释400倍后仍能与ApNMV阳性苹果叶片样品发生免疫反应.由此证明,本研究建立的ApNMV间接ELISA检测方法具有较好的灵敏性,检测效率高,能够用于田间大量样品ApNMV的诊断. 展开更多
关键词 苹果坏死花叶病毒 外壳蛋白基因 原核表达 抗血清 酶联免疫吸附分析方法
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线虫surface coat proteins提取方法的建立与双向电泳分析 被引量:1
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作者 黄薇 田宝玉 +4 位作者 郭菁 蔡婉玲 高媛媛 柯崇榕 黄建忠 《生物技术》 CAS CSCD 北大核心 2010年第2期30-33,共4页
目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础。方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋... 目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础。方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋白提取沉淀方法,进而采用SDS-PAGE电泳技术和双向电泳技术对所提蛋白进行评价。结果:通过35%乙醇结合TCA-丙酮沉淀法获得的质量较好的线虫SCPs,在12%的SDS-PAGE分析中该法提取的蛋白背景浅,蛋白条带多且清晰尖锐,含有丰富的蛋白信息量。通过双向电泳分析,可从提取的蛋白中鉴定出清晰蛋白点400多个。随机选择5个蛋白斑点,进行基质辅助激光解吸电离飞行时间质谱鉴定,鉴定得到高度匹配的已知线虫蛋白质2个。结论:所建立的方法可为今后研究线虫surface coat蛋白质组学及线虫病理生理学提供重要工具。 展开更多
关键词 线虫 SURFACE coat proteins 蛋白提取 双向电泳 MALDI-TOF质谱
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P1 of strawberry vein banding virus, a multilocalized protein, functions as a movement protein and interacts with the coat protein 被引量:1
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作者 RUI Peng-huan WANG Zhan-qi +5 位作者 SHAN Wen-shu XIA Wei-wei ZHOU Xiu-hong YANG Lian-lian JIANG Lei JIANG Tong 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2022年第4期1071-1083,共13页
Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of ea... Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus. 展开更多
关键词 strawberry vein banding virus P1 protein movement protein coat protein virus movement
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Transformation of Coat Protein Encoding Gene from Soil-Borne Mosaic Virus into Wheat
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作者 PANGJun-lan XUHui-jun +6 位作者 DULi-pu YEXing-guo LILian-cheng XINZHi-yong MAYou-zhi DIAOAi-po AdamsMJ 《Agricultural Sciences in China》 CAS CSCD 2003年第4期355-358,共4页
CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat variety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 -5 mg L-1 Bialaphos were obtained, 21 plants wer... CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat variety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 -5 mg L-1 Bialaphos were obtained, 21 plants were identified to be positive in T0 generation by PCR-Southern test, and the transformation frequency had 0.99%. T1 plants were further tested by PCR and Southern hybridization. Results demonstrated that the alien resistance gene had been integrated into the wheat genome. The segregation ratio of CP1+ to CP1- in T1 generation was 1.0 to 1. 3, and didn't agree with Mendelian rule. RT-PCR result from T2 plants showed that the alien gene CWMV-CP1 had stable expression in wheat genetic background. 展开更多
关键词 WHEAT BOMBARDMENT coat protein gene Transgenic wheat Molecular analysis
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Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus
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作者 LIU Mei LIU Li-ming +2 位作者 WU Hui-jie KANG Bao-shan GU Qin-sheng 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2020年第1期153-163,共11页
Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMM... Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP. 展开更多
关键词 Cucumber green mottle mosaic virus coat protein subgenomic promoter
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Sweet Potato Leaf Curl Virus: Coat Protein Gene Expression in <i>Escherichia coli</i>and Product Identification by Mass Spectrometry
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作者 Dina Lida Gutierrez Reynoso Rodrigo A. Valverde Norimoto Murai 《American Journal of Plant Sciences》 2015年第19期3013-3024,共12页
Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (R... Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strain XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 μg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltosebinding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of SPLCV based on the fact that the mass spectrometry elucidated the sequences corresponding to 37% of amino acid positions of the SPLCV coat protein. 展开更多
关键词 Affinity Chromatography Purification coat protein Escherichia coli Mass Spectrometry MALTOSE Binding protein Sweet Potato LEAF CURL VIRUS
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Transgenic Tobacco Lines Expressing Yam Mosaic Virus Coat Protein-Derived dsRNA Are Resistant to Yam Mosaic Virus
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作者 Effiom Eyo Ita Edak Aniedi Uyoh +1 位作者 Ikuo Nakamura Valentine Otang Ntui 《American Journal of Plant Sciences》 2020年第9期1487-1504,共18页
Yam mosaic virus (YMV), a Potyvirus, is a highly destructive pathogen of yam accounting for yield losses up to 40%. Apart from causing significant reduction in tuber size and quality, it restricts international exchan... Yam mosaic virus (YMV), a Potyvirus, is a highly destructive pathogen of yam accounting for yield losses up to 40%. Apart from causing significant reduction in tuber size and quality, it restricts international exchange of germplasms. It thus becomes crucial to get resistant or at least virus-free planting materials for farmers. This study was aimed at inducing resistance to YMV in tobacco by RNA silencing. An RNAi construct containing 161 bp fragment of <span style="font-family:Verdana;">YMV-coat protein (CP) </span><span style="font-family:Verdana;">gene was developed and used to produce transgenic tobacco lines expressing </span><span style="font-family:Verdana;">YMV-coat protein (CP)</span><span style="font-family:Verdana;"> derived </span><span style="font-family:Verdana;">double stranded RNA (dsRNA)</span><span style="font-family:""><span style="font-family:Verdana;"> via </span><i><span style="font-family:Verdana;">Agrobacterium</span></i><span style="font-family:Verdana;">-mediated transformation. Of the eight T</span><sub><span style="font-family:Verdana;">1</span></sub><span style="font-family:Verdana;"> transgenic lines inoculated with YMV, six (L1, L2, L3, L5, L7 and L8) showed immunity to YMV as no symptoms were detected, whereas two (L4 and L10) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and qPCR analyses of </span></span><span style="font-family:Verdana;">YMV-coat protein (CP)</span><span style="font-family:Verdana;">. </span><span style="font-family:Verdana;">The presence of small interfering RNAs in transgenic</span><span style="font-family:MinionPro-Capt;"> </span><span style="font-family:Verdana;">lines after virus challenge indicates</span><span style="font-family:Verdana;"> that the resistance was acquired through RNA silencing.</span> 展开更多
关键词 Yam Mosaic Virus DSRNA PTGS coat protein Gene RNAi
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Application of M13 Phage Coat Proteins
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作者 Zhi DING Ling ZHAO Xuemei MA 《Agricultural Biotechnology》 CAS 2013年第1期45-47,共3页
M13 phage'is a filamentous bacteriophage containing a circular single-stranded DNA molecular, which is surrounded by approximately 2 800 copies of protein PVIII. In addition, there are five copies of each of proteins... M13 phage'is a filamentous bacteriophage containing a circular single-stranded DNA molecular, which is surrounded by approximately 2 800 copies of protein PVIII. In addition, there are five copies of each of proteins PVII and PXI in one end, and five copies of each of proteins PVI and PUI on the other. These coat proteins have play an important role in the infection and assembly of M13 phage. With the development of phage display technology, these five coat proteins all can be used in phage display, which plays an increasingly important role in molecular detection and treatment. 展开更多
关键词 M13 phage coat protein Phage display technology
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李属坏死环斑病毒扬州分离物CP基因的原核表达及抗血清制备
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作者 王智磊 纪开燕 +5 位作者 赵景奎 丁诗文 徐小伟 秦朗 张坤 贺振 《扬州大学学报(农业与生命科学版)》 CAS 北大核心 2023年第6期140-146,共7页
李属坏死环斑病毒(prunus necrotic ringspot virus,PNRSV)严重危害蔷薇科植物,在世界范围内普遍分布。为建立快速有效的PNRSV检测方法,依据PNRSV CP基因序列设计引物,并从桃叶片上扩增PNRSV-CP片段,将双酶切产物插入原核表达载体pET28a... 李属坏死环斑病毒(prunus necrotic ringspot virus,PNRSV)严重危害蔷薇科植物,在世界范围内普遍分布。为建立快速有效的PNRSV检测方法,依据PNRSV CP基因序列设计引物,并从桃叶片上扩增PNRSV-CP片段,将双酶切产物插入原核表达载体pET28a中,获得重组质粒pET28a-CP^(PNRSV),转化大肠埃希菌Rosetta菌株,IPTG诱导重组蛋白表达。SDS-PAGE电泳检测显示,在分子量约25 ku处有蛋白质条带,符合预期大小。镍柱亲和纯化PNRSV CP蛋白,以其纯化蛋白质免疫健康新西兰白兔,制备多克隆抗血清。Western blot、ELISA、Dot blot检测结果显示,制备的抗血清能检测到PNRSV CP基因在感病植物叶片中表达,而在健康植株中未检测到;制备的抗血清滴度稀释至1∶64000时,仍能与纯化蛋白质发生特异性反应。综上,制备的PNRSV抗血清特异性强、效价高,可用于PNRSV的快速检测。 展开更多
关键词 李属坏死环斑病毒 cp蛋白 原核表达 抗血清
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地黄花叶病毒河南分离物的CP基因克隆及序列分析
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作者 冯陈尉 李正刚 +6 位作者 郭枭 庄新建 丁诗文 董卓倬 贺振 王铁霖 张坤 《扬州大学学报(农业与生命科学版)》 CAS 北大核心 2023年第4期113-118,共6页
为探究地黄花叶病毒(rehmannia mosaic virus,ReMV)在河南地黄上的发生与分布及其基因变异情况,从河南温县中草药园采集若干疑似受病毒侵染而呈现出花叶、枯萎等症状的地黄样品,用酚仿法提取地黄样品总RNA,通过PCR扩增目的基因地黄花叶... 为探究地黄花叶病毒(rehmannia mosaic virus,ReMV)在河南地黄上的发生与分布及其基因变异情况,从河南温县中草药园采集若干疑似受病毒侵染而呈现出花叶、枯萎等症状的地黄样品,用酚仿法提取地黄样品总RNA,通过PCR扩增目的基因地黄花叶病毒外壳蛋白(coat protein,CP)基因片段,经过回收纯化后,克隆至pMD19-T载体中,转化大肠杆菌DH5α,随机挑选4个阳性单克隆测序,最后将结果与NCBI数据库中已有的核苷酸序列进行比对,并结合NCBI数据库中已发布的不同地区ReMV分离物的CP基因序列,构建基于CP^(ReMV)核苷酸序列的ReMV不同地区分离物的系统进化树。结果表明:成功克隆出ReMV河南分离物的CP基因,将该序列上传至NCBI后获得GenBank登录号为OM964874;通过构建系统发育进化树,该分离物与郑州、山西、韩国报道的ReMV分离物亲缘关系较近,可能有相近传染源,而与美国、日本的分离物亲缘关系较远,存在一定的遗传分化,序列一致性分析结果与此一致。综上,这一研究扩大了ReMV群体的传播范围,丰富了其基因信息,为揭示ReMV的致病机制奠定了基础。 展开更多
关键词 地黄花叶病毒 外壳蛋白 基因克隆 进化树分析
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Genetic Characteristics of <i>Citrus Tristeza Virus</i>Isolates from Cultivated Citrus in China Based on Coat Protein Gene
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作者 Yiqun Chen Xiaoshang You +1 位作者 Bo Chen Long Yi 《Journal of Biosciences and Medicines》 2021年第10期190-200,共11页
<div style="text-align:justify;"> <em>Citrus tristeza virus</em> (CTV) is an important citrus pathogen causing considerable economic loss to citrus production. Knowledge on genetic evolutio... <div style="text-align:justify;"> <em>Citrus tristeza virus</em> (CTV) is an important citrus pathogen causing considerable economic loss to citrus production. Knowledge on genetic evolutionary of the CTV population in China remains limited. In this study, 1439 samples were collected from nine citrus-producing areas of China. The coat protein (CP) genes of CTV were amplified by RT-PCR, and sequenced to analyze the genetic evolution. Analysis of the base composition showed an AU preference pattern, with the GC content was lower than AU content. Nine CTV populations were clustered into one clade in neighbor-joining (NJ) tree, indicative of a close phylogenetic relationship among the populations in China. Analysis of molecular variation (AMOVA) revealed that 77.72% genetic variations of CTV populations were observed among populations, with an <em>F</em><sub>ST</sub> value of 0.223. The values of <em>d<sub>N</sub>/d<sub>S</sub></em> and neutrality test of <em>CP</em> gene were ranged from 0.016 to 0.082 and -1.377 to 1.456, respectively, the results suggesting that all of nine CTV populations were relatively constantly maintained under purifying selection. Our study demonstrated the genetic characteristics and molecular evolution relationship of CTV populations in China, and provided a theoretical basis for scientific control of CTV. </div> 展开更多
关键词 Citrus Tristeza Virus Cultivated Citrus coat protein Gene Genetic Evolution
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Capillary Zone Electrophoretic Separation of Basic Proteins with Coated Columns Prepared by Sol-Gel Technology
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作者 Fang LI Hui JIN +2 位作者 Ruo Nong FU Jun Ling GU Guang Ju LU(Department of Chemical Engineering & Materials Science, Beijing Institute of Technology, Beijing 100081) 《Chinese Chemical Letters》 SCIE CAS CSCD 1997年第9期793-796,共4页
Coated capillary columns were prepared by sol-gel technology and used in the separation of basic proteins with capillary zone electrophoresis. The results indicated that a significant decrease in protein adsorption wa... Coated capillary columns were prepared by sol-gel technology and used in the separation of basic proteins with capillary zone electrophoresis. The results indicated that a significant decrease in protein adsorption was obtained and EOF was also diminished to zero in the pH range of 3-10. 展开更多
关键词 Basic Capillary Zone Electrophoretic Separation of Basic proteins with coated Columns Prepared by Sol-Gel Technology
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Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells
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作者 Guo-cheng FAN Fang-luan GAO +5 位作者 Tai-yun WEI Mei-ying HUANG Li-yan XIE Zu-jian WU Qi-ying LIN Lian-hui XIE 《Virologica Sinica》 SCIE CAS CSCD 2010年第6期401-408,共8页
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8... To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells. 展开更多
关键词 外壳蛋白基因 水稻瘤矮病毒 昆虫细胞 杆状病毒转移载体 重组杆状病毒 杆状病毒表达系统 SF9细胞 荧光显微镜
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Hydrophilic Silica/Copolymer Nanoparticles and Protein-Resistance Coatings
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作者 Hongpu Huang Ling He 《Journal of Materials Science and Chemical Engineering》 2016年第1期18-23,共6页
Hydrophilic silica/copolymer nanoparticles of SiO<sub>2</sub>-g-P(PEGMA)-b-P(PEG) are prepared by silica surface-initiating atom transfer radical polymerization (SI-ATRP) of poly (ethylene glycol) methyl e... Hydrophilic silica/copolymer nanoparticles of SiO<sub>2</sub>-g-P(PEGMA)-b-P(PEG) are prepared by silica surface-initiating atom transfer radical polymerization (SI-ATRP) of poly (ethylene glycol) methyl ether methacrylate (PEGMA) and poly(ethylene glycol) methacrylate (PEG), by using Three molar ratios of SiO<sub>2</sub>-Br/PEGMA/PEG as 1/42.46/19.44, 1/42.46/38.88 and 1/42.46/77.76. Their temperature sensitive behaviour, pH response and surface properties as protein-resistance coatings are characterized. 220 nm core-shell nanoparticles as P(PEGMA)-b-P(PEG) shell grafted on SiO2 core are formed in water solution, which gained LCST at 60<sup>。</sup>C - 77<sup>。</sup>C and good dispersion in water when pH > 5.0. The water-casted films by SiO<sub>2</sub>-g-P(PEGMA)-b-P(PEG) obtain a little rough surface (Ra = 26.8 - 29.7 nm). While, the introduction of P(PEG) segments could slight increase the protein-repelling adsorption of SiO<sub>2</sub>-g-P(PEGMA)-b-P(PEG) films (△f = ?6.96 Hz ~ ?7.25 Hz) compared with SiO2-g-P(PEGMA) films (△f = ?9.5 Hz). Therefore, SiO2-g-P(PEGMA)-b-P(PEG) could be used as protein-resistance coatings. 展开更多
关键词 Silica/Copolymer Hydrophilic Nanoparticles Tem-Responsive protein-Resistance coatINGS
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利用酵母双杂交系统筛选与柑橘黄化脉明病毒CP互作的寄主因子
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作者 宾羽 张琦 +3 位作者 王春庆 赵晓春 宋震 周常勇 《中国农业科学》 CAS CSCD 北大核心 2023年第10期1881-1892,共12页
【目的】柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)是威胁我国柑橘产业稳定发展的主要病毒,但其在柑橘上的侵染和致病机制尚不清楚。本文以CYVCV的外壳蛋白(coat protein,CP)为诱饵筛选尤力克柠檬(Citrus limon Burm.... 【目的】柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)是威胁我国柑橘产业稳定发展的主要病毒,但其在柑橘上的侵染和致病机制尚不清楚。本文以CYVCV的外壳蛋白(coat protein,CP)为诱饵筛选尤力克柠檬(Citrus limon Burm.f.)c DNA文库,利用生物信息学方法分析与其互作的寄主因子在病毒侵染和致病过程中可能发挥的作用。【方法】Trizol法提取尤力克柠檬叶片总RNA,用SMART法反转录合成First-Strand c DNA,以其为模板通过Long-Distance PCR获得ds c DNA,均一化处理后与线性化p GADT7质粒重组链接构建尤力克柠檬初级c DNA文库,重组质粒转化大肠杆菌DH10B获得尤力克柠檬大肠杆菌c DNA文库并对文库质量进行鉴定;PCR扩增CYVCV的CP序列并构建到载体p GBKT7上,鉴定诱饵质粒对酵母细胞的毒性以及CP蛋白对酵母报告基因的自激活性。将尤力克柠檬c DNA文库质粒转化含有诱饵质粒p GBKT7-CP的Y2H Gold酵母菌株,共转化子依次涂布SD/-Leu-Trp、SD/-Leu-Trp-His/X-α-Gal和SD/-Leu-Trp-His-Ade/X-α-Gal平板,最终筛选蓝色且长势较好的阳性菌落,提取酵母质粒并测序比对获得候选基因,利用Uniprot在线网站的gene ontology(GO)注释候选基因,分析候选互作蛋白的生物学功能。根据分析结果,选取可能参与寄主抗病或症状发展的候选因子,扩增其CDS全长序列并构建到靶标载体p GADT7后分别与p GBKT7-CP共转化酵母细胞进行点对点酵母双杂交互作验证。【结果】尤力克柠檬大肠杆菌c DNA文库滴度为1.02×10^(8)cfu/m L,库容符合试验标准;成功构建酵母双杂交诱饵载体p GBKT7-CP,该载体没有自激活性且对酵母菌没有毒性;在SD/-Leu-Trp-His-Ade/X-α-Gal平板上筛选得到41个酵母阳性克隆,经序列相似性比对,去除重复,共筛得32个候选寄主因子;GO通路注释结果表明这些寄主因子参与多个叶绿体相关的生物过程,包括碳水化合物代谢、光合作用、光刺激反应、代谢分解和生物合成等过程;这32个寄主因子的分子功能多样,包括催化活性、水解酶活性、转移酶活性、蛋白质结合、转录因子活性和翻译因子活性等,其细胞组分涉及叶绿体、类囊体、膜、细胞质、细胞核和高尔基体等。从候选寄主因子中选取14个重要的蛋白与CP的一对一酵母双杂交验证结果表明,CP与这14个蛋白均发生互作。【结论】成功构建了尤力克柠檬c DNA文库,筛选出32个与CYVCV CP互作的尤力克柠檬寄主因子,分析重要的寄主蛋白功能,推测CYVCV CP通过与光系统Ⅱ放氧强化复合物组分蛋白(Psb P)、光系统I叶绿素结合蛋白(Lhca3)和1,5-二磷酸核酮糖羧化酶小亚基(Rbc S)等多个叶绿体相关蛋白的互作,影响光系统稳定、类囊体结构和叶绿素合成,从而导致光合作用降低以及叶绿体形态和功能受损,酵母点对点验证了CP与这些叶绿体相关因子的互作,这为揭示CYVCV CP参与病毒致病的分子机制提供了理论依据。 展开更多
关键词 柑橘黄化脉明病毒 尤力克柠檬 酵母双杂交 外壳蛋白 寄主因子
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饲粮蛋白水平和酿酒酵母对秦川肉牛血常规指标和氮磷代谢的影响 被引量:2
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作者 王晓慧 于胜晨 +5 位作者 梁成成 李炳志 王思虎 江中良 赵春平 昝林森 《西北农林科技大学学报(自然科学版)》 CSCD 北大核心 2024年第2期40-50,共11页
【目的】研究不同蛋白水平饲粮及酿酒酵母(SC)对秦川肉牛血液生理生化指标和氮磷代谢的影响。【方法】选择18头14月龄初始体质量为(298±9.78)kg/头、健康的秦川肉牛(母牛)作为试验动物,采用2种蛋白水平(13.23%(A1)和(10.59%(A2))... 【目的】研究不同蛋白水平饲粮及酿酒酵母(SC)对秦川肉牛血液生理生化指标和氮磷代谢的影响。【方法】选择18头14月龄初始体质量为(298±9.78)kg/头、健康的秦川肉牛(母牛)作为试验动物,采用2种蛋白水平(13.23%(A1)和(10.59%(A2))饲粮和3种酿酒酵母添加水平(0(B1),4×10^(8) CFU/kg(B2),8×10^(8) CFU/kg(B3))作为两因素进行饲喂试验。采用全收粪(尿)法测定各处理秦川肉牛粪便和尿液中的氮磷含量,试验结束时于晨饲前采用颈静脉穿刺法采血,测定血常规指标和酶活性,试验期55 d。【结果】①试验期11~55 d,饲粮中添加8×10^(8) CFU/kg SC显著提高了秦川肉牛的采食量(P<0.05),A1B3组秦川肉牛采食量最高,显著高于A1B1组(P<0.05)。②B3组秦川肉牛血清中甘油三酯含量显著低于B1组(P<0.05),A2B3组秦川肉牛血清中总蛋白、白蛋白、球蛋白、免疫球蛋白G和免疫球蛋白M含量均显著高于A2B1组(P<0.05)。③B3组秦川肉牛粗蛋白(CP)和中性洗涤纤维(NDF)的表观消化率较B1组分别显著提高了8.44%和16.90%(P<0.05),A2B3组秦川肉牛的粗蛋白、有机物和酸性洗涤纤维表观消化率最高。④与A2B1组相比,A2B3组秦川肉牛粪氮排放减少了31.28%,尿氮排放减少了19.67%,氮消化率提高了7.66%,氮沉积率提高了62.57%。⑤B2和B3组秦川肉牛的粪磷含量显著低于B1组,A2B3组秦川肉牛磷消化率、磷沉积率相较于A2B1组显著升高了35.96%和45.94%(P<0.05)。【结论】本试验条件下,添加8×10^(8) CFU/kg酿酒酵母能够提高秦川肉牛的采食量、免疫水平和营养成分消化率,减少氮磷排放。在10.59%粗蛋白水平下,饲粮添加8×10^(8) CFU/kg酿酒酵母饲喂秦川肉牛效果最好。 展开更多
关键词 秦川肉牛 粗蛋白水平 酿酒酵母 氮磷代谢 血清指标
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中国马铃薯Y病毒的检测鉴定及CP基因的分子变异 被引量:29
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作者 高芳銮 沈建国 +3 位作者 史凤阳 方治国 谢联辉 詹家绥 《中国农业科学》 CAS CSCD 北大核心 2013年第15期3125-3133,共9页
【目的】查明马铃薯Y病毒(Potato virus Y,PVY)病在中国的发生情况,及时、准确地鉴定出PVY并对其分子变异进行分析。【方法】采用ELISA方法对采自中国14个省(直辖市)马铃薯种植区疑似受PVY感染的样品进行了检测,并对其中的部分材料镜检... 【目的】查明马铃薯Y病毒(Potato virus Y,PVY)病在中国的发生情况,及时、准确地鉴定出PVY并对其分子变异进行分析。【方法】采用ELISA方法对采自中国14个省(直辖市)马铃薯种植区疑似受PVY感染的样品进行了检测,并对其中的部分材料镜检验证,然后根据CP基因序列设计1对简并引物针对随机选择感染PVY的14个省(直辖市)代表样品进行CP基因扩增克隆,将测序得到的序列进行分子变异分析,并使用贝叶斯法(Bayesian inference,BI)重建系统发育关系。【结果】ELISA检测结果表明,691份样品中220个样品与PVY抗体呈阳性反应,其余呈阴性反应;ELISA检测的阳性材料在透射电镜下均可观察到明显的风轮状内含体,14个PVY分离物均成功扩增出预期大小(约800 bp)的特异性片段,CP基因的核苷酸序列与已报道PVY不同株系的核苷酸序列一致性均在88%以上;在14个PVY分离物CP基因中共发现有29个多态性位点,其中6个简约信息位点,23个单一变异位点。系统发育分析结果显示,14个PVY分离物与PVYN:O株系相聚成簇,表明其在系统发育关系上,与PVYN:O株系的亲缘关系最近。【结论】PVY CP基因高度保守,但不同地区分离物也存在一定的分子变异,本研究可为今后了解PVY病毒病流行、变异趋势及其防治提供依据。 展开更多
关键词 马铃薯Y病毒 ELISA 外壳蛋白基因 分子变异 贝叶斯法
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瞬时表达比较马铃薯X病毒CP基因3种结构对RNA沉默的诱导效果 被引量:5
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作者 竺晓平 刘金亮 +3 位作者 田延平 于晓庆 李向东 刘红梅 《应用与环境生物学报》 CAS CSCD 北大核心 2007年第1期1-4,共4页
RNA沉默是植物抵御病毒侵染的一种防卫机制,病原来源的抗性(pathogen-derived resistance,PDR)被认为是通过RNA沉默起作用的.转化的核酸片段在基因组中的位置、长短和不同排列结构等均影响RNA沉默的效率.用全长马铃薯X病毒(PVX)CP基因... RNA沉默是植物抵御病毒侵染的一种防卫机制,病原来源的抗性(pathogen-derived resistance,PDR)被认为是通过RNA沉默起作用的.转化的核酸片段在基因组中的位置、长短和不同排列结构等均影响RNA沉默的效率.用全长马铃薯X病毒(PVX)CP基因构建非翻译的正义、反义和反向重复结构转基因的植物表达载体,通过农杆菌渗入法瞬时表达测试了这些转基因对RNA介导抗性的诱导效率和有效性.结果表明,反向重复的PVXCP是更为有效的RNA沉默诱导因子,同时也证明让目的基因在受试植物中瞬时表达,在转化植物之前能比较快速地检测转基因的表达情况以及表达产物的功能,从而节约时间和资源,并减少盲目性. 展开更多
关键词 RNA沉默 马铃薯X病毒(PVX) 外壳蛋白(cp)基因 农杆菌渗入 抗性
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转CP基因线辣椒对CMV和CMV-RNA的抗病性比较 被引量:10
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作者 徐秉良 商鸿生 王旭 《植物病理学报》 CAS CSCD 北大核心 2002年第2期132-137,共6页
本试验以转化 CMV- CP和 TMV- CP基因线辣椒纯合系作试材 ,比较了接种 CMV粒体和CMV- RNA后的发病特点和叶片中的病毒含量。结果表明 :转化线辣椒不仅能抵抗 CMV粒体的侵染 ,而且还能抵抗 CMV- RNA的侵染。不论接种 CMV粒体或 CMV- RNA,... 本试验以转化 CMV- CP和 TMV- CP基因线辣椒纯合系作试材 ,比较了接种 CMV粒体和CMV- RNA后的发病特点和叶片中的病毒含量。结果表明 :转化线辣椒不仅能抵抗 CMV粒体的侵染 ,而且还能抵抗 CMV- RNA的侵染。不论接种 CMV粒体或 CMV- RNA,CP(+ )线辣椒的系统症状都延迟出现 ,显症株率和病害严重度级别大幅度降低 ,病毒增殖和运转受到抑制 ,接种叶片与新生叶片中的病毒含量明显减低。这一结果证实 CMV- RNA不能克服线辣椒由 CP基因介导的抗病性。 展开更多
关键词 cp基因线辣椒 CMV CMV-RNA 抗病性 转基因线辣椒 抗病毒
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低温胁迫对黄瓜花叶病毒CMV-BG株系cp基因多态性的影响 被引量:1
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作者 张兴桃 李艳红 +4 位作者 高贵珍 曹稳根 刘小阳 王海潮 袁维风 《安徽农业大学学报》 CAS CSCD 北大核心 2011年第2期292-295,共4页
研究了低温胁迫下黄瓜花叶病毒与三生烟互作后CMV cp基因序列的变化规律,并分析了植物RNA病毒的进化机制。用黄瓜花叶病毒北京甘蓝分离物(CMV-BG)侵染三生烟不同单株,置于不同温度条件下(常温和低温)培养30 d后,对CMV cp基因进行克隆、... 研究了低温胁迫下黄瓜花叶病毒与三生烟互作后CMV cp基因序列的变化规律,并分析了植物RNA病毒的进化机制。用黄瓜花叶病毒北京甘蓝分离物(CMV-BG)侵染三生烟不同单株,置于不同温度条件下(常温和低温)培养30 d后,对CMV cp基因进行克隆、序列测定和多态性分析。结果表明,侵染低温组植物CMV的cp基因多态性(Pi=0.001 76)高于常温组(Pi=0.001 14),IFEL分析检测到低温组第48个氨基酸位点为正选择位点,初步表明低温胁迫有助于提高CMV-BG cp基因序列的多态性,温度胁迫是植物RNA病毒与宿主互作过程中基因序列变异的一种重要的作用力。 展开更多
关键词 黄瓜花叶病毒 低温 cp基因 多态性
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