共查询到19条相似文献,搜索用时 562 毫秒
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反向遗传学技术是上世纪90年代在分子病毒学研究领域兴起的新技术,通常也被称为“病毒拯救”。综述了应用反向遗传学技术“拯救”狂犬病毒的主要技术体系以及反向遗传学技术在狂犬病毒致病机理、狂犬病毒疫苗及载体研究中的应用进展。 相似文献
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为实现对RNA病毒基因分子水平上的操作与研究,可将病毒基因组RNA体外转换成cDNA,构建出cDNA克隆,通过cDNA克隆与重组技术实现对RNA病毒基因分子水平的修饰,从而为病毒基因组的结构和功能研究提供有效的方法。这种方法是现代实验病毒学非常有用的工具,称为RNA病毒感染性克隆技术或反向遗传学技术。我们对该技术及其应用进行简要综述。 相似文献
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近年来,在分子病毒学研究领域兴起一门新型技术,即病毒的全长感染性cDNA克隆技术,是一种反向遗传操作技术(reverse genetics),通常被称为"病毒拯救(the rescue of virus)",它解决了对RNA病毒基因组难以操作这一困扰研究者多年的难题.从cDNA克隆拯救出负链RNA全病毒是20世纪90年代分子病毒学研究领域最振奋人心的突破之一,它开启了人们对病毒基因组进行人工操作和详细了解病毒基因及其产物功能的大门.该技术发展迅速,倍受国内外研究者关注[1-5]. 相似文献
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口蹄疫病毒A/AKT/58株基因组全长感染性cDNA克隆的体内拯救 总被引:1,自引:0,他引:1
构建了两种口蹄疫病毒(foot-and-mouth disease virus,FMDV)基因组全长cDNA克隆DTA/FMDV和pCA/FMDV,利用体外转录和体内转录方法制备感染性病毒,并对其抗原性,乳鼠毒力(LD50)和病毒生长动力学等生物学特性进行了分析。为了鉴别野毒与重组病毒,利用融合PCR技术在两种全长cDNA基因组内分别引入了几个点突变(pTA/FMDV;T1029G,pCA/FMDV:A174G,A308G,T1029G)作为遗传标记。结果表明,两种重组病毒对3日龄乳鼠均表现致病性。但是由pTA/FMDV合成的感染性体外转录本RNA的毒力较弱(10^-6);而与pCT7RNAP质粒共转染的pCA/FMDV体内拯救病毒的毒力较强(10^-7.5),并在BHK-21细胞上表现出与野毒相似的生长特性。这一结果表明,在FMDV的拯救过程中,以pCT7RNAP为基础的体内拯救系统相比体外转录方法更为简便、实用。该系统为利用反向遗传操作技术深入研究FMDV的分子致病机制及其准种特性等奠定了良好的基础。 相似文献
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反向遗传学技术实现了在DNA分子水平上对RNA病毒基因组的人工操作,在深入阐明基因组结构与功能、筛选研制新型基因工程疫苗及基因治疗等方面显示出良好的应用前景。本文对反向遗传学技术及在肠道病毒71型病毒中的应用进行了综述。1肠道病毒71型基因组结构及复制特征肠道病毒71型(Enterovirus type 71,EV71)是小RNA病毒科肠道属成员,其感染主要引起手足口病,还可能引起严重的神经系统疾病,如脑炎、无菌性脑膜炎和脊髓灰质炎样的麻痹等。根据EV71衣壳蛋白VP1区的核苷酸序列的不同,EV71可以分为A、B、C三个基因型,其中B型和C型又 相似文献
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摘要:【目的】建立狂犬病毒的反向遗传系统,为研制不含狂犬病病毒致病性的新型安全高效的狂犬疫苗提供技术依据。【方法】本研究采用反向遗传学方法和分子克隆技术,建立了狂犬病病毒Evelyn- Rokitnicki-Abelseth (ERA)疫苗株的CMV/ T7、T7启动子病毒拯救系统,构建了表达N、P、L蛋白的辅助质粒。【结果】成功拯救出野生型病毒rERA-VC,在Vero细胞上的生长动力学特性与父母本ERA 相同,第三代可在Vero细胞上可获得很高的生长滴度。【结论】建立了狂犬病病毒的反向遗传系统,拯救出的野生型病毒生物学特性与父母本相同。 相似文献
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Although methods for reverse genetics of herpesviruses have been established in early 1980s, the steps are laborious and time-consuming. In 1997, Dr. Koszinwski's group reported a novel approach for the construction of herpesvirus mutants, based on cloning the viral genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetics modification of the viral genome in E. coli using bacterial genetics is possible, thereby facilitating the introduction of mutagenesis into herpesvirus genome. This 'BAC system' has opened new avenues for reverse and forward genetics of herpesviruses in basic research and in vector development for human therapy. Here we describe the principle of the 'BAC system' in herpesvirus researches. 相似文献
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Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples. 相似文献
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口蹄疫病毒(FMDV)导致了偶蹄动物口蹄疫的发生,它是一类有着自身特点的RNA病毒。首先,FMDV衣壳蛋白VP1识别结合宿主细胞膜上的整联蛋白等受体,以内吞的方式进入细胞,利用宿主细胞成分完成病毒蛋白的合成。这些新合成的L^pro、2C和3C^pro等病毒致病因子进一步抑制宿主基因的转录和翻译,诱导细胞凋亡和白噬,并抑制干扰素介导的一系列先天性和获得性免疫反应。宿主则在病毒侵染细胞的初期,利用病毒识别受体等来识别病毒并诱导合成干扰素等细胞因子,介导多种免疫反应以清除病毒。病毒和宿主两者在持续的利用和较量中完成疾病的发生和痊愈等。其次,不断发现的病毒受体、结合基序、致病因子及宿主细胞的多种免疫调节因子将成为相关领域新的研究内容。综上,开发高效安全疫苗、增强自身免疫力及利用RNAi直接抑制病毒RNA等便成为现代FMDV防治的主要内容。 相似文献
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D. P. Gladue V. O'Donnell R. Baker-Branstetter L. G. Holinka J. M. Pacheco I. Fernández Sainz Z. Lu X. Ambroggio L. Rodriguez M. V. Borca 《Journal of virology》2013,87(12):6794-6803
Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Aphthovirus within the Picornaviridae family. During infection with FMDV, several host cell membrane rearrangements occur to form sites of viral replication. FMDV protein 2C is part of the replication complex and thought to have multiple roles during virus replication. To better understand the role of 2C in the process of virus replication, we have been using a yeast two-hybrid approach to identify host proteins that interact with 2C. We recently reported that cellular Beclin1 is a natural ligand of 2C and that it is involved in the autophagy pathway, which was shown to be important for FMDV replication. Here, we report that cellular vimentin is also a specific host binding partner for 2C. The 2C-vimentin interaction was further confirmed by coimmunoprecipitation and immunofluorescence staining to occur in FMDV-infected cells. It was shown that upon infection a vimentin structure forms around 2C and that this structure is later resolved or disappears. Interestingly, overexpression of vimentin had no effect on virus replication; however, overexpression of a truncated dominant-negative form of vimentin resulted in a significant decrease in viral yield. Acrylamide, which causes disruption of vimentin filaments, also inhibited viral yield. Alanine scanning mutagenesis was used to map the specific amino acid residues in 2C critical for vimentin binding. Using reverse genetics, we identified 2C residues that are necessary for virus growth, suggesting that the interaction between FMDV 2C and cellular vimentin is essential for virus replication. 相似文献
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Haiwei Wang Shanshan Song Jianxiong Zeng Guohui Zhou Decheng Yang Te Liang Li Yu 《中国病毒学》2014,29(2):103-111
Infection by foot-and-mouth disease virus (FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease (FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements (VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV. 相似文献
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Chunhua Li Zhen Li Yong Zou Oliver Wicht Frank J. M. van Kuppeveld Peter J. M. Rottier Berend Jan Bosch 《PloS one》2013,8(8)
Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines. 相似文献
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Hong Yuan Pinghua Li Xueqing Ma Zengjun Lu Pu Sun Xingwen Bai Jing Zhang Huifang Bao Yimei Cao Dong Li Yuanfang Fu Yingli Chen Qifeng Bai Jie Zhang Zaixin Liu 《Virology journal》2017,14(1):233