杆状病毒Ac110蛋白的保守氨基酸位点N27对病毒在中肠建立有效感染起重要作用

杆状病毒是节肢动物,特别是鳞翅目昆虫的重要病原微生物,在生物防治中有着广泛的应用前景。其对昆虫虫体口服感染的过程中,需要一类称为杆状病毒口服感染因子的帮助才能在虫体中肠成功建立系统感染。Ac110是本实验室最新发现的口服感染因子之一,但其作用机制仍是未知。在本研究中,通过定点突变技术成功构建了两株Ac110保守氨基酸位点突变型重组杆状病毒,将病毒DNA转染昆虫细胞Sf9后,通过蛋白免疫印迹实验验证了Ac110的突变体蛋白均能够正常表达;接着通过病毒滴度测定实验证明了重组病毒均能在Sf9细胞中产生与补回型病毒相当的感染性病毒粒子,说明Ac110的这两个保守氨基酸位点的突变并不影响病毒在细胞中的复制和在细胞之间的传播;最后将纯化的病毒多角体口服感染甜菜夜蛾幼虫进行生物测定实验,发现Ac110的N27氨基酸位点的突变对昆虫幼虫的口服感染能力显著性降低,而L35氨基酸位点的突变则没有影响。     

杆状病毒包埋型病毒粒子研究进展

杆状病毒是一类专一性感染节肢动物的病原微生物,在其生活周期中产生两种不同形态的病毒粒子,即芽生型病毒粒子和包埋型病毒粒子。自然界中,杆状病毒通过经口感染的方式在其宿主的中肠中建立原发性感染,杆状病毒的包埋型病毒粒子在此过程中发挥关键作用。本文回顾了杆状病毒的基本特性,阐述杆状病毒包埋型病毒粒子的蛋白组成及分类,并着重介绍多粒包埋型病毒粒子的进化及在杆状病毒经口感染过程建立中的意义,对进一步了解杆状病毒的进化历程和侵染机制具有重要的指导意义。     

昆虫杆状病毒几丁质酶及其应用研究进展

杆状病毒几丁质酶基因（chitinase,,ChiA）是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区：N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原（pro-V-Cath）的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiA或V-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。     

杆状病毒部分功能基因的研究进展

杆状病毒以核型多角体病毒NPV的研究最为深入,到目前已有三种NPV的基因组全部测序：苜蓿银纹夜蛾核型多角体病毒（AcMNPV）［1］、黄杉古毒蛾核型多角体病毒（OpMNPV）［2］、家蚕核型多角体病毒（BmNPV）（GenBank accession no.L33180）。 　　杆状病毒基因组除编码病毒复制必需的基因及结构基因外,还编码一些有利于病毒充分增殖但对病毒复制并非一定必需的基因,这些基因在病毒的增殖过程中执行特定的功能,这类功能基因体现了杆状病毒的复制策略、病毒与细胞的相互作用关系,与杆状病毒宿主特异性的决定有关。本文综述了杆状病毒部分功能基因的研究进展。     

苜蓿丫纹夜蛾核型多角体病毒fp25k基因的改造及用于稳定转化Sf9昆虫细胞系

【目的】苜蓿丫纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)在昆虫细胞中连续传代以后,会出现从多多角体表型到少多角体表型的转变,这种转变与一个编码25 kDa蛋白的基因（few polyhedra, fp25k）突变失活有关。杆状病毒的fp25k基因突变后产生的包涵体（多角体）衍生病毒粒子变少而出芽型病毒粒子增加,会降低外源基因在杆状病毒表达系统中的表达。本研究拟改造fp25k并构建能持续表达FP25K蛋白的转基因昆虫细胞,以克服杆状病毒, fp25k基因易突变导致的表达系统缺陷。【方法】本实验通过改造杆状病毒, fp25k基因在细胞传代过程中容易产生突变的位点,得到 mfp25k,并将mfp25k构建到pIZT/V5-His载体上,重组载体转染Sf9细胞,通过Zeocin抗性筛选逐步淘汰未成功转化的Sf9细胞。【结果】成功改造AcMNPV的, fp25k基因的TTAA位点,得到pIZT-mfp25k重组载体。重组载体成功转染Sf9细胞,通过Zeocin抗性筛选后获得基因组中带有mfp25k的Sf9-mfp25k稳定的转基因细胞系。用AcMNPV的fp25k突变型病毒AcP2感染转基因Sf9-mfp25k昆虫细胞系与正常Sf9细胞,发现转基因Sf9-mfp25k昆虫细胞系表达的FP25K蛋白可弥补病毒, fp25k基因突变的缺陷。【结论】建立的Sf9-mfp25k转基因昆虫细胞系通过细胞表达FP25K蛋白,可以弥补因杆状病毒fp25k基因突变产生的缺陷。研究结果为构建稳定的杆状病毒 昆虫细胞表达系统提供了新途径。     

杆状病毒在载体疫苗中应用的研究进展

目前利用来源于哺乳动物的病毒作病毒载体介导动物和人体的免疫应答存在诸多问题,如预存免疫和高致病力重组病毒的出现等。将自然条件下宿主为非哺乳类动物的病毒发展为载体的过程中,由于杆状病毒内在的免疫刺激活性、广泛的组织趋向性及生物安全性使杆状病毒成为疫苗载体发展中的有效工具。随着分子生物学的深入发展,国内外学者应用基因工程技术对杆状病毒转移载体进行了深入研究,并取得丰富的研究成果。对杆状病毒作为转移载体在未来载体疫苗发展中的应用进行综述。     

粉纹夜蛾颗粒体病毒增强蛋白锌离子结合域定点突变

粉纹夜蛾颗粒体病毒（Trichoplusia ni granulovirus, TnGV）增强蛋白（enhancin）具有增强病毒感染力的作用。该蛋白包含一个多种杆状病毒增强蛋白都具有的保守结构域HELGH,是典型的金属蛋白酶锌离子结合域,但该结构域对增强蛋白生物活性的重要性尚未得到研究。本研究通过定点突变构建了该结构域的5个氨基酸分别突变为2种不同氨基酸的共10种增强蛋白突变体基因,并用杆状病毒载体进行了重组表达。活性测定发现,10种突变型增强蛋白大部分都丧失了野生型增强蛋白所具有的降解粉纹夜蛾幼虫围食膜粘蛋白的生物学功能,只有1种（第4位G突变为A）保留该生物学活性。这一结果表明锌离子结合域对增强蛋白生物活性具有重要作用,也提示增强蛋白确是一种金属蛋白酶。     

杆状病毒的分子生物学研究进展

杆状病毒科(Baculoviridae)是昆虫病毒中最大的一科,数百种病毒多为鳞翅目、双翅目及膜翅目昆虫的致病原。这一科病毒共分三个亚组:A亚组,具有巨大包含体的核多角体病毒(Nuclear Polyhedrosis Virus,NPV);B亚组,颗粒体病毒(Gtallulin Virus,GV);C亚组,无包含体的独角仙杆状病毒(Oryctes)。昆虫病毒中,杆状病毒是发现最     

杆状病毒生态学研究进展

杆状病毒是一类寄生于鳞翅目、膜翅目和双翅目昆虫及其它节肢动物的病原体,是具有囊膜的双链环状DNA病毒。目前至少已从600多种昆虫中发现有杆状病毒的感染,其中一部分能够引起宿主种群不同程度的流行病。杆状病毒在分类上独立为杆状病毒科,其典型的结构特征是病毒粒子包埋于蛋     

杆状病毒侵染的分子基础*

相状病毒是昆虫的专一性寄生病毒,可作为一种有效的生物杀虫剂。杆状病毒侵染寄主表现在其基因的功能上。侵染是一个复杂的过程,包括许多不同基因的表达、调控等,以苜蓿丫纹夜蛾核多角体病毒（Autographa califormica mucleopolyhedrovirus,AcMNPV）为例,从分子水平上概述了与杆状病毒侵染有关的基因及蛋白质的结构特性与功能,为进一步了解杆状病毒侵染寄主的机制,从而有效的改良杆状病毒,扩大杀虫谱,提高其杀虫效果提供参考。     

杆状病毒DNA聚合酶基因的研究概况

杆状病毒DNA聚合酶基因属于杆状病毒早期基因,是杆状病毒复制的必需基因。它编码病毒诱导的DNA聚合酶,能与其它复制因子一起与杆状病毒DNA的同源区和非同源区的顺式作用元件相互作用起始DNA复制。此基因作为杆状病毒系统发育分类的依据,较之包涵体蛋白、egt基因有更大的优势。     

杆状病毒基因组DNA复制相关基因的研究进展

综述了与杆状病毒DNA复制相关基因的研究进展.杆状病毒表达系统是最重要的四大基因工程表达系统之一,杆状病毒还具有作为生物杀虫剂的潜能.DNA复制是杆状病毒复制循环的中心环节.     

Studies of the silencing of baculovirus DNA binding protein

Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.     

Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures

The aim of our study was to establish an efficient system for thein vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.Abbreviations Ac-NPV Autographa californica Nuclear Polyhedrosis Virus - BV Baculovirus - MOI Multiplicity Of Infection - ECV Extracellular Virus     

PCR analysis of insertion site specificity, transcription, and structural uniformity of the Lepidopteran transposable element IFP2 in the TN-368 cell genome

The IFP2 element is a unique Lepidopteran transposon that has been associated with spontaneous Baculovirus mutants isolated following passage of the virus in the TN-368 cell line. Independent genomic representatives of IFP2 from TN-368 cells show little sequence divergence, suggesting that IFP2 was recently introduced into this genome and is highly stable. IFP2 is inserted within AT-rich regions of the TN-368 genome and targets TTAA sites. The specificity for TTAA target sites during transposition is not limited to the movement of IFP2 during an active Baculovirus infection, but is a property of its movement in uninfected cells as well. The exact origin of IFP2 remains obscure since it is found in two independently established Trichoplusia ni cell lines but not in three others, and we have not yet identified any IFP2 sequences in either field collected larvae or laboratory colonies.     

Replication patterns and cytopathology of cells infected with baculoviruses

Conclusion In vitro studies have contributed greatly to an understanding of viral cytopathology, molecular biology, and pathogenesis. A model of the role of baculoviruses in a host-parasite relationship is developing which reveals the virus as gaining control of many aspects of host cell biology including control of the cell replication machinery (apoptotic response, macromolecular synthesis), the cytoskeletal structure, the nuclear membrane and intranuclear architecture. Baculovirus replication is a collection of independent but inter-related processes which work within the framework of the host cell, with the in vivo goal of maximizing production of progeny virions. Further molecular dissection of baculovirus replication should yield insight into the processes and principles of viral and host regulatory systems, perhaps facilitating development of new generations of high efficiency sub-viral expression vector systems and the development of genetically improved strains of virus safe for field use in ecologically based pest management strategies.     

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction.

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell.     

Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wild-type virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.     

BHK cells expressing Sindbis virus-induced homologous interference allow the translation of nonstructural genes of superinfecting virus.

The process by which Sindbis virus excludes superinfecting homologous virus was investigated with the use of temperature-sensitive mutants. Mutants in two RNA-negative complementation groups were found to be defective in their ability to establish interference at the nonpermissive temperature. These mutants were unable to establish interference in a mixed infection (complementation), suggesting that both were defective in a common gene product. Homologous interference was found to block the replication of superinfecting virus after attachment, penetration, and translation of the nonstructural genes encoded in the virus RNA. The production of nonstructural gene products of superinfecting wild-type virus was found to enhance the replication of certain RNA- temperature-sensitive interfering viruses at the permissive and the nonpermissive temperature. The ability of certain RNA- mutants to establish homologous interference and to demonstrate enhanced growth after superinfection with wild-type virus was interpreted to produce a model implicating both virus and host components in the establishment of homologous interference and in the replication of Sindbis virus RNA.