柞蚕核多角体病毒核多角体基因的定位和克隆

通过Southern转印杂交证明,柞蚕核多角体病毒(Antheraea pernyi nuclear polyhedrosis virus,ApNPV)核多角体基因位于该病毒基因组DNA Bam HⅠ D和E片段上,我们巳将这两个片段分别克隆到pAT153质粒中,并用末端杂交法确定了ApNPV核多角体基因的方向,对含有这一基因的片段进行了限制性内切酶图谱分析,进而对这一基因部分编码区进行了核苷酸序列分析,在用ApNPV这一段序列(222bp)与其他昆虫核多角体病毒AcNPV(AutograPha californica NPV,苜蓿丫纹夜蛾NPV);BmNPV(Bombyx mory NPV,家蚕NPV);OpNPV(Orqyia Pseudotsugata NPV,黄杉毒蛾NPV)核多角体基因相应区段相比较分析中,发现它们之间的同源核苷酸序列比率分别为77.5％、84％和80％。     

两株蓖麻蚕核型多角体病毒的比较研究

本文描述来源不同的两株菌麻蚕多角体病毒的形态特征和理化特性。一株为较长期饲喂马桑叶的蓖麻蚕从自然罹死的幼虫和蛹中分离的多角体病毒（简称ArscsNPV);另一株为饲喂蓖麻叶的蓖麻蚕从幼虫分离的核型多角体病毒（简称ArscsNPV);另一株为饲喂蓖麻叶的蓖麻蚕从幼虫分离的核型多角体病毒（简称ArNPV)。两株核型多角体病ArNPV多角体大小约1．2－2．0μm最大的可达2．9μm。两株NPV病毒粒子均为杆状,ArscsNPV病毒粒子大小平均为310×50nm;ArNPV病毒粒子大小为350×50nm。两株NPV均为多粒包埋型。两株NPV的多角体蛋白均为单一组分,ArscsNPV多角体蛋白分子量为27．5kd;ArNPV多角体蛋白分子量为28kd。两株NPV的病毒粒子结构多肽均含有21条多肽,其中各多肽分子量有所差异。ArscsNPV的病毒粒子多肽分子量范围为11－130kd;ArNPV病毒粒子多肽分子量范围为11－96kd,其中有11种多肽了量彼此相同包括两种主要多肽（54kd和33kd）。用SDS－苯酚提取的病毒核酸,经实验证明均为双链DNA型使用几种内切酶酶解,求得两株NPV的核酸分子量,ArscsNPV为52．4×10^6d;ArNPV为73．5×10^6d。     

蓖麻蚕核型多角体病毒与苜蓿银纹夜蛾核型多角体病毒多角体蛋白基因同源性的研究

本文报道以苜蓿银纹夜蛾核型多角体病毒多角体蛋白基因mRNA的cDNA的重组质粒PMA-Ⅵ DNA转化E.coli RR_1。采用了多种筛选方法,包括抗菌素抗性筛选,菌落杂交及电泳等方法。快速地筛选出含有PMA-Ⅵ质粒的菌株。并以蓖麻蚕NPV-DNA作探针,通过Southern杂交,表明蓖麻蚕NPV基因组中具有与苜蓿银纹夜蛾NPV多角体蛋白基因同源性的核苷酸序列。     

两株蓖麻蚕核型多角体病毒DNA同源性的研究

两株不同来源的蓖麻蚕核型多角体病毒(ArscsNPV和ArNPV)经提纯后,使用SDS—苯酚抽提病毒核酸,并使用限制性内切酶EcoRI,BamHI酶解后,用分子杂交方法与缺口平移标记的ArscsNPV-DNA探针杂交,分析了两株蓖麻蚕NPV病毒核酸的同源性。EcoRI酶解的ArNPV-DNA产生8个片段,其中5个片段能与ArscsNPV-DNA探针杂交。BamHI酶解ArNPV-DNA产生7个片段,其中6个片段能与ArscsNPV-DNA探针杂交。结果表明:两株蓖麻蚕NPV之间病毒核酸具有很高的同源性。使用斑点杂交方法分析了ArscsNPV与ArNPV,柞蚕NPV及家蚕NPV之间的核酸同源性,结果表明:ArscsNPV与ArNPV,柞蚕NPV具有同源性。而与家蚕NPV无核酸同源性。     

柞蚕核型多角体病毒多角体蛋白基因上游5''''端核苷酸序列分析

核型多角体病毒(Nuclear Polyhedrosis Virus,简称NPV)的核多角体蛋白(Polyhedrin)基因具有一个非常强的启动子和基因调控序列。目前利用这一基因的上述序列已组建了多种表达载体,高效地表达了十几种外源基因产物,成为当前最有前途的新的表达系统。但是,在组建这一病毒载体过程中,为了使插入的外源基因靠近病毒启动子序列,各     

~(35)S-α-dATP用于缺口转移标记探针DNA及蓖麻蚕核型多角体病毒(ArNPV)多角体蛋白基因的初步定位

本文用苜蓿银纹夜蛾核型多角体病毒的多角体蛋白基因mRNA的cDNA为探针,用~(35)S-α-dATP为标记化合物,经缺口转移体外标记探针DNA,用旋转柱层析法分离出标记探针,由膜上DNA-DNA杂交,将ArNPV的多角体蛋白基因定位,确定是在其EcoRⅠ-Ⅱ片段上。     

家蚕核型多角体病毒可视化快速核酸检测方法

家蚕核型多角体病在广西多批次的养蚕生产中非常普遍,严重影响蚕农的经济效益。根据家蚕核型多角体病毒(Bm NPV)广西分离株的多角体蛋白基因(polh)序列设计4条用于环介导核酸等温扩增(LAMP)特异性引物,以感染Bm NPV的5龄家蚕血淋巴的多角体DNA为模板,通过优化LAMP反应体系,确定Mg2+、d NTPs、甜菜碱最适终浓度分别为8 mmol/L、1.4 mmol/L和0.8 mol/L,外引物和内引物浓度为0.2μmol/L和1.6μmol/L,在64℃恒温条件下反应1 h;在反应管中加钙黄绿素,通过肉眼观察颜色变化直接判定结果实现可视化检测。该检测方法的Bm NPV最低模板DNA为25 fg/μL,其敏感度为常规PCR l00倍,兼具简便、快速、灵敏等优点,可用于家蚕核型多角体病的早期诊断及流行病学调查。     

棉铃虫核型多角体病毒多角体蛋白聚集体特性及与其它包涵体蛋白的血清学关系

纯化的多角体碱解释放多角体蛋白,经等电点沉淀和柱层析对多角体蛋白进行分离纯化,结合SDS-PAGE、免疫双向扩散、免疫电镜等方法,证明棉铃虫核型多角体病毒(HaNPV)的多角体蛋白以聚集体形式存在。用ELISA法检测包涵体蛋白之间的血清学关系,结果表明,与黄地老虎颗粒体病毒(AsGV)和粘虫颗粒体病毒(PsGV)颗粒体蛋白相比较,HaNPV多角体蛋白与葡萄天蛾核型多角体病毒(ArNPV)和黄地老虎核型多角体病毒(AsNPV)多角体蛋白之间的血清学关系更为密切。     

家蚕核型多角体病毒的PCR检测及其部分序列分析

为研究应用PCR技术进行家蚕核型多角体病毒广东株的敏感性检验以及探讨不同地理株系的基因水平的相互关系,本文通过对家蚕核型多角体病毒BmNPV广东株的人工繁殖与纯化,引用了一对根据多角体蛋白基因设计的引物phy35/phy36,对BmNPV的基因组模板DNA进行了PCR扩增,并对其产物进行测序分析.结果显示,PCR技术均可扩增检测出3×108个/mL至3×102个/mL不同浓度的BmNPV模板DNA,特异目标片段大小约为680 bp,且扩增带的亮度随着病毒液浓度的降低而减弱,说明应用引物phy35/phy36进行PCR方法可以有效地应用于检测BmNPV病毒感染的家蚕.同时,测序获得了BmNPV广东株多角体蛋白polyhedrin基因674 bp大小的片段,GC含量为46.4%.经过BLAST比对分析,与BmNPV泰国株的相似性为99%,暗示家蚕BmNPV广东株与泰国株的BmNPV (登录号AY779044)亲缘关系非常相近,两者可能属于BmNPV的不同地理株系.通过系统发育树的进一步分析发现,家蚕核型多角体病毒广东株polyhedrin基因部分序列与家蚕NPV分离株S9多角体蛋白基因(DQ231336)关系很近.     

一株雀纹天蛾核型多角体病毒的鉴定及其基因组序列分析

从自然死亡的雀纹天蛾幼虫分离到一株雀纹天蛾核型多角体病毒。通过扫描及切片透射电镜发现,该病毒为单粒包埋型核型多角体病毒,命名为ThjaSNPV(Theretra japonica single nucleopolyhedrovirus)。病毒全基因组重测序后拼接显示,该病毒基因组全长134 899 bp,GC含量37.28%,与豆天蛾单粒包埋型核型多角体病毒株DZ1基因组序列相似性高达96.24%。ThjaSNPV含有131个开放阅读框（ORF）其中55个为正链基因,76个为负链基因,与宿主同为天蛾科的豆天蛾单粒包埋型核型多角体基因组相比,雀纹天蛾单粒包埋型核型多角体病毒新注释到7个基因：chitin-binding protein(ORF9),ODV-E18(ORF11),lef-11(ORF27),hypothetical protein(ORF41),lef-10(ORF42),pif-6(ORF66),P6.9(ORF88)。ThjaSNPV的DNA光裂酶基因（DNA photolyase,ORF53）中不具有豆天蛾NPV中的1bp碱基的缺失,只编码一个大的完整DNA裂合酶。3...     

家蚕NPV SOD基因序列和在大肠杆菌中表达

通过ＰＣＲ 克隆了家蚕核型多角体病毒（ＢｍＮＰＶ） ＳＯＤ基因, 并在大肠杆菌中进行表达, 证明了Ｂｍ ＮＰＶＳＯＤ基因产物确有ＳＯＤ活性, 其活力单位约为５７６ ｕ／ｍＬ 培养液。ＤＮＡ 测序结果表明Ｂｍ ＮＰＶ ＳＯＤ 基因编码１５１ 个氨基酸, 与人的ＳＯＤ１ 基因的核苷酸同源性为５６ ％ , 与ＡｃＮＰＶ 拟为的ＳＯＤ基因同源性为９７ ．２％ 。     

Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene.     

美国白蛾核型多角体病毒超氧化物歧化酶基因的序列分析和表达

根据测序结果 ,HcNPVsod的核苷酸序列与BmNPVsod的完全一致 ,与AcNPVsod的核苷酸序列相比 ,同源性达到 97 2 % ;推测HcNPVsod编码 1 51个氨基酸 ,与BmNPVsod的完全一致 ,与AcNPVsod编码的氨基酸相比 ,有三个氨基酸的差别。按基酸序列分析表明 ,HcNPVSOD蛋白中含有对SOD结构和活性必需的氨基酸残基 ,在HcNPVsod中均是保守的。SOD活性测定表明酶活为 1 47 0 9U/mL菌液     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression

The four main gene expression systems currently used to produce recombinant proteins are the prokary-otic, yeast, insect cell, and mammalian cell expression systems. The baculovirus expression vector system (BEVS) is a protein production system which uses a recombinant baculovirus harboring a foreign gene of interest to produce recombinant protein in an insect or its cultured cells. BEVS has many advantages: (i) BEVS requires less time to establish the production system than is needed in a…     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus.

Homologous regions (hrs) (hr1,hr2-left,hr2-right,hr3,hr4-left,hr 4-right, and hr5) similar to those found in the Autographa californica nuclear polyhedrosis virus (AcNPV) genome were found in the Bombyx mori NPV (BmNPV) genome. The BmNPV hrs contained two to eight repeats of a homologous nucleotide sequence which were on average about 75 bp long. All of these homologous sequence repeats contained a 26-bp-long palindrome motif with an EcoRI or EcoRI-like site at its core. The consensus sequence of the BmNPV hrs showed 95% conservation with respect to those found in AcNPV. Nucleotide sequence analysis indicated that hr2-left and hr2-right of BmNPV evolved from an ancestor similar to hr2 of AcNPV by inversion, cleavage, and ligation. The polarities of the BmNPV and AcNPV hrs were conserved except for that of hr4-left. Within hr4-right of BmNPV, four repeats of a previously underscribed palindrome motif were found. Bmhr5D, a BmNPV mutant which lacked hr5, replicated at a rate similar to that of wild-type BmNPV in BmN cells and silkworm larvae, indicating that hr5 was not essential for viral replication. After ten passages of Bmhr5D in BmN cells, no detectable changes in its genome were observed by restriction endonuclease analysis. The evolution and divergence of the BmNPV genome are also discussed.     

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV.

We have isolated hybrid baculoviruses of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV) capable of replicating in both BmN (not susceptible to AcNPV) and SF-21 (not susceptible to BmNPV) cells (A. Kondo and S. Maeda, J. Virol. 65:3625-3632, 1991). Repeated backcross infection of one of these recombinant isolates with AcNPV generated eh-AcNPV, a virus with restriction endonuclease patterns of genomic DNA nearly identical to those of AcNPV but capable of replicating in both BmN and SF-21 cells, i.e., host range expanded. Expanded host range viruses were also isolated following cotransfection of AcNPV DNA with eh-AcNPV DNA cleaved with either HindIII or PstI. Subsequent cotransfection of AcNPV DNA with plasmids from an eh-AcNPV DNA fragment library identified an 11-kbp HindIII fragment that could expand the host range of AcNPV. Subcloning and cotransfection analyses localized a 572-bp SacI-HindIII fragment within this 11-kbp fragment which could alone expand the host range of AcNPV. Mapping and nucleotide sequencing analysis revealed that this fragment was identical to the corresponding 572-bp fragment (BmScH) of BmNPV. Furthermore, this fragment originated from the coding region of the putative DNA helicase gene. Cotransfection of AcNPV DNA with BmScH also generated a host range-expanded virus, eh2-AcNPV. These results indicated that the expanded host range characteristics of eh2-AcNPV were solely the result of recombination within the coding region of the putative DNA helicase gene.     

Isolation of p10 gene fromBombyx mori nuclear polyhedrosis virus and study of its promoter activity in recombinant baculovirus vector system

A homologue ofAutographa californica NPV (AcNPV) p10 gene was identified and cloned fromBombyx mori NPV (BmNPV). BmNPV p10 gene encodes truncated protein of 70 amino acid residues that lacks carboxyl terminus comparing with the p10 protein encoded by AcNPV. The putative TATA box sequence and the ATAAG motif which is the consensus sequence of baculovirus very late promoter were conserved. A transfer vector, pBNT1, which includes the p10 promoter region of BmNPV for foreign gene expression was constructed. By using pBNT1, a recombinant BmNPV, Bmp10-Luc, in which the p10 gene was replaced by the firefly luciferase gene, was obtained. We also obtained another recombinant virus, BmPH-Luc, in which the polyhedrin gene was replaced by the luciferase gene. The luciferase activity detected in BoMo-15AIIc insect cells infected with Bmp10-Luc was approximately 50% of that infected with BmPH-Luc, suggesting that although both the p10 and polyhedrin promoters of BrnNPV are effective in high-level expression of foreign gene, the p10 promoter is not so strong as the polyhedrin promoter.     

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.