油桐尺蠖核多角体病毒多角体蛋白基因定位与克隆

以[~(32)P]-dATP标记含AcNPV DNA的EcoRI-I片段的重组质粒为探针,在35℃条件下对油桐尺蠖核多角体病毒(BsNPV)多角体蛋白基因进行了定位,将其分别定位在BamH Ⅰ-A,Bgl Ⅰ-A,Bgl Ⅱ-F,EocR Ⅰ-R,Hind Ⅲ-A,Kpn Ⅰ-Ⅰ,Pst Ⅰ-D,Xba Ⅰ-A(或B),Xho Ⅰ-F和G片段上,并以M13mp18为载体,克隆了Kpn Ⅰ-Ⅰ片段。     

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％。     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

含有昆虫杆状病毒多角体蛋白基因的转运质粒的构建

为了利用苜蓿尺蠖核多角体病毒作为表达外源基因的运载体,我们将含有该病毒多角体蛋白基因的EcoR I I片段克隆在大肠杆菌质粒pBR325中。并对这一片段进行改建,构建成两个可以将外源基因置于多角体蛋白基因启动子控制下的表达质粒。     

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。     

银染色测定粘虫核多角体病毒多角体基因序列

LsMNPV DNA用EcoRV酶切进行基因组克降,用AcMNPV的部分多角体基因顺序DNA片段作探针,菌落原位杂交法结合测序筛选到分别含LsMNPV部分多角体基因的重组质粒pLsEV1和pLsPH5。用银染色PCR线性扩增双脱氧法测序,发现LsMNPv的完整基因即位于这两个片段上。LsMNPV多角体基因长741bp,编码区碱基同源性与AcMNPV和MbMNPV分别为80.0％和97.0％,氨基酸同源性分别为89.8和97.5％。氨基酸组成中以谷氨酸(Glu)含量最高,谷氨酰胺和色氨酸含量最低。密码子选用以第三个碱基为嘧啶的密码子频率最高。多角体蛋白N端有一类似信号肽结构的26个氨基酸的疏水区。     

蓖麻蚕核型多角体病毒多角体蛋白基因的克隆和核苷酸序列分析

蓖麻蚕(Attacus ricini)是我国特有蚕种,以其核多角体病毒(ArNPV)为载体有可能发展成为新的基因工程表达系统,我们建立了ArNPV基因库,并亚克隆了含多角体蛋白(Ph)基因DNA片段。对该1.1kb全长DNA片段进行序列分析,确定ArNPV Ph结构基因全长735bp,与苜蓿尺蠖NPV(AcNPV)、家蚕NPV(BmNPV)同源性分别为76%和81%,ArNPV 5'端调控结构Rohrmann box与各类NPV的Ph基因相似,但3'下游序列几无同源,显示了ArNPV Ph基因结构的特征性。同时,我们还对Ph基因启动子的其它结构特点作了剖析。     

马尾松毛虫质型多角体病毒多角体蛋白基因的表达与定位

为了表达马尾松毛虫质型多角体病毒(DpCPV)多角体蛋白基因(S10片段)并探讨多角体蛋白在真核细胞中的定位,从DpCPV中分离出S10,与pET-28a载体连接成重组表达质粒pET28-S10;将S10克隆到杆状病毒转座载体pFASTBACHTb中,依次筛选出重组转座质粒pFASTBACS10,重组穿梭质粒BacmidS10,重组杆状病毒AcS10。多角体蛋白基因表达后,用SDS-PAGE、Western-blot和免疫荧光技术对表达产物进行了检测。结果表明:S10原核表达质粒、重组杆状病毒成功获得;在昆虫细胞中表达的质型多角体蛋白主要定位于细胞质,同时有少量产物定位于细胞核。     

马尾松毛虫质型多角体病毒多角体蛋白基因的表达与定位

为了表达马尾松毛虫质型多角体病毒(DpCPV)多角体蛋白基因(S10片段)并探讨多角体蛋白在真核细胞中的定位,从DpCPV中分离出S10,与pET-28a载体连接成重组表达质粒pET28-S10;将S10克隆到杆状病毒转座载体pFASTBAC HTb中,依次筛选出重组转座质粒pFASTBAC S10,重组穿梭质粒Bacmid S10,重组杆状病毒Ac S10.多角体蛋白基因表达后,用SDS-PAGE、Western-blot和免疫荧光技术对表达产物进行了检测.结果表明S10原核表达质粒、重组杆状病毒成功获得;在昆虫细胞中表达的质型多角体蛋白主要定位于细胞质,同时有少量产物定位于细胞核.     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini-F replicon、一个卡那霉素抗性基因和一个lacZα基因8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内,构建了既能在E.coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid-HZ8).另外将HaSNPV的多角体蛋白基因和P10启动子序列取代pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列,构建插入HaSNPV多角体蛋白基因和 P10启动子序列的HapFastBacPhP10供体质粒.利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上,随后将含有eGFP基因的重组HaBacmid DNA转染至HZAm1细胞内.转染5d后,细胞核内能形成典型的多角体,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光.结果证明我们构建的HaBac to Bcac 表达系统能有效的表达外源基因.     

以杆状病毒为载体表达可溶性55kD人肿瘤坏死因子受体

从培养的HeLa细胞中提取总RNA,通过反转录PCR技术, 从该总RNA中扩增了约530 bp的shTNFR55基因的cDNA,将cDNA克隆至转移载体pAcGp67B的多角 体蛋白基因启动子的下游与转移载体构建成重组转移质粒pAcTNFR。pAcTNFR与杆状病毒AcNP V的DNA共转染昆虫细胞sf9,通过同源重组形成含有shTNFR55基因的重组病毒。经空斑分析 和DNA斑点杂交获得了纯化的重组病毒AcNPVTNFR。采用肿瘤坏死因子(TNF)敏感的L929细 胞检测表达产物的生物学活性。结果表明表达产物可以中和TNF对L929的细胞毒性。蛋白配 基印迹(Ligand blot)分析表明表达产物分子量约在20～25kD之间,有三条带。     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。     

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.     

柞蚕核型多角体病毒(ApNPV)转移载体质粒pAp M2614的组建

自从美国科学家G.Smith等首次建立苜蓿尺蠖核型多角体病毒(AcNPV)转移载体表达系统以来,已被广泛用于外源基因的表达,成为世界上一新的具有巨大潜力的载体表达系统。为了进一步提高表达产量,降低成本,日本科学家前田进建立了家蚕核型多角体病毒(BmNPV)载体表达系统,并获得了高效表达。柞蚕是我国特产,以蛹滞育越冬,保存时间长,个体大,可工厂化生产。因此,组建柞蚕NPV转移载体,进而建立该载体表达系统,是目前利用昆虫活体为宿主进行外源基因表达较理想的昆虫杆状病毒载体表达系统。     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.     

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.     

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.