一种新型家蚕核多角体病毒Bac to Bac系统的构建

基因编辑技术在家蚕育种中的应用指南概述：随着生物技术的快速发展，基因编辑技术在农业领域的应用越来越广泛。

家蚕作为一种重要的农业害虫，其良好的育种性状一直是育种学家们关注的重点。

基因编辑技术的出现为家蚕育种带来了新的思路和工具。

本文将介绍基因编辑技术在家蚕育种中的应用指南，包括基因编辑技术的原理、方法和在家蚕育种中的具体应用。

一、基因编辑技术原理概述基因编辑技术是一种通过人工修改生物体DNA序列的技术，其中最常用的工具是CRISPR-Cas9系统。

CRISPR-Cas9系统能够精确地定位到DNA序列中的目标位点，并在该位点引入修饰性的DNA改变。

通过这种方式，可以实现增加、删除或修饰目标基因的功能。

二、基因编辑技术的方法1. CRISPR-Cas9系统的设计与构建CRISPR-Cas9系统由CRISPR RNA (crRNA)、转录 RNA（tracrRNA）和Cas9蛋白质组成。

首先，需要确定目标基因的DNA序列，并设计与其互补的crRNA。

然后，将crRNA和tracrRNA序列合成，形成sgRNA（single-guide RNA）。

最后，将sgRNA与Cas9蛋白质形成复合物，用于靶向编辑基因。

2. DNA修饰使用CRISPR-Cas9系统进行基因编辑，可以实现三种主要的DNA修饰方式：- Knockout：通过在目标基因中引入缺失、插入或替换等突变，达到基因失活的目的。

- Knockin：在目标基因中插入外源基因序列，以增加或修饰目标功能。

- Base editing：基于CRISPR-Cas9系统的改进版本，可以实现直接编辑基因碱基，而无需引入外源DNA。

三、基因编辑技术在家蚕育种中的应用1. 抗虫性育种家蚕常常受到棉铃虫等害虫的威胁，而抗虫基因的引入能够提高家蚕对害虫的抵抗能力。

通过基因编辑技术，可以将抗虫性基因导入到家蚕基因组中，实现对虫害的抗性改良。

2. 抗病性育种家蚕容易感染各种疾病，如家蚕核多角体病毒（BmNPV）等。

BmBDV、BmCPV反向遗传学系统的建立及不同抗性家蚕对BmCPV的感染应答家蚕浓核病毒(Bm BDV)能特异性地侵染家蚕中肠组织的柱状细胞,是家蚕软化病的病原。

Bm BDV的基因组由两种不同的单链DNA,VD1(6,543 nts)和VD2(6,022 nts)构成,病毒粒子中只含有两种DNA分子中的一种。

目前还没有一种细胞系可感染Bm BDV,也不能通过基因操作改变Bm BDV的基因研究病毒基因的功能。

家蚕质型多角体病毒(Bm CPV)基因组由10个分节ds RNA片段组成,每个ds RNA片段含有一个完整的开放阅读框(ORF)。

由于Bm CPV为ds RNA,且缺乏Bm CPV的敏感细胞系,无法通过基因敲除或敲入研究Bm CPV基因功能,导致Bm CPV基因功能研究几无进展。

不同的家蚕品种对Bm CPV的抗性存在差异,但品种间的抗性差异机制仍有许多不明之处。

基于目前的研究现状和存在问题,我们开展了基于Bm BDV基因组DNA质粒拯救病毒研究、基于体外转录的病毒RNA转染细胞拯救Bm CPV研究,期望为Bm BDV 和Bm CPV基因的功能研究提供新的系统;同时,利用RNA-Seq技术,研究了不同抗性品种家蚕感染Bm CPV后,中肠组织基因组转录水平的变化,期望为理解家蚕对Bm CPV的感染应答及抗Bm CPV的分子机制提供新的线索。

1、Bm BDV反向遗传学系统的研究为了提供Bm BDV基因功能的研究平台,将含有Bm BDV VD1基因组的重组质粒p GEM-VD1转染家蚕卵巢(Bm N)细胞,发现细胞表现出明显的细胞病变;Western blotting与免疫荧光皆能检测到Bm BDV基因的表达。

同时,我们通过Bac-to-Bac载体表达系统构建了装载VD1全基因组的重组杆状病毒Bm Bac-VD1,该重组病毒基因组DNA转染Bm N培养细胞,可观察到细胞出现明显病变,将培养上清转接正常Bm N细胞,表现出相同的病变;Westernblotting与免疫荧光试验可检测到Bm BDV病毒结构蛋白的表达;透射电镜可同时观察到球形病毒粒子与杆状病毒粒子,通过差速离心将两种病毒粒子分离,分子生物学鉴定结果显示,球形病毒样颗粒中包裹有重组病毒基因组。

家蚕核型多角体病毒与家蚕抗病毒相关的蛋白质组
分析的开题报告
标题：家蚕核型多角体病毒与家蚕抗病毒相关的蛋白质组分析
背景：家蚕是中国的重要养殖物种之一，但是它会受到很多疾病的影响，其中核型多角体病毒是一种致命的疾病。

目前尚不清楚家蚕与核型多角体病毒之间的交互作用及相关的蛋白质组成。

研究目的：本研究旨在探究家蚕核型多角体病毒与家蚕抗病毒相关的蛋白质组成，以期为防控家蚕核型多角体病毒提供新的理论和实践基础。

研究方法：选取健康家蚕和被核型多角体病毒感染的家蚕为研究对象。

首先，通过蛋白质分离技术分离出病毒和宿主细胞蛋白质。

然后，采用质谱技术分析分离出的蛋白质，筛选出与抗病毒相关的蛋白质。

研究内容：本研究将从以下几个方面进行探究：
1. 核型多角体病毒的生长特征及病毒颗粒的形态结构。

2. 家蚕细胞抗核型多角体病毒的机制及其相关蛋白。

3. 宿主细胞中核型多角体病毒蛋白的功能及其对宿主细胞的影响。

预期结果：通过对家蚕和核型多角体病毒之间交互作用的探究，我们可以深入了解家蚕核型多角体病毒的生物特性及其与宿主细胞的相互作用。

同时，我们也可以筛选出一些与抗病毒相关的蛋白质，这些结果有助于进一步的研究和发现新的防治方法。

结论：本研究将为家蚕核型多角体病毒的防治提供新的思路和理论基础，丰富我们对病毒感染机制以及抗病毒免疫的认识。

家蚕核型多角体病毒Bm94基因的特性分析的开题报告一、研究背景家蚕是我国重要的经济昆虫，对于家蚕疾病的防治具有重要意义。

家蚕核型多角体病毒（BmNPV）是家蚕常见的病毒之一，主要引起家蚕多角体病，严重影响家蚕的养殖和产业发展。

在BmNPV中的Bm94基因编码一个较短的基因产物，其功能尚未完全阐明。

因此，对Bm94基因进行特性分析，可为深入了解BmNPV的基因组结构和生物学特性提供参考。

二、研究目的本研究旨在对家蚕核型多角体病毒Bm94基因进行特性分析，包括其基因结构、编码产物的生物学功能以及其在BmNPV的生命周期中的作用等方面。

三、研究内容1. Bm94基因的克隆和序列分析；2. 基于不同亚型间的序列比对，分析Bm94基因的进化关系及保守区域；3. 分析Bm94基因编码产物的生物学功能和结构特点；4. 研究Bm94基因在BmNPV的感染与复制过程中的作用，揭示Bm94基因在宿主细胞中的表达规律。

四、研究意义1. 有助于进一步了解BmNPV的基因组结构与生物学特性，为BmNPV疾病的防治提供科学依据；2. 为相关基因的深入研究提供参考，有助于加深病毒生态学和宿主免疫学等方向的研究；3. 为其他昆虫病毒的基因功能和作用研究提供借鉴。

五、研究方法1. 采用PCR技术从BmNPV中克隆Bm94基因，并进行序列分析、构建系统进化树和比对分析；2. 结合生物信息学和生物化学方法，预测和分析Bm94基因编码产物的性质、功能及三维结构特征、信号转导通路等；3. 采用实时荧光定量PCR、Western blotting技术等研究Bm94基因在BmNPV感染过程中的表达规律，并结合细胞培养和动物模型体外体内实验证明其功能。

六、研究进度安排1. 第一周：查阅相关文献，确定研究方向；2. 第二周至第四周：采用PCR技术克隆Bm94基因，并进行测序、序列分析和构建系统进化树；3. 第五周至第七周：预测和分析Bm94基因编码产物的性质、功能及三维结构特征、信号转导通路等；4. 第八周至第十周：采用实时荧光定量PCR、Western blotting技术等研究Bm94基因在BmNPV感染过程中的表达规律；5. 第十一周至第十二周：结合细胞培养和动物模型体外体内实验证明其功能；6. 第十二周至第十四周：分析实验结果，撰写论文并进行汇报。

由家蚕核型多角体病毒（Bombyx mori nucle-opolyhedrovirus,BmNPV ）引起的家蚕核型多角体病毒病是蚕业生产上最常见危害最为严重的一类蚕病，当前在全国各大蚕区均有发生且呈蔓延趋势。

据报道，我国每年因该病造成产茧量减产15%～20%，给蚕桑生产带来巨大的经济损失。

多年来，蚕业科研人员一直致力于筛选抗BmNPV 家蚕品种资源、阐明抗性分子机制和发现抗性基因，以期通过传统常规育种或现代分子育种手段选育出对BmNPV 具有较高抗性的家蚕新品种。

目前，相关研究已取得了一定进展。

1抗BmNPV 家蚕品种资源的筛选和鉴定我国家蚕品种资源丰富，从中筛选到对BmN-PV 高抗的家蚕品种，是推进家蚕抗病育种研究的重要物质基础。

张远能等[1]研究了33个家蚕品种对NPV 、CPV 、FV 和微粒子等6种蚕病的抗性差异，挖掘了一批单抗或兼抗的抗病品种。

陈克平等[2]在对我国家蚕种质资源344个品种进行抗Bm-NPV 鉴定后，发现了1个对BmNPV 高抗的地方资源品种，其LC 50高达6.39×108个/mL 。

陆瑞好、黄平等[3,4]也先后对广西地区和云南省保存的家蚕品种品系进行了BmNPV 经口感染抵抗性的比较试验，分别发现了一批抗性较高的地方品种，为实用家蚕抗性品种的选育提供了良好的育种素材。

2家蚕对BmNPV 抵抗性的遗传规律研究家蚕不同品种对BmNPV 的抗性存在很大的差异，表明这一抗逆性状主要还是由遗传因素决定的。

中外学者对其抗性遗传规律作了深入的研究，但结果却不相一致。

荒武义信[5]认为杂交F 1代对NPV 的抵抗性受母体影响，F 2代也未见偏父遗传现象。

孟智启等[6]的研究结果则表明家蚕经口感染NPV 后，其杂交后代（F 1、F 2）的抵抗性受父本影响，有偏父遗传的现象。

另外，荒武义信通过试验推断家蚕对NPV 的抵抗性受多因子影响，孟智启则认为家蚕对NPV 病毒经口感染的抵抗性遗传受一对主效显性基因和若干微效基因控制。

V IROLOGICA S INICA, June 2007, 22 (3):0218-225Received: 2006-11-13, Accepted: 2007-01-30* Foundation items: 973 (2003CB114202); Programme Strategic Scientific Alliances between China and the Netherlands (2004CB720404); National Natural Fundation of China project (30630002)** Corresponding author. Tel/Fax: 86-27-87197180, E-mail: huzh@HUANG et al.Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru 219(BmNPV), are most widely used to express foreign proteins. More than a thousand genes have been cloned and expressed through recombinant AcMNPV and BmNPV, ranging from the components of transcription machinery to pharmaceutical products (1). However, the traditional preparation of recombi- nant baculovirus to express foreign genes is very time consuming, because multiple rounds of purification and amplification of recombinant viruses are needed. Recently, the newly developed Bac-to-Bac TM system of AcMNPV has overcome this drawback. The AcMNPV bacmid can autonomously replicate in E. coli as a large plasmid at a low copy number, and the recombinant virus can be generated by the site specific transposition in Escherichia coli (E. coli)and used to infect insect cells. Since this system eliminates multiple rounds of purification and amplification of virus, recombinant viruses can be selected and purified within 7-10 days.BmNPV, a member of the family Baculoviridae, is a natural pathogen of the mulberry silkworm Bombyx mori. Though the BmNPV genome is over 90% identical to the genome of AcMNPV (7), its host specificities is very narrow. Unlike AcMNPV which can infect more than 30 lepidopteran insects (2, 8), the BmNPV can only infect silkworm and its cell lines. Since it was first used to express α-interferon in 1985 (15), the BEVS of BmNPV has been used for the expression of many foreign proteins either in a cell culture system or in a insect larvae system (1).B. mori BEVS are particularly suitable for the large-scale manufacture of foreign proteins, as the protein expression using silkworm or pupae is 10- to 100-fold higher than from B. mori cells. Silkworms are safe to the environment, and easy to breed with low cost. There is a long history of raising silkworms in China. All these make B. mori BEVS one of the most optimal systems for mass production of recombi- nant proteins. Several B. mori BEVS have been developed in past years (5, 10,11, 26, 27), however, the applications were limited due to the time con- suming process or low infectivity of the virus to silkworm.In this report, we described the construction of a bacmid of BmNPV, BmBacJS13, using in vivo recom- bination. To study the infectivity of the bacmid, the polyhedrin gene was inserted into the bacmid generating recombinant virus BmBacJS13-ph. The infectivity of BmBacJS13-ph was demonstrated by growth curve analysis of budded viruses and bioassay in B. mori larvae. The results indicated that BmBac- JS13-ph is a functional virus with similar infectivity to that of wt BmNPV. The results also showed that infective recombinant viruses could be generated with BmBacJS13 by site specific transposition in E. coli, indicating a functional Bac-to-Bac system of BmNPV was constructed.MATERIAL AND METHODSInsect, virus and cell lineLarvae of Bombyx mori, were reared on an artificial diet at 27℃(4) and third or fifth instars larvae were used in the experiments. BmN cells were cultured in TC-100 (JRH) insect medium supplemented with 10% fetal bovine serum (GIBCO/BRL) at 28℃ using standard techniques (17).The wt BmNPV Shaanxi strain, collected in Shaanxi province, China, were propagated on silkworm larvae (3).Construction of transfer vectorAccording to the sequence of the BmNPV T3 strain (GenBank accession number L33180), a 1.5 kb segment upstream of polyhedrin gene was amplified220 V IROLOGICA S INICA V ol.22, No 3by PCR from the wt BmNPV genome DNA using primers(5’-GGGCCGCGGGCGTAGAGATTCGAC GAAAGC-3’ and 5’-GCGCTGCAGATAATTACAA ATAGGATTGAGGCC-3’) and cloned into the Sac II and Pst I sites of pBluescriptKS II+ (Stratagene). A 1.7 kb segment downstream of the polyhedrin gene was PCR amplified using forward primer (5’-GGGG AATTCCCTGAGGTAAGCGTTAGATTCTGTGCG TTTG-3’) with Eco R I and Bsu 36I sites, and reverse primer (5’-GCGGGTACCTGACGAATCGTAAATA TGAATTCTGT -3’) with Kpn I site. The product was cloned into the plasmid containing the upstream segment to generate pKS-USR-DSR. The mini-F replicon, kanamycin resistance gene and the Tn7 target sites were cut out as an 8.6 kb Bsu 36I fragment from plasmid pBAC-Bsu 36I (18) and inserted into the Bsu 36I site of pKS-USR-DSR to generate transfer vector pBmTV (Fig. 1).Recombination and identification of the bacmid in vivoNewly molted fifth instar larvae were co-trans- fected with 20 μL DNA mixture per larva (including 0.45μg linearized plasmid pBmTV DNA, 0.15 μg wt BmNPV viral DNA and 3μL lipofectin (Invitrogen)) through subcutaneous injection (25). The haemolymph of the larvae were collected at 4 days post transfection and budded viruses (BVs) were retrieved from the supernatant of haemolymph after centrifuging. TheDNA of BVs was extracted and transformed into E. coli DH10B cells (GIBCO/BRL), and colonies were selected in the presence of kanamycin and X-Gal. The colonies were further analyzed by PCR and REN digestion. One of the positive clones, BmBacJS13 which had similar REN profiles to that wt BmNPV, was chosen for further analysis. Construction of BmBacJS13-phThe Polyhedrin gene of BmNPV was amplified by forward primer (5’-GCGGGATCCTGTCGACAAGC TCTGTCCGTTT-3’) with Bam HI (underlined), and reverse primer (5’-GGGGAATTCTTAATACGCCG GACCAGTG-3’) with Eco R I (underlined). The Bam H I site of the fragment was blunted and the fragment was inserted into Bst 1107 I and Eco R I sites of pFast bac Dual (Invitrogen) to generate pFast- DUAL-ph . The pFast bac Dual-ph was then used to produce a recombinant BmBacJS13-ph via trans- position in E. coli according to the Bac-to-Bac TM system manual. The bacmid DNAs of the recombinant were extracted and were used to transfect the BmN cells. Transfection of BmN cellsBacmid DNA of BmBacJS13-ph was isolated by methods developed for large plasmids (instruction manual of Bac-to-Bac TM system/Life Technologies). The DNA was used to transfect BmN cells with lipofectin, and genomic DNA of wt BmNPV was usedas a control. The supernatant was collected from theFig. 1. Schematic of transfer vector pBmTV . The 1.5kb upstream sequence and 1.7kb downstream sequence are homologous arms,through recombination the polh was replaced with kanamycin cassette, lacZ: attTN7: lacZ and mini-F replicon.HUANG et al.Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru 221transfected BmN cells at 96 hrs post transfection (h.p.i.) and was used to infect BmN cells. The supernatants of the infected cells were collected at 96 h.p.i and TCID50 was determined using end-point dilution method.Electron microscopeBmN cells (1×106 ) were infected with wt BmNPV or BmBacJS13-ph with a multiplicity of infection (MOI) of 5. The infected cells were harvested at 72 h.p.i. The samples were processed for electron microscopy examination as described by Van Lent et al. (22).Comparison of the growth curves between wt BmNPV and BmBacJS 13-phBmN cells were infected with BV of Bm- BacJS13-ph or wt BmNPV at an MOI of 5. At the appropriate time points post infection, the supernatants were collected and the titers were detected using the end-point dilution method, Each virus infection was repeated five times and the data were statistically analyzed with one-way ANOV A (SPSS); polyhedral inclusion body (PIB) was used as the marker during the assay.BioassayThe third instars of B. mori larvae were used in oral infection assays to determine the infectivity of Bm- BacJS13-ph and wt BmNPV. The larvae (n=50 per viral dose) were fed with artificial diet which contain- ned different concentrations of viruses (3×107 PIB/ mL, 107 PIB/mL, 3×106 PIB/mL, 106 PIB/mL, 3×105 PIB/mL and 105 PIB/mL, respectively) for twenty- four hrs. Then the larvae were transferred to fresh diet in plates and reared at 27℃to investigate the mortality. The infectivity of BmBacJS13-ph and wt BmNPV was determined by probit analysis with SPSS10.0 and LC50 values of the viruses were further compared with a two side’s z-test (21).RESULTSIdentification of the bacmid BmBacJS13After the construction of transfer vector pBmTV (Fig. 1), the pBmTV DNA was co-transfected with BmNPV DNA into newly molted fifth instar larvae. The hemolymphs of the larvae were collected 4 days post transfection, and BV DNAs were retrieved to transform E. coli DH10B cells. Colonies were chosen randomly and bacmid DNAs were digested with Hin d III. By comparison with the wt BmNPV profiles, several variations were found in different colonies (data not shown). One of the colonies, BmBacJS13, which had similar REN profiles to that of wt BmNPV, was analyzed with additional restriction enzymes. As s h o wn i n F i g.2,c o m p a r i s o n w i t h t h e wt BmNPVindicates that the Xba I profile of BmBacJS13 lacked B (19.3 kb)，D (15.8 kb),G (7.6 kb) and H (6.3 kb) bands, and contained five additional bands, D+H (22.2 kb), B’ (19.0kb), G’ (9.1 kb), 4.9 kb and 1.6 kb. Due to the deletion of the polyhedron gene, the B band in wt BmNPV became a shorter band B’ in BmBacJS13. The 4.9 kb and the 1.6 kb bands were from the inserted 8.6 kb Bsu36I fragment which contains three Xba I sites. The remaining Bsu36I fragment (2.1kb) was linked with the remainder of G the fragment (7.0 kb) to generate G’(9.1 kb). The D+H band in BmBacJS13, however, was a sub-molecular band of the D and H bands in the wt BmNPV. These were the expected changes for the bacmid. Therefore BmBacJS13 was constructed correctly and was used for further investigation Biological activity of BmBacJS13-ph222 V IROLOGICA S INICA V ol.22, No 3To test the oral infectivity of BmBacJS13, the poly-hedrin gene was repaired into the bacmid, generatingBmBacJS13-ph. The BmBacJS13-ph was identified tobe correct by PCR and REN analysis (data not shown),and transfected into BmN cells to generate BVs.BmN cells were infected with BmBacJS13-ph at anMOI of 5, occlusion bodies were observed in thenucleus of cells by optical microscopy from 48 hrs p.i,and the detachment of the cells were observed at thesame time. These cytopathic effect (CPE) were similarto that of wt BmNPV infected cells. Electronmicroscopy analysis showed that the occlusion bodieswere formed in the nuclei. The polyhedra and ODVsof BmBacJS13-ph had a similar shape and size asFig. 2. Xba I digestion profiles and linearized physical maps of BmBacJS13 and wt BmNPV. A. Xba I digestion profile of BmBacJS13 and wt BmNPV. 1, λ DNA digested by Bam H I, Eco R I and Hin d III; 2, BmBacJS13; 3, wt BmNPV. B. Linearized Xba I physical maps of BmBacJS13 and wt BmNPV. The restriction sites are indicated in kb from the zero point. The genome size in kb is shown on a scale at the top. The location of polh and 8.6kb Bsu36I fragment are shown.Fig. 3. EM pictures of BmN cells infected with wt BmNPV (A, B) and BmBacJS13-ph (C, D).HUANG et al.Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru 225those of wild type BmNPV (Fig. 3).One-step growth curve analysis indicated that BmBacJS13-ph BV had similar replication dynamics to that of wt BmNPV (Fig. 4). The titers of the two viruses were very similar during the early infection, but the titers of BmBacJS13-ph were slightly lower than that of wt BmNPV during 24 to 96 h.p.i. Statistical analysis indicated that the differences were not significant except those for 72 h.p.i..The bioassay result showed that LC50 of BmBac- JS13-ph against 3th larvae was 3.9x106 PIB/ml, which was not significantly different from of that of wt Bm- NPV (3.7x106 PIB/ml) (z=0.3276, P>0.05) (Table 1).DISCUSSIONA functional Bac-to-Bac system should possess the following characteristics: first, the bacmid should be able to autonomously replicate in the bacteria; second, a foreign gene can be inserted into the bacmid via transposition, and lastly, the backbone virus must have similar biological properties as the wt virus, in both cells and insects. Our study showed that BmBacJS13 could autonomously replicate in the bacteria, and a foreign gene (ph) was introduced into the Tn7 site ofFig. 4. Growth curves for BmBacJS13-ph and wt BmNPV. BmN cells were infected with BmBacJS13-ph or wt BmNPV at an MOI of 5. The supernatants were harvested at different times post infection and the BV titers were analyzed by EPDA. The average titer from five independent TCID50 assays were shown with the bars indicating standard errors. Table 1. LC50 of wt BmNPV and BmBacJS13-ph in early third instar B. mori larvae95% confidence limits VirusLC50(PIB/mL)Lower Upper wt BmNPV 3669441a 654072 28459424 BmBacJS13-ph3876462a 1400386 25047426aNo significant difference.BmBacJS13 with the aid of the helper plasmid. The recombinant virus BmBacJS13-ph had similar replication dynamics to that of wt BmNPV and bioassay results showed that its in vivo infectivity was similar to that of wt BmNPV. Therefore a functional Bac-to-Bac system of BmNPV has been constructed, and BmBacJS13 can be used to construct BmNPV recombinants for expressing foreign proteins. During our experiments, a similar Bac-to-Bac system of BmNPV was reported by Motohashia et al(16). In their system, a bacmid of the BmNPV T3 strain was constructed, and it could express enhanced green fluorescence gene (egfp) in larvae and pupae. As we have used a local strain from China (Shaanxi strain), it would be interesting to compare the properties between BmBacJS13 and the T3 bacmid.The Bac-to-Bac system can also be used for functional genomics studies of BmNPV. Bacmids have been widely used to delete genes by site-specific recombination in E. coli in AcMNPV and Helicoverpa armigera NPV (6, 9, 13, 12, 14, 19, 24, 23).. The target genes were deleted and subsequently repaired by transposition to determine the function of the genesin viral life cycle. Currently we are studying the functions of several BmNPV genes using BmBacJS13 and the results will be reported in the future. AcknowledgmentsWe thank Prof. Just M. Vlak of Wageningen University for kindly providing pBAC-Bsu36I and223222 V IROLOGICA S INICA V ol.22, No 3Prof. Chuanxi Zhang of Zhejiang University for BmN cell line. We thank Prof. Hanzhong Wang of Wuhan Institute of Virology for technical assistance. We thank the Animal Center of Wuhan Institute of Virology for providing B. mori larvae. We also thank the grants of 973 (2003CB114202), Programme Strategic Scientific Alliances between China and the Netherlands (2004CB720404), National Natural Fundation of China project (30630002) for financial support.References1.Acharya A, Sriram S, Saehrawat S. 2002. Bombyx morinucleopolyhedrovirus: molecular biology and biotech- nological applications for large-scale synthesis of recombi- nant proteins. Curr. Sci,83: 455-465.2.Adams J R, McClintock J T.1991. Baculoviridae.Nuclearpolyhedrosis Viruses. Boca Raton, FL: CRC Press, p87-204.3.Arakawa T.2002. Promotion of nucleopolyhedrovirusinfection in larvae of the silkworm, Bombyx mori (Lepidoptera: Bombycidae) by flufenoxuron. Appl En- tomol Zool, 37: 7-11.4.Choudary P V, Kamita S G, Maeda S. 1995. Baculovirusexpression protocols. Totowa: Humana press, NJ. p243- 264.5.Deng X Z, Zhu Y D, Zhen Y, et al. 2000. Construction ofa novel BmNPV Bac to Bac system. Acta MicrobiologicaSinica, 40:155-160.(in Chinese)6.Dong C S, Li D, Long G,et al. 2005. Identification offunctional domains required for HearNPV P10 filament formation. Virology, 338: 112-120.7.Gomi S, Majima K, Maeda S.1999. Sequence analysisof the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol, 80: 1323-1337.8.Granados R R, Williams, K A. 1986. In vivo infectionand replication of baculoviruses. Boca Raton, FL: CRC Press. I: p89-108.9.Hou S W, Chen X W, Wang H Z,et al. 2002. Efficientmethod to generate homologous recombinant baculovirus genomes in E. coli. Biotechniques, 32: 783-789.10.Je Y H, Chang J H, Kim M H, et al.2001. The use ofdefective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation ofocclusion-positive and occlusion-negative expression vectors.Biotechnol Lett,23:1809-1817.11.Kondo A, Maeda S. 1991. Host range expansion byrecombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica Nuclear Polyhedrosis Virus. J Virol,65: 3625-3632.12.Lin G, Blissard G W. 2002. Analysis of an Autographacalifornica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication.J Virol, 76: 2770- 2779. 13.Lin G, Blissard G W. 2002. Analysis of an Autographacalifornica multicapsid nucleopolyhedrovirus lef-6-null virus: LEF-6 is not essential for viral replication but appears to accelerate late gene transcription. J Virol, 76: 5503-5514.14.Lung O, Westenberg M, Vlak J M, et al. 2002.Pseudotyping Autographa californica multicapsid nucleo- polyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64. J Virol, 76: 5729-5736.15.Maeda S, Kawai M, Obinata H, et al. 1985. Productionof human alpha-interferon in silkworm using a baculovirus.Nature, 315:592-594.16.Motohashia T, Shimojimab T, Fukagawac T, et al. 2005.Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mor i nuclear polyhedrosisvirus bacmid system. Biochem Biophys Res Commun, 326: 564-56917.O'Reilly D R, Miller L K, Luckov V A. 1992.Baculovirus Expression Vectors. A Laboratory Manual.New York: Oxford University Press, NY. p216-229.18.Pijlman G P, Dortmans J F M, Vermeesch A M G,et al.2002. Pivotal roal of the non-hr prigin of DNA replication in the genesis of defective interfering baculovirus. J Virol, 76: 5605-5611.19.Smith G E, Fraser M J, Summers M D. 1983. Molecularengineering of the Autographa californica nuclear polyhed- rosis virus genome: deletion mutations within the polyhe- drin gene. J Virol, 46: 584-593.20.Smith G E, Summers M D, Fraser M J. 1983.Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol, 3: 2156-2165.21.Snedecor G W, Cochran, W G. 1989. Statistical methods.8th Edition, Iowa: Iowa State University Press, p503.22.van Lent J W M, Groenen J T M, Klingge-Roode E C,et al. 1990. Localization of the 34 kDa polyhedron224HUANG et al.Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru 225envelope protein in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus.Arch Virol, 111: 103-114.23.Wang H Z, Deng F, Pijlman G P, et al.2003. Cloning ofbiologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by usinga bacterial artificial chromosome. Virus Research, 97: 57-63.24.Wu D, Deng F, Sun X L, et al. 2005. Functional analysisof FP25K of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. J Gen Virol, 86: 2439-2444.25.Wu X F, Cao C P, Kumar V S, et al. 2004. An innovativetechnique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin. Research in Microbiology, 155: 462-466.26.Wu X F, Cao C P, Xu Y X, et al. 2004. Construction of ahost range-expanded hybrid baculovirus of BmNPV and AcMNPV, and knockout of cysteinase gene for more efficient expression. Science in China Ser. C Life Science, 47: 406-415.27.Wu X F, Yang G Z, Hu J X. 1998. A recombinantrescue linearizable BmNPV baculovirus BmBacPAK [Patent]. China Patent: application No. 98110963.2.。

家蚕Polh^+ Bac-to-Bac杆状病毒表达系统的构建曹翠平;兰丽盼;姚慧鹏;何芳青;郭爱芹;吴小锋【期刊名称】《蚕业科学》【年(卷),期】2008(34)2【摘要】为利用杆状病毒的强启动子——多角体启动子而构建的重组杆状病毒一般是多角体缺失型(polh-)病毒。

为了解决家蚕生物反应器规模化生产中病毒必需经皮接种而效率低下的问题,在建立家蚕Bac-to-Bac快速表达系统的基础上,构建了能形成多角体的家蚕Polh+ Bac-to-Bac表达系统。

通过该系统能够产生表达外源目的基因的重组病毒,获得的该重组病毒能在培养细胞内表达,也能通过经口添食感染表达。

利用EGFP报告基因分析了该系统的表达效果,构建的重组病毒不仅有效表达了EGFP蛋白,还在细胞核中形成了大量多角体。

该系统较好解决了重组病毒必需经皮接种感染的缺陷,提高了生产效率,拓宽了杆状病毒在生物杀虫剂、基因治疗等领域的应用前景。

【总页数】7页(P237-243)【关键词】家蚕;杆状病毒表达系统;多角体;经口感染【作者】曹翠平;兰丽盼;姚慧鹏;何芳青;郭爱芹;吴小锋【作者单位】浙江大学动物科学学院【正文语种】中文【中图分类】S884.5;Q78【相关文献】1.利用Bac-to-Bac杆状病毒系统超量表达家蚕 let-7簇microRNAs [J], 何婷;尹权;王伟;黄亚玺;吴小燕;夏庆友;刘仕平2.家蚕核型多角体病毒orf90基因在Bac-to-Bac家蚕杆状病毒系统中快速表达[J], 王强;陈克平;郭忠建;姚勤;王海燕;陈慧卿3.利用家蚕杆状病毒Bac-to-Bac系统表达大肠杆菌L-天冬酰胺酶Ⅱ [J], 费建明;吴岩;占鹏飞;施国方;王文兵4.零背景转座技术高效构建重组家蚕杆状病毒表达系统 [J], 姚伦广;张红玲;冯娟;张二辉;文祯中5.利用细菌转座子构建适于家蚕的Bac-to-Bac快速基因表达系统 [J], 吴小锋;曹翠平;鲁兴萌因版权原因，仅展示原文概要，查看原文内容请购买。

专利名称：一种扩繁家蚕核型多角体病毒的方法专利类型：发明专利
发明人：蔡国祥,林水中,刘桂州,茆迎春,杨俊生申请号：CN201711500879.6
申请日：20171218
公开号：CN108220250A
公开日：
20180629
专利内容由知识产权出版社提供
摘要：本发明公开一种扩繁家蚕核型多角体病毒的方法，包括毒源病毒的制备、病毒的临时保存病毒的人工接种、病毒的采收几个步骤，接种病毒前首先对毒源病毒提纯，病毒的临时保存方法为病毒多角体配制成1.2×10个/毫升～1.4×10个/毫升浓度，按病毒液∶10％氟派酸∶10％多菌灵1∶0.8～1.2∶0.8～1.2的配比，24～26℃室温条件保存1～2小时，接种病毒的时间选择为家蚕五龄饷食后4～8小时，接种病毒后90～100小时采收病毒。

本申请提供的扩繁家蚕核型多角体病毒的方法，生产工艺简单，生产成本低，扩繁效益高，对环境的要求也低，是生产家蚕血液型脓病灭活疫苗首选的病毒扩繁方案。

申请人：蔡国祥
地址：224002 江苏省盐城市亭湖区文港北路33号1幢504室
国籍：CN
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快速敲除补回家蚕核型多角体病毒ORF29王蓉【摘要】利用Red重组系统成功敲除家蚕核型多角体病毒ORF29基因(BmNPV ORF29,简称Bm29),并利用Bac-to-Bac系统构建补回型的Bm29基因.通过PCR鉴定敲除和补回均成功,为后续的Bm29基因功能研究奠定基础.【期刊名称】《浙江农业科学》【年(卷),期】2014(000)010【总页数】3页(P1624-1626)【关键词】家蚕核型多角体病毒;ORF29基因;Red重组;Bac-to-Bac【作者】王蓉【作者单位】浙江理工大学,浙江杭州310018【正文语种】中文【中图分类】S884.5杆状病毒是已知昆虫病毒中类群最大，且具有较大实用意义的昆虫病毒［1］。

目前，已有29种昆虫杆状病毒的基因组被完全测序［2］。

基因敲除技术是研究基因功能的一种常用手段。

传统的构建重组杆状病毒方法是利用转移载体与野生型病毒DNA共转染昆虫细胞，通过同源交换和空斑纯化获得重组病毒，但该方法不仅重组效率低，而且耗时耗材［3］。

近年来，Red重组系统，因其准确率高、操作简便的优点逐渐成为新型的基因敲除方法［4］。

Red重组系统属于λ噬菌体的重组系统，它的编码基因exo，bet和gam置于L-阿拉伯糖启动子控制下，在L-阿拉伯糖诱导下能够表达重组所需的酶，从而介导线性化片段与染色体的特定片段进行同源重组，进而利用外源基因替换靶基因［5］。

目前，已经有报道利用Red重组成功敲除了杆状病毒基因lef-6，l ef-8和lef-10［6-8］。

家蚕核型多角体病毒（BmNPV）基因组已经被测序完全，含有136个完整的开放阅读框［9］。

BmNPVORF29，简称Bm29，全长654 bp，位于BmNPV基因组T3株的26449～27102 bp处，表达产物预测分子量为22.0 ku。

该基因功能的研究还一直未见报道，本文首次利用Red重组系统在大肠杆菌中成功地对Bm29基因进行了定点敲除，并利用Bac-to-Bac系统补回缺失型的Bm29基因，为后续基因功能的研究奠定基础。

一种新型家蚕核多角体病毒Bac to Bac系统的构建邓小昭;朱应;刁振宇;齐义鹏;周宗安【期刊名称】《微生物学报》【年(卷),期】2000(040)002【摘要】用家蚕核型多角体病毒的全基因组DNA与Ac-BacmidDNA共转染家蚕BmN细胞,构建转座一穿梭载体Bm-Bacmid.另一供体质粒以转座方式将乙肝病毒e抗原基因HBeAg整合到Bm-Bacmid的attTn7位点上成为重组rBmHBe.结果表明Bm-Bacmid既能在大肠杆菌中以质粒的形式复制,又能在家蚕BmN细胞和草地夜蛾Sf9细胞中复制,形成感染性病毒粒子.Southernblotting证实重组病毒的构建是正确的.BmN细胞能正确识别与切割HBeAg信号肽序列,SDS-PAGE 表明HBeAg基因在家蚕细胞中高效表达,ELISA测定培养上清中HBeAg效价达1:32000,细胞内HBeAg效价为1:2000,培养液及细胞内的HBcAg含量极低(<1:160).【总页数】6页(P155-160)【作者】邓小昭;朱应;刁振宇;齐义鹏;周宗安【作者单位】南京军区军事医学研究所分子生物学实验室,南京,210002;武汉大学病毒研究所,武汉,430073;南京军区军事医学研究所分子生物学实验室,南京,210002;武汉大学病毒研究所,武汉,430073;南京军区军事医学研究所分子生物学实验室,南京,210002【正文语种】中文【中图分类】S884.5【相关文献】1.利用家蚕杆状病毒Bac-to-Bac系统表达大肠杆菌L-天冬酰胺酶Ⅱ [J], 费建明;吴岩;占鹏飞;施国方;王文兵2.甜菜夜蛾核多角体病毒BAC-TO-BAC外源基因表达系统的建立 [J], 杨凯;庞义3.利用细菌转座子构建适于家蚕的Bac-to-Bac快速基因表达系统 [J], 吴小锋;曹翠平;鲁兴萌4.Bac-to-Bac系统的研究进展及在家蚕中的应用 [J], 吴小锋;岳万福;刘剑梅;李广立;缪云根5.家蚕Polh^+ Bac-to-Bac杆状病毒表达系统的构建 [J], 曹翠平;兰丽盼;姚慧鹏;何芳青;郭爱芹;吴小锋因版权原因，仅展示原文概要，查看原文内容请购买。