以家蚕核型多角体病毒为载体高效表达天花粉蛋白基因

本文报道以家蚕核型多角体病毒为载体,在家蚕体内高效表达天花粉蛋白基因的结果。天花粉蛋白基因是用ＰＣＲ技术从栝楼基因组中分离的,该基因被插入到家蚕核型多角体病毒转移载体质粒ｐＢｍ-１的多角体蛋白基因启动子下游,构建成重组质粒ｐＢｍＴＣＳ。将重组质粒ＤＮＡ和野生型ＢｍＮＰＶＤＮＡ共转染家蚕培养细胞,通过在家蚕培养细胞中进行同源重组和筛选,获得了无多角体的重组病毒ＢｍＴＣＳ。采用ＰＣＲ技术对重组病毒进行了鉴定,证实重组病毒合天花粉蛋白基因。重组病毒对家蚕的感染性不及野生病毒,提示表达产物对病毒的增殖有抑制作用。对重组病毒感染的家蚕血淋巴进行了ＳＤＳ-ＰＡＧＥ和免疫印迹分析,结果显示在蚕体血淋巴中的表达产物天花粉蛋白占总蛋白的５％。本实验为利用基因工程方法大量生产天花粉蛋白提供了又一条新的途径。     

茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。     

家蚕核型多角体病毒对小鼠的感染性研究

家蚕核型多角体病毒是一类双链闭合环状DNA病毒 ,该病毒被作为载体应用在家蚕生物表达系统 ;为确定该病毒的安全性 ,观察了该病毒对小鼠瘤细胞以及小鼠的感染性 ,结果显示芽生型家蚕核型多角体病毒在人DC杂交瘤细胞株和HL60细胞中无增殖 ;不同剂量的包埋型家蚕核型多角体病毒经口灌胃给小鼠 ,在小鼠肾、肝病理切片及电镜观察中未见病毒颗粒 ,用免疫组化试验检测小鼠肾、肝组织中的多角体病毒也为阴性。     

家蚕BmNPV多角体蛋白与外源蛋白共包埋入多角体的研究

为了探索家蚕核型多角体病毒多角体的包装特性,构建了一种不形成多角体但能大量表达绿色荧光蛋白(EGFP)的重组病毒vBmBac(polh-)-5B-EGFP,将其与野生型BmNPV共同感染BmN细胞,于荧光显微镜下观察到EGFP与多角体可以在同一细胞中同时表达。从感染的BmN细胞中收集纯化多角体,观察到多角体能被激发出绿色荧光,进一步利用Western blot证实多角体中含有EGFP。上述结果表明,多角体可以将自身病毒粒子以外的其他病毒粒子的成分包装进入多角体,表明多角体的包装机制中存在非特异性识别机制。     

转座子介导的多角体基因表达及提高病毒粒子的感染效率

现行的杆状病毒表达外源基因的方法是将外源基因取代病毒中的多角体基因,因而得到的重组杆状病毒感染活体时不能经口感染,只能进行针刺注射,效率低且易引起活体感染其他疾病。将家蚕核型多角体病毒(Bombyx mor inucleopolyhedrovirus,BmNPV)中的多角体基因(polyhedrin,poly)及其启动子片段克隆到转座子载体pigA3GFP中,将其与辅助质粒pHA3PIG利用脂质体介导法导入家蚕细胞中,经过多次筛选获得稳定的转基因家蚕细胞。之后先将BmPAK6(含LacZ)及BmGFP(含GFP)重组病毒分别感染转基因细胞,再将得到的重组病毒经口感染5龄家蚕幼虫。结果显示,重组杆状病毒可以经口感染家蚕幼虫。这些研究表明来自于转基因家蚕细胞的poly基因表达产物可以提高重组杆状病毒经口感染家蚕率,为解决杆状病毒表达系统中重组病毒不能经口感染家蚕幼虫的问题提供新思路。     

增强蛋白与多角体蛋白共包埋的研究

颗粒体病毒的增强蛋白（enhancin)是一种能显著提高核型多角体病毒（NPV）对昆虫感染力的病毒蛋白。构建了一种不形成多角体但表达粉纹夜蛾颗粒体病毒增强蛋白的重组病毒AcBBH-TnEn,将它与野生型AcMNPV共同感染SF21细胞,经SDS－PAGE、免疫印迹分析、荧光免疫等方法检测证实,增强蛋白与多角体可在同一细胞中同时表达,而且发现所形成的病毒多角体带有增强蛋白。这表明,可以通过混合感染的方式生产带有增强蛋白的病毒多角体。     

家蚕核型多角体病毒同源重复区hr5的结构和功能

家蚕核型多角体病毒(BmNPV)和首蓿尺蠖核型多角体病毒(AcMNPV)是宿主不同的同类昆虫杆状病毒.AcMNPV有5个同源重复区,hr5具较强增强子功能,最近被发现可能参与病毒DNA复制.Maeda等曾将BmNPV hrs图谱定位,最近又报道了BmNPV hr s的结构,但功能研究至今未见报道.与Maeda等同时,从野生型BmNPV基因组中克隆了hr 5区,进行了全序列测定,与Maeda等的报道有一定差别.功能分析证实,含BmNPV hr 5的质粒在辅助病毒存在下,不仅在宿主细胞(BmN)中能进行复制,而且在非宿主细胞 Sf 21中亦能进行复制.此外,不仅完整的BmNPV hr 5(含 8个重复单位),而且部分序列(含6个重复单位)亦显示上述功能.以上研究结果,并对hr 5在病毒 DNA复制过程中可能有的功能进行了探讨.     

重组家蚕核型多角体病毒表达乙肝病毒S区抗原

报道了将乙型肝炎病毒（ａｄｒ亚型）Ｓ区基因（共６８１ｎｔ）克隆于家蚕核型多角体病毒的基因组中,并将其导入家蚕细胞,通过多轮筛选得到纯的重组家蚕杆状病毒,用该病毒接种家蚕,初步证明能够得到乙肝表面抗原的高效表达,在家蚕幼虫的血淋巴和蛹体中其用ＰＲＨＡ法检测的滴度可达１∶４０９６。     

家蚕核型多角体病毒蛋白组份检测方法建立

研制了兔抗家蚕核型多角体病毒蛋白组份的多克隆抗体,用间接法建立了家蚕核型多角体病毒蛋白的dot-ELISA检测方法。此法检测多角体病毒蛋白的灵敏度达10ng,与人血清和兔血清无交叉反应,可用于家蚕生物反应器生产的目的蛋白中多角体病毒蛋白组份的检测。     

家蚕核型多角体病毒对小鼠的易感性及其蛋白组份的检测

家蚕核型多角体病毒(Bm-NPV)是一类环状DNA病毒。该病毒作为载体,应用在杆状病毒-昆虫真核生物表达系统。为确定家蚕基因工程表达系统产品对人的安全性,观察了该病毒对哺乳动物细胞株及对小鼠的易感性,建立了检测Bm-NPV蛋白组份的dot-ELISA方法,可用于生物表达器生产的产品中残存多角体病毒组份的检测。结果表明,芽生型多角体病毒(BV)在杂交瘤细胞和HL60细胞中不增殖。不同剂量包埋型多角体病毒(OV)灌胃感染小鼠,小鼠肝、肾组织切片的电镜观察及免疫组化染色未见到病毒颗粒。     

人促红细胞生成素基因在家蚕体中的高效表达

人促红细胞生成素(EPO)是一种调控红系干细胞增殖、分化和成熟的糖蛋白激素。将合成的EPO cDNA插入杆状病毒转移载体pBlueBacⅢ,使其置于Ph基因强启动子控制之下,获得了转移载体pBlueBacEPO。将pBlueBacEPO DNA与野生型BmNPV DNA共转染BmN细胞,经空斑纯化,获插入EPO cDNA的重组病毒rBmNPVEPO。经Sonthern杂交和PCR扩增鉴定证明人EPO基因已正确组建于BmNPV的预定位置。将重组病毒rBmNPVEPO穿刺接种5龄幼虫和蛹,收集感染第3～5d的幼虫血淋巴和3～6.5d蛹血淋巴。用ELISA检测幼虫血淋巴中EPO表达量高达62800u/mL,蛹血淋巴中表达量达74000u/mL。Western blot结果显示幼虫血淋巴和蛹血淋巴均有一条明显的免疫杂交带,分子量均约为26kD。用TF1细胞对幼虫表达产物进行了生物活性测定,每毫升血淋巴中EPO活性约为63000u。     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus(BmNPV),a transfer vector was constructed which contained an Escherichia coli(E.coli)mini-F replicon and a lacZ:attTN7:lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene.B.mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo.The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E.coli DH10B.Recombinant bacmids were screened by kanamycin resistance,PCR and restriction enzyme(REN)digestion.One of the bacmid colonies,BmBacJS13,which had similar REN profiles to that of wild-type BmNPV,was selected for further research.To investigate the infectivity of BmBacJS13,the polyhedrin gene was introduced into the bacmid and the resultant recombinant(BmBacJS13-ph)was transfected to BmN cells.The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells.Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV.Bio-assays indicated that BmBacJS13-ph was also infectious to B.mori larvae.     

Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.     

High-frequency homologous recombination between baculoviruses involves DNA replication

We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4% when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1.4 kb, the mean frequency of recombination was 2.7%. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was 相似文献   

Abnormal formation of polyhedra resulting from a single mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.     

HcNPV半胱氨酸蛋白酶,几丁质酶基因失活分析

将含有美国白蛾核型多角体病毒（Ｈｙｐｈａｎｔｒｉａ ｃｕｍｅａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＨｃＮＰＶ）半胱氨酸蛋白酶基因（ＣＰ）的自然和几丁质酶基因（ＣｈｉＡ）的片段克隆进ＰＣＲⅡ,构建了转移载体ｐＨｃＣＶｄｅｌ;将含有ＨｃＮＰＶ多角体蛋白全基因（ｐｏｌｈ）序列的片段插入到ｐＨｃＣＶｄｅｌ的ＥｃｏＲＩ位点,得到重组转移载体ｐＨｃＣＶｐｏｌｈ。通过重组转移载体与含有家蚕促     

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体,与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN,经200μg/ml zeocin抗生素筛选一个月,成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒,其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染,结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主,却并不在宿主体内形成包涵体,从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题,通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。