杆状病毒

• The recombinant meq - baculovirus was obtained by co - transfecting the insect sf9 cells with pblubac4 - meq and linearised bac - n - blue dna . the recombinant baculovirus was selected by plaque assay and confirmed by pcr technique and sequencing of the inserted gene
  应用重组痘病毒表达的meq制备的单抗23b46对重组杆状病毒感染的sfg细胞及其裂解物分别进行间接免疫荧光试验、 westemblot和免疫沉淀试验的检测。
• A general introduction of the research on baculovirus is present in chapter one , including the overview of baculovirus , taxonomy and structure of baculovirus and the life cycle of the phenotype virus ( autographa californica multiple - nucleocapsid nucleopolyhedrovirus , acmnpv ) of baculovirus
  本论文共包括4章第一章首先对杆状病毒的研究历史作了综述性报道，包括病毒的分类学研究，病毒结构和感染循环等。
• The angiostatin baculovirus transfer vector was co - transfected with viral dna into sf9 cells according to the manufacturers protocol . to purify the recombinant virus , we used the plaque assay to screen the pure recombinant plaque and amplify it to generate p - 1 stock
  构建重组病毒：用已经构建好的angiostatin杆状病毒转移载体pbluebachiszb和病毒dna共同转染sfg细胞，通过蚀斑实验筛选出纯的重组斑点并扩增产生p二病毒贮存液。
• Ii ) the renaturation of synthetic hpab was carried out in glutathione oxidation / reduction solution ( l : 10 ) . the renatured synthetic hpab could kill escherichia coli , pseudomonas aeruginosa and staphylococcus aureus on lb agar plates . thirdly , construction of artificial mutants of hpab
  3 .突变体的设计及其重组杆状病毒构建:采用同源模建法分析hpab一p的空间结构，并以此结构为基础，设计hpab一p突变体hpab一p38和hpab一p34 。
• There are 3 chapters in this thesis : chapter one is a general introduction of two kinds of baculoviral envelope fusion proteins ( efp ) , gp64 and f proteins . the structure and functions of these proteins and the application of gp64 for surface display as well as the substitution of several viral efps were summarized
  第一章对杆状病毒的膜融合蛋白的研究进展作了综述性介绍，包括gp64与f蛋白的结构与功能，几种病毒膜融合蛋白的替换关系， gp64表面展示的应用。
• To obtain large amounts of appa phytase , the appa gene was subcloned into the prokaryotic expression vector pet - 28a ( + ) and baculovirus transfer vector pvl - 1393 under the control of the lac and polyhedrin promoter , respectively . the appa phytase was overexpressed in e . coli strain bl21 induced by lactose
  为了大量表达appa植酸酶，我们将appa基因分别克隆至原核表达载体pet - 28a ( + )和杆状病毒转移载体pvl - 1393中，将其分别置于lac和polyhedrin启动子控制之下。
• In order to get the soluble recombinant eo protein and inspect the protein expression status convinently , the egfp and eo gene were ligated into baculovirus transfer vector . with the co - transfecting sf9 cells of baculovirus recombinant transfer vector and linearized viral dna , and plaque purification in the posttransfection procedure , the pure recombinant baculovirus were harvested , which infected the sf9 cells for amplifying to generate a p - l stock . . in the meantime , the fluorescence microscopy detection indicated expressed egfp protein to confirm the heterogenous protein expression of recombinant baculovirus . the pi - stock from a pure plaque was used to generate a high liter p - 2 stock , which was determined in liter as 1 . 14 107pfu / ml by performing a plaque assay . when a volume of p - 2 stock infected the sf9 cells with moi 5 - 10 for expression , the strong fluorescence was obeserved on the day 3 of postinfection
  此外，为了得到可溶性重组eo蛋白并便于观察重组蛋白的表达情况，我们将egfp基因与eo基因相连插入昆虫杆状病毒转移载体中，与线性杆状病毒dna共转染sf9细胞后通过噬斑纯化得到纯的重组杆状病毒，将其感染sf9细胞制备p1种子液，同时用荧光显微镜观察绿色荧光蛋白的表达情况剔除表达效果差的重组杆状病毒。再用p1种子液感染sf9细胞制备高效价的p2种子液。通过病毒液的梯度稀释和噬斑测定，确定p2种子液的病毒滴度达1 . 14 10 ~ 7pfu ml 。
• The reasults are summed up as following : 1 the study on chromosomes and mitoses of bmn cells the cell line , bmn , is a silkworm cell line widely used in silkworm molecular genetics , cell engineering , gene engineering and baculovirus expression system but whose genetics and cytobiology studies are nearly untouched . the chromosomes and mitoses of the bmn cells are researched by the air - drying method and culturing cells on cover glasses
  同时，还通过原代培养实验对新的家蚕胚胎细胞系的建立进行了探索和尝试，并对家蚕胚胎原代培养过程中出现的细胞和组织类型进行了观察、探讨与研究。 1bmn细胞有丝分裂及染色体研究bmn细胞是家蚕分子遗传学，细胞工程、基因工程和杆状病毒表达系统中广泛应用的家蚕细胞，但其遗传学和细胞生物学背景知之甚少。
• The interest gene was inserted in the - tha l . tho 1 multiple cloning sites of donor plasimd pfastbachtb of baculovirus expression system . after analysis by restriction endonuclease and pcr , the recombinant donor plasmid gpl - fast was transforn1ed to the competent celi dhi0bac which contalns the bacmid and the helper plasmid , the recombinant bacmid gpl - bac was acquired which would express the vpl of gpv strain h l
  同时将该目的基因插入到杆状病毒表达系统的供体质粒pf _ ( ast ) b _ ( ac ) htb的xba 、 xho多克隆位点间，经酶切、 pcr鉴定后，将重组的供体质粒gp1 - fast转化到含有杆状病毒和辅助质粒的dh10b _ ( ac )感受态细胞中，获得了表达gpvh1株vp1的重组杆状病毒gp1 - bac 。