新加坡石斑鱼虹彩病毒ORF162蛋白的表达、纯化及抗体制备Expression,Purification and Antibody Preparation of Singapore Grouper Iridovirus ORF162
夏立群,梁海鹰,江韶英,张尧清
摘要(Abstract)：
将新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)的ORF162的开放式阅读框插入pET-32a表达载体T7启动子控制下的6-His·Tag编码基因上游,构建SGIVORF162原核表达质粒pET-ORF162。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达SGIV ORF162融合蛋白。对IPTG浓度、诱导温度、诱导时间等诱导表达条件进行优化后,确定在0.7mmol/LIPTG、16℃条件下诱导14h时可溶性SGIV ORF162重组蛋白占重组蛋白总量的95%。经镍琼脂糖凝胶纯化,获得纯度为90%以上的SGIV ORF162蛋白。用纯化的SGIV ORF162蛋白免疫小鼠,获得高效特异的SGIV ORF162多克隆抗体。
关键词(KeyWords)： 新加坡石斑鱼虹彩病毒;原核表达;多克隆抗体
基金项目(Foundation): 广东省自然科学基金(06104920)
作者(Author): 夏立群,梁海鹰,江韶英,张尧清
参考文献(References)：
- [1]Qin Q W,Lam T J,Sin Y M,et al.Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper,Epinepheous tauvina[J].J Virol Methods,2001,98(1):17-24.
- [2]Qin Q W,Chang S F,Ngoh G H,et al.Characterization of a novel ranavirus isolated from brown-spotted grouper,Epinephelus tauvina[J].Dis Aquat Organ,2003,53(1):1-9.
- [3]Song W J,Qin Q W,Qiu J,et al.Functional genomics analysis of Singapore grouper iridovirus:complete sequence determination and proteomic analysis[J].J Virol,2004,78(22):12576-12590.
- [4]Song W,Lin Q,Joshi S B,et al.Proteomic studies of the Singaporegrouper iridovirus[J].Mol Cell Proteomics,2006,5(2):256-264.
- [5]Teng Y,Hou Z,Gong J,et al.Whole-genome transcriptional profiles of a novel marine fish iridovirus,Singapore grouper iridovirus(SGIV)in virus-infected grouper spleen cell cultures and in orange-spotted grouper,Epinephulus coioides[J].Virology,2008,377(1):39-48.
- [6]D'Costa S M D,Yao H,Bilimoria S L.Transcription and temporal cascade in Chiloiridescent virus infected cells[J].Arch Virol,2001,146(11):2165-2178.
- [7]Sanfilippo C M,Blaho J A.ICP0gene expression is a herpes simplex virus type1apoptotic trigger[J].J Virol,2006,80(14):6810-6821.
- [8]Castillo J P,Yurochko A D,Kowalik T F.Role of human cytomegalovirus immediate-early proteins in cell growth control[J].J Virol,2000,74(17):8028-8037.
- [9]Eaton H E,Metcalf J,Penny E,et al.Comparative genomic analysis of the family Iridoviridae:re-annotating and defining the core set of iridovirus genes[J].Virol J,2007,4(11):1-17
- [10]Pallister J,Goldie S,Coupar B,et al.Promoter activity in the5’flanking regions of the Bohle iridovirus ICP18,ICP46and major capsid protein genes[J].Arch Viro.2005,150(8):1911-1919.
- [11]Kinchington PR,Fite K,Seman A,et al.Virion Association of IE62,the varicella-zoster virus(VZV)major transcriptional regulatory protein,requires expression of the VZV open reading frame66protein kinase[J].J Virol.2001,75(19):9106-9113.
- [12]Moss B,Shisler JL.Immunology101at poxvirus U:immune evasion genes[J].Semin Immunol.2001,13(1):59-66.
- [13]Hobbs WE,DeLuca NA.Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0[J].J Virol.1999,73(10):8245-8255.