表达O型口蹄疫病毒VP1基因的重组病毒BHV-1的构建与鉴定  

Construction and identification of recombinant BHV-1 expressing foot and mouth disease virus VP1 gene

作　　者：任宪刚[1] 薛飞[1] 朱远茂[1] 童光志[1] 王延辉[1] 冯军科[1] 祖立闯[1] 李娇[1] 史鸿飞[1] 高欲燃[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所大动物传染病研究室,兽医生物技术国家重点实验室,哈尔滨150001

出　　处：《微生物学报》2009年第5期677-682,共6页Acta Microbiologica Sinica

基　　金：国家“863计划”(2006AA10A204)~~

摘　　要：【目的】为了构建表达口蹄疫病毒(O/China/99)VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒(CMV)启动子之下构建gE基因缺失转移载体。【方法】利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1。【结果】PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。【结论】本研究成功地构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。[Objective] In order to construct the recombinant bovine hepervirus-1 （BHV-1） which expressed foot and mouth disease virus （FMDV）VP1 gene, we constructed a BHV-1 gE gene transfer vector by inserting the synthetic VP1 gene of FMDV （O/China/99） under the immediate-early promoter of cytomegalovirus. [ Methods] The mixtures of parental virus （BHV-1/gE^- / LacZ^＋ ） DNA and transfer vector was transfected into bovine turbinate ceils using calcium phosphate-mediated transfection. Then the propagated viruses were harvested. The recombinant BHV-1 （designated BHV-1/gE-/VP1 ） was obtained by selection for white virus plaques. [ Results] PCR results showed that VP1 gene was successfully inserted into the genome of BHV-1/gE^- . The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting. [ Conclusion] The research provided a basis for development of BHV-1 vector vaccines for FMD and other important bovine infectious diseases.

关 键 词：口蹄疫病毒 VP1基因 牛疱疹病毒1型 磷酸钙介导转染 

分 类 号：S852.5[农业科学—基础兽医学]