表达虎源H_5N_1亚型禽流感病毒NP蛋白重组犬2型腺病毒的构建及鉴定  

Construction and identification of expression NP protein of AIV H_5N_1 subtype from tiger recombinant canine adenovirus type 2

作　　者：彭广能[1,2] 耿长国[1,2] 何春燕[1,2] 汪开毓[1] 夏咸柱[2] 高玉伟[2] 杨松涛[2] 刘益新[1,2] 

机构地区：[1]四川农业大学动物医学院动物疫病与人类健康四川省重点实验室,四川雅安625014 [2]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2010年第12期1623-1628,共6页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划资助项目(2010BAD04B01);教育部"长江学者和创新团队发展规划"创新团队资助项目(IRT0848)

摘　　要：将A/Tiger/Harbin/132/2007(H5N1)的NP基因克隆入pVAX1载体中,然后将含有NP基因的表达盒(CMV+NP+PolyA)克隆入pVAXE3的SspⅠ酶切缺失处,获得含有NP基因表达盒的穿梭载体pVAXΔE3-NP。用SalⅠ+NruⅠ分别对pVAXΔE3-NP和pPoly-2-CAV-2进行双酶切,将含有NP基因表达盒的片段定向克隆入pPoly-2-CAV-2,获得在E3区缺失处插入NP基因表达盒的重组质粒pCAV-2-NP。释放CAV-2-NP重组基因组,脂质体介导转染DK细胞,获得了能使DK细胞产生典型腺病毒样细胞病变的CAV-2-NP重组病毒。从形态学、基因组水平、NP基因的转录、NP蛋白的表达以及重组病毒的生长特征等方面进行鉴定。结果表明,CAV-2-NP具有典型的CAV-2形态特征,在繁殖过程中没有对H5N1NP表达盒片段进行缺失或重排,并且能够转录H5N1NP基因的mRNA,ELISA和免疫荧光分析表明重组表达产物可被流感病毒NP基因单克隆抗体1G6G4所识别。NP genes of A/Tiger/Harbin/132/2007（H5N1） were cloned into pVAX1 carrier,and the expression cassette containing NP genes（CMV＋NP＋PolyA）was cloned into the Ssp Ⅰ enzyme lossed division of pVAXE3,then obtaining shuttle vector（pVAXΔE3-NP） containing NP expression cassette.Using SalⅠ ＋ NruⅠdouble digestion for pVAXΔE3-NP and pPoly-2-CAV-2,the fragment containing NP expression cassette was directionally cloned into pPoly2-CAV-2 and obtaining recombinant plasmid（pCAV-2-NP） of NP expression cassette inserted into the E3 lacking area.After releasing CAV-2-NP recombination genome and transfecting DK cells by liposomes mediate,the CAV-2-NP recombination virus were obtained,which typically made the DK cells adenovirus-like cytopathic.From morphology,genomics level,NP genes transcription,NP protein expression and the growth characteristics of recombinant virus,etc,the recombinant virus was indentified.The results showed that CAV-2-NP has typical morphologic characteristics of CAV-2,and didn＇t delete or rearrange H5N1 NP expression cassette fragments and transcribed mRNA of H5N1 NP in the process of breeding.The results from ELISA and immunofluorescence analysis indicated that recombinant expression product can be recognized by influenza virus NP monoclonal antibody 1G6G4.

关 键 词：虎源H5N1亚型禽流感病毒 NP基因 犬2型腺病毒 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]