重组虎源H5N1流感病毒HA基因犬2型腺病毒的构建与实验免疫研究  

Construction and experimental immunity of recombinant replication-competent Canine adenovirus type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus

作　　者：高玉伟[1] 夏咸柱[1] 王立刚 刘丹 黄耕[1] 

机构地区：[1]军事医学科学院军事兽医研究所,长春130062 [2]黑龙江省东北虎林园,哈尔滨150027

出　　处：《微生物学报》2006年第2期297-300,共4页Acta Microbiologica Sinica

基　　金：全军医学科研基金(04Z016);国家自然科学基金(30471Z95)~~

摘　　要：H5N1流感病毒可以对虎和猫产生致死性感染，为研制可用于预防猫科动物流感的新型疫苗，构建了重组虎源H5N1流感病毒HA基因的犬2型腺病毒。将A／Tiger／Harbin/01／2003（H5N1）的HA基因克隆人pVAX1载体中，然后将含有HA基因的表达盒（CMV＋HA＋PolyA）克隆人pVAXAE3的SSPⅠ酶切缺失处，获得含有HA表达盒的穿梭载体pAEHA。用SalⅠ＋NruⅠ分别对pAEHA和pPoly-2-CAV2进行双酶切，将含有HA表达盒的SalⅠ＋NruⅠ片段克隆人pPoly2-CA V2，获得了在E3区缺失处插入HA表达盒的重组质粒pCAV-2／HA。释放CAV-2／HA重组基因组转染MDCK细胞，获得了重组活病毒CAV2／HA，经Western blot分析表明重组表达产物可被流感病毒HA单克隆抗体3A13所识别。使用该重组病毒免疫猫可以产生效价为1：8—1：16的抗H5亚型流感病毒血凝抑制抗体。H5N1 highly pathogenic avian influenza virus was highly pathogenic and sometimes even fatal for tigers and cats. To develop a new type of vaccine for Felidae influenza prevention, recombinant replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus was constructed. Ntiger/Harbin/O1/2003（H5N1） HA gene was cloned into PVAX1. The HA expression cassette which included CMV and HA and PolyA was ligated into the E3 deletion region of pVAXAE. The recombinant plasmid was named p△EHA. The p＆ EHA and the pPoly2-CAV2 were digested with Nru Ⅰ/Sal Ⅰ, respectively. The purified Nru Ⅰ/Sal Ⅰ DNA fragment containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the recombinant plasmid pCAV-2/HA. The recombinant genome was released from pCAV-2/HA, and was transfected into MDCK cells by Lipofectamine. The recombinant virus named CAV2/HA was gained. Anti-HSN1 influenza virus HI antibody （1：8 - 1：16） was detected in the cat immunized with CAV-2/HA.

关 键 词：HPAIV 血凝素基因 犬2型腺病毒 

分 类 号：S852.65[农业科学—基础兽医学]