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

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基因克隆入pVAX1载体中,然后将含有HA基因的表达盒(CMV HA PolyA)克隆入pVAXΔE3的SSPⅠ酶切缺失处,获得含有HA表达盒的穿梭载体pΔEHA。用SalⅠ NruⅠ分别对pΔEHA和pPoly-2-CAV2进行双酶切,将含有HA表达盒的SalⅠ NruⅠ片段克隆入pPoly2-CAV2,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。

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

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. A/tiger/Harbin/01/2003 (HSN1) 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 pVAXdeltaE. The recombinant plasmid was named pdeltaEHA. The pdelta 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-H5N1 influenza virus HI antibody (1:8 - 1:16) was detected in the cat immunized with CAV-2/HA.