禽流感病毒核蛋白基因在重组杆状病毒中的表达

Expression of Avian Influenza Virus Nucleoprotein Gene in Recombinant Baculovirus

利用ＲＴ－ＰＣＲ方法成功地扩增了我国禽流感病毒分离株Ａ／Ｘｉｎｇｊｉａｎｇ／１／９６（Ｈ１４Ｎ５）的核蛋白（ＮＰ）基因，其限制性内切酶图谱和核苷酸序列与鸭源的标准Ｈ１４Ｎ５毒株几乎完全一致，与其它毒株则有较大差异，说明该鸡源分离株与鸭源毒株有非常近的亲缘关系。将ＮＰ基因定向克隆到杆状病毒转移载体ｐＶＬ１３９３中，再与杆状病毒线性ＤＮＡ（ＢＡＣ－Ｎ－ＢｌｕｅＤＮＡ）共转染于Ｓｆ９昆虫细胞中，经过三次蚀斑筛选，获得重组病毒ｒＢ２。用其细胞表达产物裂解后作ＳＤＳ－ＰＡＧＥ蛋白电泳、Ｗｅｓｔｅｒｎ－ｂｌｏｔ和ｄｏｔ－ＥＬＩＳＡ，结果表明ＮＰ基因在杆状病毒系统中获得了表达。同时用表达产物作琼脂扩散试验，结果表明表达产物与现行标准禽流感琼扩抗原具有相同的生物学活性。

Nucleoprotein(NP) encoding cDNA of an avian influenza virus(AIV) A/Xingjiang/1/96(H14N5) was amplified and cloned into pUC119 plasmid.The NP gene cDNA then was subcloned into a baculovirus transfer vecter pVL 1393 plasmid.Sf9 insect cells were co transfected with the recombinant plasmid and linearized Bac N Blue TM DNA.The expression of NP protein in Sf9 cells was confirmed by Western blot,dot ELISA and AGP test with ployclonal antibodies.The results from AGP test indicate that the recombinant NP protein has the same biological activities as the ordinary AGP antigens