人β－干扰素在杆状病毒载体与Sf细胞体系中的表达

Expression of human beta-interferon gene in system of haculovectors and Sf cells

采用苜蓿尺蠖核多角休病毒（Ａｕｔｏｇｒａｐｈａｃａｌｉｆｏｒｎｉｃａｎｕｃｌｅａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ，ＡｃＮＰＶ）ＤＮＡ中多角体蛋白基因启动子及其前后序列作为转移载体，将人β－干扰素（ＨｕＩＦＮ－β基因插入转移载体ｐＵＡｃ－５，构建成ｐＡｃ－ＩＦＮ－β载体。该质粒ＤＮＡ与野生型ＡｃＮＰＶＤＮＡ经ｌｉｐｏｆｅｃｔｉｎ共转染秋粘虫（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）传代细胞（Ｓｆ９细胞），利用重组病毒不产生多角体蛋白的特征，进行病毒空斑筛选。用核酸杂交方法鉴定出携带ＨｕＩＦＮ－β基因的主组病毒。用重组病毒感染Ｓｆ９细胞，可表达ｒＨｕＩＦＮ－β。在１．０×１０￣６细胞／ｍｌ感染上清中测定ｒＨｕＩＦＮ－β最高活性为３．０×１０￣６ＩＵ／ｍｌ，抗天然ＨｕＩＦＮ－β抗体能完全中和ｒＨｕＩＦＮ－β的抗病毒活性。

A DNA fragment containing polyhedrin promoter and its 5'and 3'sequences of A Autographacalifornica nuclear polyhedrosis viruses (AcNPV)was used as the transfer vector. Human IFN-β gene was in-serted into transfer vector pUAC-5,to build the pAc-IFN-βvector plasmid.Spodoptera frugiperda(Sf)cellswere cotransfected with the transfer vector plasmid DNA and wild type AcNPV genomic DNA using lipofectinreagent. The recombinant viruses were isolated and purified by the virus plaque assay several times. Dot blot andSouthern blot hybridization revealed that the recombinant viruses Ac-IFN-β DNA contained HuIFN-β gene se-quence. Biological active interferon was produced in Sf 9 cells infected with recombinant viruses and secreted intomedia, A maximum of 3.0 × 10￣6 IU / ml of interferon activity of rHuIFN-β was produced in 1.0×10￣6 cells/mlof Sf cells infected with Ac-IFN-β.Antiviral activity of rHuIFN-βmight be neutralized with the antibody of na-tive HuIFN-β.