Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.
Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.
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利用昆虫细胞和蚕幼虫通过两种不同的杆状病毒基因表达系统生产具有生物活性的重组牛干扰素-γ。
作者：
K. Murakami;A. Uchiyama;T. Kokuho;Y. Mori;H. Sentsui;T. Yada;M. Tanigawa;A. Kuwano;H. Nagaya;S. Ishiyama;H. Kaki;Y. Yokomizo;S. Inumaru
将含分泌信号肽编码区的牛干扰素γ (bIFN-gamma)全长cDNA克隆到杆状病毒转移载体pAcYM1和pBm050上。将这些载体分别与加利福尼亚自签名核多角体病毒（AcNPV）或家蚕核多角体病毒（BmNPV） DNA共转染到狐尾蛾细胞（SF21AE）和家蚕细胞（BmN）中。重组病毒，命名为acbifn - γ和bmbifn - γ，然后恢复。重组bifn - γ (rbifn - γ)在acbifn - γ感染的ni毛癣细胞和bmbifn - γ感染的家蚕幼虫培养液中积累。经SDS-PAGE和tunicamycin处理证实，这些rbifn - γ形式被证明是糖基化的20和22 kDa蛋白。这些产物对半胱氨酸蛋白酶敏感。通过水疱性口炎病毒的斑块减少试验和对牛巨噬细胞的MHC II类抗原诱导，两种rbifn - γ蛋白均显示出高水平的生物活性。
The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.