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将克隆的鸡γ-干扰素(Ch IFNγ)基因亚克隆至酵母表达载体p PICZα-A中,构建重组质粒p PIC-Ch IFNγ。将鉴定正确的质粒p PIC-Ch IFNγ线性化,通过电转化整合到毕赤酵母菌株X-33基因组中,得到阳性菌株。经1%甲醇连续诱导4 d,表达产物经SDS-PAGE检测,结果表明Ch IFNγ在酵母中获得了分泌型表达,表达产物分子量约为20 ku。表达产物粗提后以细胞病变抑制法检测其活性,效价为107.69U/m L;在鸡胚中能有效抑制H9N2亚型禽流感病毒的增殖;动物攻毒保护试验结果显示,表达的蛋白具有较好的抗病毒活性。
The cloned Chicken Interferon-γ (Ch IFNγ) gene was subcloned into the yeast expression vector p PICZα-A to construct the recombinant plasmid p PIC-Ch IFNγ. The correct plasmid p PIC-Ch IFNγ was linearized and transformed into the Pichia pastoris strain X-33 genome by electrotransformation to give a positive strain. After continuous induction with 1% methanol for 4 days, the expressed product was detected by SDS-PAGE. The results showed that Ch IFNγ was secreted in yeast and its molecular weight was about 20 ku. After the crude product was extracted, its activity was detected by cytopathic effect inhibition assay with the titer of 107.69U / m L. The chicken embryo could effectively inhibit the proliferation of H9N2 subtype avian influenza virus. The results of animal challenge protection showed that the expressed protein Has a good antiviral activity.