鸡γ-干扰素在毕赤酵母中的分泌表达及其抗病毒活性研究

Expression of Chicken Interferon-γ in Pichia pastoris and Its Antiviral Activity

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 gene of chicken interferon gamma （ChIFNγ）was amplified by reverse transcription-polymerase chain reaction （RT-PCR）from SPF chicken blood genome and cloned. The coding region （495 bp）was subcloned into the expression vector pPICZα-A. The recombinant plasmid pPIC-ChIFNγ was identified by enzyme digestion. After linearized by Sac I ,the plasmid was transferred into Pichia pastoris strain X-33. The recombinant strain was isolated and identified by PCR. The recombinant strain was induced by 1% methanol for 4 days, the production of recombinant ChIFNγ was detected by SDS-PAGE. The assay results showed that the protein weight was approximate 20ku,and the recombinant protein was secreted effectively in Pichia pastoris. Antiviral activity of the recombinant ChIFNγ was detected by the classical experiment of cell pathological effect inhibition assay. The results showed the titer was about 10^7.69 U/mL; The expressed protein could also inhibit subtype H9N2 avian influenza virus replication in chick embryo and SPF chick; Animal experiment indicated that recombinant protein had good antivirus activity.