功能性猪α干扰素基因的表达及活性检测

目的]检测克隆猪α干扰素基因（IFN-α基因）在毕赤酵母中的表达。方法]经植物血凝素（PHA）诱导后,提取猪外周血淋巴细胞,并用RT-PCR方法获得IFN-α基因,将该基因与真核表达质粒pPIC9K连接,并电转入毕赤酵母GS115菌中,经甲醇诱导表达后,SDS-PAGE电泳检测IFN-α蛋白表达,Western-blot分析IFN-α蛋白活性。结果]重组转化菌株经甲醇诱导后能表达相对分子质量约为19kD的蛋白质,该蛋白质能与相应抗体产生免疫反应。结论]实现了猪功能性IFN-α基因表在毕赤酵母中表达,为进一步探讨IFN-α的生物学活性研究奠定了基础。

Expression and Activity Deter mination of Functional Porcine Interferon-α Gene

Objective]To clone Porcine Interferon-α(IFN-α)gene and determine its activity in yeast.Methods]Interferon were induced and produced using phytohemagglutinin(PHA)in porcine peripheral blood lymphocytes.IFN-α gene was amplified by RT-PCR,and cloned into pPIC9K,the recombinant expression vetors were transformed into host strain Pichia pastoris GS115 by eletrization.After induced with methanol,The expression of IFN-α gene.was detected by SDS-PAGE and Western-blot.Result]The porcine IFN-α gene were produced by RT-PCR and expressed in Pichia pastoris GS115 induced by methanol and had antigen specificity with IFN-α antibody.The molecular weight of this protein was 19 kD.Conclusion]The functional porcine IFN-α gene were expressed in Pichia pastrois GS115.This study has paved the way for further exploring the IFN-α biological activity.