藏獒IFN-γ基因在毕赤酵母中的表达与抗病毒活性分析

Expression and antiviral activity of Tibetan mastiff interferon-gamma gene in Pichia pastoris

采用毕赤酵母表达系统表达藏獒γ-干扰素(Tibetan mastiff interferon gamma,TmIFN-γ)并分析其生物活性,为抗病毒生物制品的研发与临床应用奠定基础。将构建的TmIFN-γ的真核表达质粒pPIC9k-TmIFN-γ转化至毕赤酵母GS115细胞,用不同浓度的G418筛选得到多拷贝重组菌株;利用甲醇进行诱导表达,以镍层析柱纯化目的蛋白,对表达产物进行SDS-PAGE和Western blotting鉴定;通过CCK-8试验检测重组TmIFN-γ蛋白的细胞毒性作用,利用VSV-MDBK系统检测其抗病毒生物活性。结果显示,成功筛选得到高拷贝pPIC9k-TmIFN-γ重组转化毕赤酵母菌株,利用甲醇诱导可获得TmIFN-γ重组蛋白,该蛋白对细胞无毒性,具有显著抗病毒活性,其抗病毒生物效价为1.727×10~5IU/mL。本研究获得了具有抗病毒活性的TmIFN-γ,有助于进一步探索TmIFN-γ的功能和开发新型基因工程抗病毒药物。

Tibetan mastiff interferon gamma(TmIFN-γ)with higher biological activity was preparaed by P.pastoris expression system,so as to provide basis for development and application of antival products.The pPIC9k-rTmIFN-γeukaryotic expression plasmid was constructed with a Kozak sequence and His tag.This plasmid was then transformed into P.pastoris GS115 cells through electroporation,and multi-copy recombinant strains were screened using the G418 resistance gradient method.The identification of the recombinant GS115/pPIC9k-TmIFN-γwas confirmed with RCR by vector and TmIFN-γgene primers.The recombinant transformant was induced with methanol.The recombinant TmIFN-γprotein was purified using affinity chromatography and verified through SDS-PAGE and Western blotting.Subsequently,the cytotoxicity of TmIFN-γwas assessed by CCK-8 test,and its bioactivity was evaluated in the VSV/MDBK test system.The multi-copy recombinant transformants was obtained,the protein was expressed by the induction of methanol.The expressed TmIFN-γprotein had no cytotoxic effects and the bioactivity of TmIFN-γwas 1.727×105 IU/mL in the VSV/MDBK system.TmIFN-γprotein with antiviral activity is obtained by P.pastoris expression system,which can provide a basis for the development of novel genetically engineered antiviral drugs.