猪IFN-α在巴斯德毕赤酵母中的分泌表达

The Secretive Expression of Porcine Alpha-Interferon Gene in Pichia pastoris

为了获得高效抗病毒活性的猪α-干扰素蛋白,采用PCR法扩增获得猪α-干扰素基因完整的开放阅读框(ORF),构建重组表达质粒pPICZαC-IFN,电击转化毕赤酵母感受态细胞X33.挑阳性菌落诱导表达,离心取上清,SDS-PAGE和Western-blotting分析,可见1条相对分子质量约19 400的清晰蛋白条带,与理论值相符,表明表达产物为猪α-干扰素.在Vero细胞上检测该干扰素抗VSV的活性为4.04×106IU/L.

In order to get alpha-interferon（INF-α） with high level secretive expression and high antiviral activities, the porcine alpha-interferon gene was amplified by PCR, and a DNA fragment of 501 bp, the open reading framework （ORF） was obtained. Then, the target gene and pPICZαC were digested with EcoRI/XbaI and were linked. The recombinant plasmid of pPICZctC-IFN was linearized by SacI and electroporated into Pichia pastoris X-33. PCR assay was used to identify colonies. The high copy recombinat strains were screened and induced by regulation of methanol utilization. IFN-α protein was detected by SDS-PAGE and Western-blotting analysis. The result showed the IFN-α protein with a raletive molecular mass of 19 400 was expressed in Pichia pastoris X-33. The antiviral activity of IFN-α against vesicular stomatitis virus（VSV） was investigated on the Veto cell and the result indicated that IFN-α could inhibit VSV and the activity was 4. 04 × 10^6 IU/L.