鹅干扰素-α的毕赤酵母表达及其生物学活性分析

Expression of goose interferon-alpha in Pichia pastoris and analysis of its antiviral activity

为在毕赤酵母表达系统中表达无冗余氨基酸且具有抗病毒活性的鹅干扰素-α(IFN-α)蛋白,采用融合PCR法扩增获得鹅干扰素-α基因片段,将此片段连入pPICZαA载体,构建重组表达质粒pPICZαA-GoIFN-α,电击转化毕赤酵母感受态细胞X33,挑取阳性重组菌用甲醇进行诱导表达,对表达产物进行West-ern-blot和ELISA分析,并测定重组IFN-α的生物活性。结果显示,融合基因获得高效表达,表达的蛋白以可溶形式存在,且相对分子质量较理论值稍大。经GEF-GPMV系统检测,证实表达的蛋白具有生物学活性,约为6.55×105 U/mL,比活力为1.80×105 U/mg。结果表明,利用毕赤酵母表达系统规模化生产具有生物学活性的鹅IFN-α有广阔的应用前景。

In order to get alpha-interferon（IFN-a） with high level secretive expression and antiviral activities,the goose IFN-a gene and the a-factor signal sequence of pPICZaA were amplified by fusion PCR. The target gene and pPICZaA were digested with HindBI -kKpn I and then were ligated. The recombinant plasmid of pPICZaA- GoIFN-a was linearized by Sac I and electrotratnsformation into Pichia pastoris X- 33. PCR assay was used to identify colonies. The positive recombinants were screened and induced by addi- tion of methanol. GoIFN-a protein was detected by SDS-PAGE and Western-blotting analyses. The result showed the IFN-a protein with a molecular mass of 25--35 ku was expressed in Pichia pastoris X-33. The antiviral activity of IFN-a against GPMV was investigated on the GEF cell and the result indicated that IFN-a could inhibit GPMV.