突变型干扰素-τ的表达、纯化及其抗乙型肝炎病毒作用

Expression,Purification and Anti-HBV Effect of Mutant Interferon-τ

目的在毕赤酵母中表达突变型干扰素-τ(IFN-τ),并检测其生物学活性及抗乙型肝炎病毒作用。方法将绵羊IFN-τ序列中6个氨基酸突变为人IFN-α相应的氨基酸,将合成的基因克隆至pPICZα质粒,构建突变型IFN-τ表达质粒pPICZα-IFN-τ,转化巴斯德毕赤酵母中进行高密度发酵。发酵上清液经超滤浓缩、离子交换层析和分子筛层析纯化后,检测其生物学活性及抗乙型肝炎病毒作用,并与市售IFN-α2a进行比较。结果发酵上清液中突变型IFN-τ的表达量为400～500mg/L,占上清总蛋白的40%以上;纯化收率达31.4%,纯度约为95%;比活为2.1×107IU/mg;对乙型肝炎病毒的抑制作用与IFN-α2a相当,但细胞毒性显著低于后者。结论已在毕赤酵母中高效表达了突变型IFN-τ,表达的蛋白具有较高的生物学活性,对乙型肝炎病毒有良好的抑制作用,且细胞毒性较低。

Objective To express mutant interferon-τ （IFN-τ） in Pichia pastoris, purify the expressed product and study its anti-HBV effect. Methods Substitute the 6 amino acids in sheep IFN-τ sequence to the responding amino acids in human IFN-α, and clone the synthetic gene to plasmid pPICZα. Transform the constructed recombinant plasmid pPICZα-IFN-τ for expression of mutant IFN-τ to Piehia pastoris, for high density fermentation. The fermentation supernatant was concentrated by ultrafiltration, purified by ion exchange and molecular chromatography and determined for biological activity, Evaluate the anti-HBV effect of expressed protein and compare with that of commercial IFN-α2a. Results The expression level of IFN-τ in fermentation supernatant was 400 - 500 mg/ L. The expressed product contained more than 40% of total protein in supernatant and reached a recovery rate of 31.4%, a purity of about 95% and a specific activity of 2.1×10^7IU/mg. The anti-HBV effect of the expressed IFN-τ was equal to, while the cytotoxicity was lower than that of commercial IFN-α2a. Conclusion Mutant IFN-τ with high biological activity was successfully expressed in Pichia pastoris,, which showed significantly inhibitory effect on HBV and low cytotoxicity.