犬IFN-α优化基因的真核表达及抗病毒活性的研究

本研究根据酵母系统密码子的偏嗜性,对成熟蛋白基因核苷酸序列进行密码子偏嗜性改造,利用毕赤酵母分泌型表达载体pPICZαC来表达犬IFN-α成熟蛋白基因。并采用非洲绿猴肾细胞(Vero) -水疱性口炎病毒(VSV)系统以细胞病变抑制法为基础来测定重组犬α干扰素的效价,同样方法测定了表达产物对PRV和CDV的抑制作用。本试验实现了犬α干扰素在酵母中的高表达,表达量高达58 mg/L,SDS-PAGE检测大小约为24 ku,比预测分子质量大,推测发生了糖基化。表达产物具有较强的种属特异性,在Vero细胞上对PRV具有较高的抗病毒活性,达到了3.6×10⁷ U/L,对CDV也有很好的抑制作用。本研究为犬IFN-α以后的临床应用研究奠定基础。

The Research of Expression of Optimized Gene of Canine Alpha-Interferon in Pichia pastoris and its Antiviral Activity

According to the yeast system codon partial tropism,modifying the nucleotide sequence of the mature protein co-don,this research used the Pichia pastorios secretion expression vectors pPICZαC to express canine interferon alpha maturedon,this research used the Pichia pastorios secretion expression vectors pPICZαC to express canine interferon alpha mature protein.CaIFN-α was measured by cytopathic effects with Vero-VSV system,the same methods to measure inhibitory effect on PRV and CDV.The results showed that the canine IFN-α got a high expression in yeast.SDS-PAGE showed the protein got 24 ku,larger than the expected size.The CaIFN-α had species specificity,it had a high antiviral activity to PRV on vero cell,reached 3.6 × 107 U/L,and also had a good inhibition to CDV.Its expression got 58 mg/L.The study provided the base for further study of CaIFN-α activity and its clinical application.