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 pPICZaC to express canine interferon alpha matureprotein. CalFN-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.%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)一水疱性口炎病毒(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.
展开▼
