重组犬α干扰素的制备及生物学活性分析

Preparation of recombinant canine interferon α and a comparative study of its biological activity

目的用毕赤酵母表达犬α干扰素并与诱生的犬天然白细胞干扰素作生物学活性比较。方法优化合成犬α干扰素基因,构建质粒pPIC9K-CaIFN-α,转入酵母SMD1168,筛选阳性克隆后诱导表达干扰素并纯化;并用犬白细胞诱生犬天然白细胞干扰素。用WISH-VSV方法检测两种干扰素的效价。结果表达的重组犬α干扰素效价约为0.65×105IU/ml,与犬白细胞干扰素效价(0.81×105 IU/ml)相近。结论成功利用毕赤酵母表达了与犬天然白细胞干扰素活性相近的重组犬α干扰素,为干扰素的工业化制备提供了种质资源。

Objective To develop recombinant canine alpha-interferon with Pichia pastoris. Methods A canine alpha interferon gene was optimized and synthesized. The plasmid pPICgK-CaIFN-α was constructed, and then the plasmid pPIC9K-CalFN-α was transformed into P. pastoris SMDl168. Positive clones were selected, interferon protein was in duced and purified, and canine spontaneous leukocyte interferon was produced by stimulation with the Newcastle disease virus （NDV）. The titer of two forms of interferon was determined with a WISH-VSV system. Results The antiviral activity of recombinant canine alpha-interferon （0.65 ×10^5IU/ml） was similar to that of canine spontaneous leukocyte in- terferon （0.81 ×10^5IU/ml）. Conclusion Recombinant canine alpha-interferon, whose antiviral activity resembles that of canine spontaneous leukocyte interferon, was successfully expressed by P. pastoris. This study lays the foundation for the industrial production of interferon.