犬干扰素α1基因的修饰及在毕赤酵母中的表达

Modification and Expression of Canine Interferon α1 Gene in Pichia pastoris

目的修饰犬干扰素α1(Canine interferon alpha1,CaIFNα1)基因,提高其在毕赤酵母中的表达水平及抗病毒活性。方法根据巴斯德毕赤酵母表达系统对密码子的偏爱性,将CaIFNα1基因改造后,克隆至分泌表达载体pPICZαC中,电转化巴斯德毕赤酵母X33,甲醇诱导表达,表达产物经SDS-PAGE鉴定和氨基酸测序后,检测重组蛋白的表达量及抗水疱性口炎病毒(Vesicular stomatitis virus,VSV)的活性。结果重组表达质粒pPICZαC-CaIFNα1经双酶切及测序证明构建正确;表达的重组CaIFNα1蛋白相对分子质量约为24 000,蛋白氨基酸序列与天然的CaIFNα1氨基酸序列相同,3批重组酵母菌表达产物的蛋白含量分别为0.253、0.198和0.224 mg/ml,在Vero细胞上均无抗VSV活性,在MDCK细胞上的抗VSV活性分别为1.58×108、1.30×108和1.50×108 U/mg。结论 已成功改造了CaIFNα1基因,并在毕赤酵母中实现了高效表达,为大量发酵生产干扰素奠定了基础。

Objective To modify canine interferon α1（CaIFNα1） gene so as to increase its expression level in Pichia pastoris and its antiviral activity.Methods According to the codon bias of P.pastoris,CaIFNα1 gene was modified and synthesized and cloned into secretory expression vector pPICZαC.The constructed recombinant plasmid pPICZαC-CaIFNα1was transformed to P.pastoris X33 by electrotransformation for expression under induction of methanol.The expressed product was identified by SDS-PAGE and subjected to amino acids sequencing,then determined for expression level and activity against vesicular stomatitis virus（VSV）.Results Both restriction analysis and sequencing proved that recombinant plasmid pPICZαC-CaIFNα1 was constructed correctly.The amino acid sequence of expressed recombinant CaIFNα1,with a relative molecular mass of about 24 000,was identical to that of natural CaIFNα1.The protein contents of three batches of expressed products in P.pastoris were 0.253,0.198 and 0.224 mg / ml respectively.All the three batches of expressed protein showed no anti-VSV activities in Vero cells,of which the activities in MDCK cells were 1.58 × 108,1.30 × 108 and 1.50 × 108 U / mg respectively.Conclusion CaIFNα1 gene was successfully modified and highly expressed in P.pasoris,which laid a foundation of large-scale production of interferon by fermentation.