猪干扰素α1与胸腺肽α1融合蛋白原核表达系统的建立

Establishment of Prokaryotic Expression System of Porcine IFNα1-THYα1 Fusion Protein

依据GenBank已发表的猪干扰素α1（poIFN-α1）成熟肽基因序列,胸腺肽α1（THY-α1）蛋白序列及Escherichia coliB密码子使用频率表,优化并人工合成poIFN-α1和THY-α1基因,利用SOE-PCR法在poIFN-α1和THY-α1之间设计1个13肽Linker。将融合基因克隆至pRSFDue-t 1表达载体,转化BL21（DE3）感受态,37℃培养至菌液OD600为0.6~0.8,IPTG（0.5 mmol/L）诱导,20℃培养12h,SDS-PAGE和Western blot鉴定。结果表明：目的蛋白为菌体内可溶性表达,经强阴离子交换层析和疏水层析纯化获得高纯度poIFNα1-THYα1融合蛋白。

The combined application of interferon and thymosin is significant in antiviral effect.According to the genic sequences of porcine interferon-α1 in GenBank,the protein sequences of thymosin-α1 and the codon bias in Escherichia coli B,the poIFN-α1 and THY-α1 genes were optimized and synthesized.A linker containing 13 amino acids was designed between poIFN-α1 and THY-α1 by SOE-PCR.The poIFNα1-THYα1 fusion gene was cloned into pRSFDuet-1 expression plasmid and transformed into BL21（DE3）,and then cultured at 37 ℃ and induced with IPTG（0.5 mmol/L） for 12 hours while its OD600 got up to 0.6—0.8,SDS-PAGE and Western blot results indicated the soluble poIFNα1-THYα1 fusion protein was successfully expressed.The high-purity poIFNα1-THYα1 fusion protein was obtained by ion exchange chromatography and hydrophobic chromatography,which made a important sense of prevention and treatment of porcine viral diseases.