鸡IFN-α基因的多点突变及其在毕赤酵母中的高效表达

Multipoint Mutagenesis of Chicken IFN-α Gene and its Efficient Expression in Pichia pastoris

根据毕赤酵母基因的密码子选择偏爱性,在不改变其编码的氨基酸序列的前提下,对鸡IFN-α序列进行PCR介导的定点突变优化,将鸡IFN-α成熟肽序列中136～143位相近的4个Arg密码子和N端的6个稀有密码子突变为毕赤酵母高表达优越密码子,构建了含正确突变和未突变序列的克隆载体pMD-IFNm、pMD-IFN和酵母表达载体pPICZ-IFNm、pPICZ-IFN,电击转化毕赤酵母X-33菌株,经筛选、鉴定表明鸡IFN-α基因已整合到酵母基因组中。通过表达产物的抗病毒活性测定,筛选出突变与未突变高表达酵母转化子各1株。经密码子优化的突变重组酵母菌株PP-IFNm于BMMY培养基中诱导72h上清中抗病毒活性单位可达410U/mL,其活性比未优化重组酵母株PP-IFN(48.33U/mL)提高了约10倍。

According to bias on codon usage of Pichia pastoris,tbe chicken IFN-α gene was multipoint-mutated to optimized codes without changing its amino acid sequence. The pMD-IFN^m plasmid with mutated ChIFN-α gene was confirmed by sequencing. The expression plasmid pPICZ-IFN^m was constructed and transformed into X-33 strain by electroporation. Positive transformants of the chromosomes integrated with ChIFN-α gene were identified by screening and analysis. The antiviral activity of expression product was tested,strain PP-IFN^m with codons optimized reached 4^10 U/mL in BMMY medium after being induced for 72h,which was about 10 times of the original strain PP-IFN.