[目的] 为了进一步提高毕赤酵母表达生产人胰岛素前体的产量,[方法] 将构建的表达载体pPICZα-IP 电转至毕赤酵母X-33中,在100 μg/mL zeocin的YPDS抗性平板筛选获得过量表达人源胰岛素前体(IP)的重组菌株B4和S6.在此基础上,以过量表达胰岛素前体的B4和S6为出发菌株,以SacI线性化的表达载体pPICZα-IP进行重复电转化,并在1000 μg/mL zeocin的YPDS平板上筛选获得一株拷贝数为7的重组毕赤酵母2B4.[结果] 该菌株在5L规模发酵罐上,人源胰岛素前体产量是低拷贝数菌株B7的2.7倍,且菌体并未因拷贝数高而表现出生长不良的现象.实时定量PCR检测后,发现2B4菌株胰岛素前体基因的转录水平是B7的2倍.[结论] 综上结果表明,采用抗性筛选和重复电转相结合的高拷贝重组毕赤酵母构建策略,能有效促进目的基因的转录水平,最终显著提高目标蛋白的产量.%[Objective] The aim of this study was to further increase the yield of insulin precursor by Pichia pastoris.[Methods] For this,we transformed the expression vector pPICZα-IP into P.pastoris X-33 using electroporation and screened two mutant strains B4 and S6 on the YPD plate containing 100 μg/mL zeocin.Both could overexpress human insulin precursor.Taking B4 and S6 as start strains,we repeatedly transformed SacI linearizing pPICZα-IP into P.pastoris X-33 by electroporation,then screened a new mutant strain 2B4 (with 7 copies) on the 1000 μg/mL zeoein YPD plate.[Results] After cultivation,the human insulin precursor yield of 2B4 strain was 2.7 fold higher than that of B7.Meanwhile,the cell growth was not inhibited.The target gene transcription level of 2B4 was 2 fold higher than that of B7 by real-time quantification PCR.[Conclusion] The strategy of combining resistance screening and repeated electroporation was efficient to increase the copy number of target gene,so as to facilitate higher transcription level and enhance objective recombinant protein yield.
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