中性纤维素酶外切葡聚糖酶CBHⅡ的异源表达

摘要

Using polymerase chain reaction ( PCR) the whole sequence of cellobiohydrolase H gene, CBH H , was cloned from Humicola insolens strain EIM-50 preserved in the laboratory. The CBH II gene was about 1 586 bp. It was cloned into the expression vector of pPIC9K, and the recombinant plasmid pPIC9K-CBH II was thus constructed successfully. The expression vector was transformed to Pichia pastoris by electro-transformation to carry out heterogenic expression. The result of SDS-PAGE silver staining showed that the recombinant plasmid gained heterogenic expression. Analysis with gel pro Analyzer software showed the apparent molecular weight of expressed protein was 62. 548 ku. And analysis with BandScan software the protein expressed amount was 2.3% , I. E. 0.738 (μg/mL (mg/L).%利用聚合酶链式反应(PCR)技术,从本实验室保存的1株特异腐质霉EIM-50上克隆到中性纤维素酶外切葡聚糖酶基因( CBHⅡ)全长序列,大小约为1 586 bp.将其克隆到pPIC9K上,成功构建重组质粒pPIC9K-CBHⅡ,并经电转化引入毕赤酵母(Pichia pastoris)GS115,进行异源表达.SDS-PAGE银染结果表明,该重组质粒在毕赤酵母中获得了异源表达.gel pro Analyser软件分析其表达蛋白的表观分子量约为62.548kuo用BandScan软件分析其蛋白表达量为2.3％,即0.738 μg/mL(mg/L).