摘要
用逆转录－聚合酶链反应（RT-PCR），以白假丝酵母(Candida albicans )总RNA为模板，扩增了几丁质酶（chitinase）的cDNA，重组到T7启动子控制下的表达载体pET23b及巴斯德毕赤酵母AOX1基因启动子控制下的表达载体pPIC3.5K，得到重组质粒pET23b-Chi及pPIC3.5K-Chi，并分别转化至大肠杆菌及巴斯德毕赤酵母。
SDS-PAGE分析表明，大肠杆菌表达菌株经1m mol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导4小时后，可表达一分子量为50KD的蛋白质。表达量约为20mg/L。对羟基苯甲酸酰肼（PAHBAH）法测定表明表达产物具有酶活性。
毕赤酵母经甲醇培养基诱导表达，检测破菌后上清液中的酶活性，并经SDS-PAGE检测，未发现外源蛋白的表达。
The cDNA encoding Candida albicans chitinase was amplified from Candida albicans total RNA by RT-PCR. The chitinase cDNA was inserted into expression vector pET23b and pPIC3.5K. The recombinant plasmids pET23b-Chi and pPIC3.5K-Chi were transformed into Escherichis coli and Pichia pastoris.
The recombinant pET23b-Chi was induced with IPTG to express chitinase in E.coli BL21(DE3) strain. SDS-PAGE gel analysis showed that the molecular mass was estimated to be 50KD. The activity of chitinase was detected by method of PAHBAH.
The recombinat strain of P.pastoris was cultured in the inducing medium. The expression of chitinase was not detected by activity assay and SDS-PAGE.
引文
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