绿木霉ZY-01的外切葡聚糖酶CBHⅠ基因的克隆及在E.coli中的表达

摘要

以前期分离筛选得到的高产纤维素酶菌株Trichoderma virens ZY-01的总RNA为模板合成cDNA,经PCR克隆得到外切葡聚糖酶(cellobiohydrolase,CBH)Ⅰ基因;并构建了pET-32a-cbh1表达载体,通过转化至E.coli BL21(DE3)中,获得CBH Ⅰ重组表达系统,成功表达出可溶性CBH Ⅰ蛋白.结果表明,Trichoderma virens ZY-01 CBH Ⅰ碱基序列与Trichoderma viride AS 3.3711 CBHⅠ基因(GenBank:AY368686.1)序列相似度高达91.95%,氨基酸同源性高达99.22%;通过SDS-PAGE检测,表明该基因在E.coli中得到可溶性表达,分子量约为53 kDa,与基因翻译出的氨基酸序列相一致.该研究为CBH Ⅰ基因的工程化及应用提供了基础.%CBH Ⅰ gene was cloned by PCR with synthetic cDNA using the total RNA of Trichoderma virens ZY-01,which is a high-yield cellulase strain and screened in our previous work,as a template.The expression vector pET-32a-cbh1 was constructed and transformed into E.coli BL21(DE3) to obtain the CBH Ⅰ recombinant expression system.And the soluble CBH Ⅰ protein was successfully expressed.The results showed that,the base sequence similarity of CBH Ⅰ to Trichoderma viride AS 3.3711 CBH I gene (GenBank:AY368686.1) was as high as 91.95% and the amino acid homology was 99.22%.The result of SDS-PAGE indicated that the expressed protein was soluble in E.coli and had a molecular weight of about 53 kDa,which was consistent with the amino acid sequence translated from the gene.This work provides the basis for the industrial application of cellobiohydrolase CBH Ⅰ gene.