中文摘要：

以康宁木霉AS3．2774mRNA为模板，通过RT—PCR技术，扩增了1413bp的CBHⅡ全长基因，利用T—A克隆，将CBHⅡ克隆至克隆载体pMD18-TSimpleVector，构建质粒pMD18-T—CBHⅡ。以Sinai和SphI双酶切pMD18-T—CBHⅡ和以胸苷酸合成酶基因fthymidylatesynthase，thyAl为选择压力的非抗生素抗性的穿梭表达栽体pW425t，将纯化的CBHⅡ亚克隆到pW425t中，得到原核表达重组质粒pW425t—CBHⅡ，可在乳酸菌与大肠杆菌之间穿梭表达。将pW425t—CBHⅡ转化在thyA基因缺陷型的大肠杆菌感受态E．coliX13中，通过质粒提取、PCR鉴定、酶切鉴定、分析测序以及生长功能弥补筛选阳性克隆。SDS—PAGE分析，约49．6kD的蛋白可见，刚果红染色显示重组大肠杆菌可产生明显的水解圈，并用pNPC法测得重组大肠杆菌的CBHⅡ酶活力在温度50cc、pH值5．0时达到最大。

英文摘要：

The full CBH Ⅱ gene of Triehoderma koningii AS3.2774 was amplified by the reverse tran- seription-polymerase chain reaetion（RT-PCR）, the product was a 1 413 bp eDNA fragment. Using T-A eloing technique, the PCR product was cloned into pMD18-T Simple Vector. Reeombinant plasmid pMD18-T-CBH Ⅱ and pW425t, the prokaryotie expression shuttle vector between E. eoli and Laetoba- eillus, was digested with SmaI and SphI enzymes respectively. The purified CBH Ⅱ gene was subeloned into the expression vector pW425t. Thus, the recombinant pW425t-CBH Ⅱ was eonstrueted, then was transformed into the competence thyA gene-mutant E.eoli X13.SDS-PAGE analysis, about 49.6 kD pro- tein had been produced. CBH H transformants can produce clear hydrolysis halos on the Congo-Red- CMC plate, and optimum enzyme activity was achieved at 50 ℃ and pH 5.0 by the pNPC method.