嗜热子囊菌光孢变种cbh1基因的cDNA克隆及在毕赤酵母的高效表达

以嗜热子囊菌光孢变种(Thermoascus aurantiacus var. levisporus)总RNA为模板，通过RT-PCR克隆出外切纤维二糖水解酶基因cbh1片段，采用RACE方法获得全长cDNA克隆，其全长为1 710 bp，编码一种由457个氨基酸组成的单肽，推导的氨基酸序列中1～19位为信号肽序列，GenBank的登录号为AY840982。将该片段克隆到毕赤酵母(Pichia pastoris)分泌型表达载体pPIC9K上，获得表达重组质粒pPIC9K/cbh1，转化毕赤酵母GS115，所得重组子经PCR验证后进行诱导表达，筛选出一重组子GSp-15,经144 h诱导后，外切纤维二糖水解酶表达量为1.17 mg/mL，产酶活力为20.3 U/mL。

cDNA Cloning of the Cellobiohydrolase Gene cbh1 from Thermoascus aurantiacus var. levisporus and Its Expression in Pichia pastoris

The cellobiohydrolase gene cbh1 fragment (GenBank Accession No. AY840982) was amplified by RT-PCR from thermophilic fungus Thermoascus aurantiacus var. levisporus. RACE was used to obtain its full-length cDNA (1710 bp), encoding 457 amino acids. The first 19 amino acids of the deduced amino acid sequence were presumed to be a signal peptide. The fragment encoding mature cellobiohydrolase was inserted into Pichia pastoris vector pPIC9K to construct recombinant plasmid pPIC9K/cbh1. The pPIC9K/cbh1 was then introduced into Pichia pastoris GS115 and 61 transformants were obtained, After comfirmed by G418 risistance and PCR, and induced expression, one clone GSp-15 was selected from the 61 transformants. The expression level of GSp-15 was 1.17 mg/mL after induction for 144 h in methanol, and its activity was 20.3 U/mL with p-NPC as substrate.