【摘要】 以嗜热子囊菌光孢变种(Thermoascusaurantiacusvar.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1710bp,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(Pichiapastoris)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144h诱导后,外切纤维二糖水解酶表达量为1.17mg/mL,产酶活力为20.3U/mL。更多还原

【Abstract】 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 confirmed 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.更多还原