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2025年8月18日 星期一
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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
陈静;李多川;张玉芹;杜欣可
山东农业大学植物保护学院环境生物系, 泰安 271018
cDNA Cloning of the Cellobiohydrolase Gene cbh1 from hermoascus aurantiacus var. levisporus and Its Expression in Pichia pastoris
 
CHEN Jing;LI Duo-chuan;ZHANG Yu-qin;DU Xin-ke
Department of Environmental Biology, College of Plant Protection,Shandong Agricultural University, Tai’an 271018, China
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摘要 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。
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陈静
李多川
张玉芹
杜欣可
关键词 嗜热子囊菌光孢变种 外切纤维二糖水解酶 cDNA克隆 RACE 毕赤酵母    
AbstractThe 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.
Key wordsThermoascus aurantiacus var. levisporus    cellobiohydrolase    cDNA cloning    RACE    Pichia pastoris
收稿日期: 2005-05-30     
通讯作者: 李多川   
引用本文:   
陈静;李多川;张玉芹;杜欣可. 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[J]. , 2006, 14(3): 406-411.
CHEN Jing;LI Duo-chuan;ZHANG Yu-qin;DU Xin-ke. cDNA Cloning of the Cellobiohydrolase Gene cbh1 from hermoascus aurantiacus var. levisporus and Its Expression in Pichia pastoris
 
. , 2006, 14(3): 406-411.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/      或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2006/V14/I3/406
 
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