哈茨木霉HP37-4纤维二糖水解酶Ⅰ基因的克隆及表达  

Cloning and Expression of Gene Encoding CellobiohydrolaseⅠfrom Trichoderma harzianum Strain HP37-4

作　　者：吴柳[1] 张政[1] 卢业飞[1] 秦秀林[1] 刘君梁[1] 冯家勋[1] 

机构地区：[1]亚热带农业生物资源保护与利用国家重点实验室,广西大学生命科学与技术学院,广西南宁530005

出　　处：《西南农业学报》2013年第5期1847-1851,共5页Southwest China Journal of Agricultural Sciences

基　　金：广西科技攻关项目(桂科攻11107008-5)

摘　　要：从哈茨木霉(Trichoderma harzianum)HP37-4中克隆得到纤维二糖水解酶Ⅰ基因的全长DNA序列,并从HP37-4总RNA中通过RT-PCR扩增得到该基因全长cDNA序列。序列测定分析表明该基因含有3个外显子和2个内含子,编码含505个氨基酸的多肽。利用生物信息学方法对该蛋白质的结构进行了分析,推测了其主要功能区的位置及结构特征。将该基因cDNA序列克隆到大肠杆菌表达载体pET30a(+)中,在大肠杆菌BL21(DE3)中诱导表达该基因。SDS-PAGE分析表明该基因能在大肠杆菌中过量表达,但以不溶性的包涵体形式存在,经包涵体变性和多肽复性后,纯化得到了表达的重组蛋白。In this study,the full-length gene encoding cellobiohydrolase Ⅰ was cloned from Trichoderma harzianum HP37-4 and the fulllength cDNA sequence was cloned from the total RNA of the strain HP37-4 through RT-PCR.Sequence analysis showed that this gene contained three exons,two introns and encoded a polypeptide with 505 amino acids.This protein structure was modeled by bioinformatic analysis to predict the main functional domain positions and structural characteristics.The cDNA sequence of the gene was inserted into expression vector pET30a（＋） and expressed in Escherichia coli BL21 （DE3）.SDS-PAGE analyses showed that the recombinant protein was expressed as insoluble inclusion body.The proteins was purified after denaturation of the inclusion body and renaturation of the polypeptide.

关 键 词：哈茨木霉 纤维二糖水解酶 基因克隆 表达 

分 类 号：S216.4[农业科学—农业机械化工程]