摘要
为了研究全新几丁质酶基因Chit37(DQ647957)的特性与功能,将该基因在大肠杆菌中进行了外源表达.通过构建哈茨木霉(Trichoderma harzianum)菌丝生长期的cDNA文库及对部分表达序列标签序列的测定与生物信息学分析,成功获得了基因Chit37的全长cDNA序列.该基因的编码框长度为1032bp,编码343个氨基酸,理论分子量为36.6kDa.采用PCR扩增的方法克隆该基因并将其构建到原核表达载体pET28(a+)上,构建了原核表达载体pET-Chit37并转化宿主菌BL21(DE3),菌落PCR及酶切鉴定均证实转化子的正确性,重组蛋白经IPTG诱导后获得表达.优化的表达条件为终浓度0.1mM IPTG诱导培养4h.
To study the character and function of a novel chitinase gene Chit 37 （ DQ647957 ）, its prokaryotic expression was carried out. The full-length eDNA of gene Chit 37 was obtained successfully by constructing the T. harzianum myeelium eDNA library, determining the expressed sequence tags and conducting the bioinfor- matic analysis of the random selection clones. The results indicated that the ORF of Chit 37 was 1 032 bp encoding a protein of 343 amino acids and the deduced molecular weight was 36.6 kDa. The gene was cloned by PCR and was inserted into the expression vector pET28 （a^＋ ） , and thereby transformed to the E. coli BL21 （DE3）. The transformants were verified by clone PCR and enzymes digest, and the fusion protein was expressed by IPTG induction. The optimal expression condition was induced for 4 h by 0. 1 mM IPTG （final concentration）.
出处
《哈尔滨工业大学学报》
EI
CAS
CSCD
北大核心
2008年第10期1609-1612,共4页
Journal of Harbin Institute of Technology
基金
国家高技术计划基金资助项目(2003AA241140)
关键词
哈茨木霉
几丁质酶
表达
Trichoderma harzianum
chitinase
expression
