哈茨木霉几丁质酶基因的cDNA克隆及表达

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.

Cloning and expression of the cDNA of chitinase gene from Trichoderma harzianum

To study the character and function of a novel chitinase gene Chit37 (DQ647957), its prokaryotic expression was carried out. The full-length cDNA of gene Chit37 was obtained successfully by constructing the T. harzianum mycelium cDNA library, determining the expressed sequence tags and conducting the bioinformatic analysis of the random selection clones. The results indicated that the ORF of Chit37 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 0.1mM IPTG (final concentration).