哈茨木霉Chiv基因的原核表达及优化.pdfVIP

Vol40 No.8 第40 卷第8 期 哈尔滨工业大学学报 2008 年8 月 JOURNAL OF HARBIN INSTITUTE OF TECHNOLOGY Aug. 2008 晗茨木霉 Chiv 基因的原核表达及优化 刘圣钢，杨谦，王奇慧 (哈尔滨工业大学生命科学与工程系，哈尔滨 15α阳， E-mail: lpgkevin@ ) 摘 要:通过筛选哈茨木霉的 DNA 文库，克隆到 Chiv 基因的 cDNA 全长序列.以连接有哈茨木霉 Chiv 基因 的质粒 pBK902 为模板，设计含有 EcoRI 和 XhoI 酶切位点引物，采用 PCR 方法扩增得到具有完整开放阅读 框的目的基因;将目的基因连接到表达载体 pET28 上并转化大肠杆菌 BUl 菌株中，成功地获得了大量的阳 性转化子.质粒通过 EcoRI 和 XhoI 酶切鉴定及测序鉴定，证明 Chiv 基因成功地转化到大肠杆菌中.重组大肠 杆菌经 I町、G 诱导表达及 SDS检测后，发现特异的蛋白表达条带，其分子量为 50 kDa 左右.通过对诱 导剂量与诱导时间等因素的优化，结果显示凡丁质酶的最佳诱导时间为 13 h ，最佳诱导剂浓度为 1 mM，为几 丁质酶的工业化生产奠定基础. 关键词:哈茨木霉;几丁质酶 v 基因;原核表达;优化 中图分类号: Q938.1 文献标识码:A 文章编号: 0367 -6234(2∞8)08 -1252-04 Prokaryotic expressing and optimization of gene from Trichoderma harzianum UU Pi-gang ,YANG Qian ,WANG Qi-hui (Dept. of Life Science and Engineering , Harbin Institute of Technology , Harbin 15ω01 ， China , E-mail: lpgkevin@ 回hu. com) Abstracl: A full -length cDNA of class v chitinase ( Chiv ) w臼 cloned by screening the cDNA lihrary con- structed from Trichoderma harzianum mycelium. The final DNA sequence was amplified hy PCR using pBK902 as the template hy designed primers according to the sequence of Chiv . 白le final gene was linked to the expressing vector pEτ28 and they were transformed into E. coli BUl cells , then several positive clones were obtained. 币le inserts of transformants were successfully confinned by electrophoresis after digested by EcoRI and XhoI and then were sequenced. The transformants were înduced by IPTG and the recombinant fu- sion protein with MW 50 kDa wωdetected hy SDS. 四e chitinase reache