哈茨木霉Chiv基因的原核表达及优化

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BL21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IPTG诱导表达及SDS-PAGE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,结果显示几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.

Prokaryotic expressing and optimization of gene from Trichoderma harzianum

A full-length cDNA of class v chitinase(Chiv) was cloned by screening the cDNA library constructed from Trichoderma harzianum mycelium.The final DNA sequence was amplified by PCR using pBK902 as the template by designed primers according to the sequence of Chiv.The final gene was linked to the expressing vector pET28 and they were transformed into E.coli BL21 cells,then several positive clones were obtained.The inserts of transformants were successfully confirmed by electrophoresis after digested by EcoRI and XhoI and then were sequenced.The transformants were induced by IPTG and the recombinant fusion protein with MW 50 kDa was detected by SDS-PAGE.The chitinase reached the peak expression after the transformants were cultured for 13 h.The optimal density of IPTG was 1 mM.The study provides the basic methodology for further industrial production of chitinase.