含正确木聚糖酶基因转化子的活性筛选  

An Activity-Screening Method for Transformants Containing Accurate Xylanase Gene

作　　者：刘亮伟[1] 李运峰[1] 郭晓丹[1] 

机构地区：[1]河南农业大学生命科学学院,郑州450002

出　　处：《中国农学通报》2010年第13期67-70,共4页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金项目"基于pH相关氨基酸定位分析的双功能木聚糖酶耐酸性突变研究"(30972123);河南省重点科技攻关项目"饲料用葡聚糖酶-木聚糖酶双功能酶的耐酸性分子改造"(092102110102)

摘　　要：为有效地筛选到带有正确基因的转化子,探讨了活性筛选方法。将木聚糖酶基因与pET20b重组,转化BL21(DE3)大肠杆菌,选取转化子,放入0.5mLLB培养基过夜培养,补加9.5mL培养基2.5h后诱导,收集并冻融裂解细胞,检测酶活性。通过对10个转化子的筛选,其中7个转化子酶活性接近或高于阳性对照菌落,认定为阳性转化子,经DNA测序证明基因序列正确。这个过程只要2～3天,具有快速简便特点。与以往筛选方法中针对转化子中的基因不同,作者直接检测转化子酶活性,筛掉不能表达活性酶的突变基因;其次,用冻融裂解细胞方法简化裂解过程,便于自动化高通量操作;最后,通过比较初筛酶活性,可以筛选高酶活转化子。In enzyme engineering, screening for transformant was labor-intensive and time-consuming. To select transformant containing accurate gene efficiently, we introduced an activity-screening method. Xylanase gene was cloned into pET20b and was used to transform E. coli strain BL21（DE3） competent cells. 10 transformants were inoculated in 0.5 mL LB for culturing overnight, 9.5 mL LB was re-added and cultured for 2.5 h. After induction for 5 h, cells were harvested and lyzed by freezing-thawing procedure in a simplified lysis buffer. For having activities closer to or higher than that of the positive control, 7 transformants were regarded as positive, thereafter, plasmid DNA was sequenced to confirm gene＇s accuracy. The procedure lasted for 2 to 3 days, having characteristic of high-throughput for assaying transformant＇ s enzyme activity in parallel. Differing from screening transformant for gene of previous method, the present method screened transformant for enzyme activity, which eliminated those nonsense mutations. The preliminarily assayed activities were compared for finding out transformant having high activity.

关 键 词：转化子 活性检测 木聚糖酶 

分 类 号：Q556[生物学—生物化学]