内切葡聚糖酶基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Endoglucanase Gene

作　　者：苗会[1] 陈光[1] 孙旸[1] 王刚[1] 张明磊[1] 

机构地区：[1]吉林农业大学生命科学学院,长春130118

出　　处：《吉林农业大学学报》2016年第5期538-542,共5页Journal of Jilin Agricultural University

基　　金：吉林省高等学校秸秆综合利用高端科技创新平台[吉高平合字(2014)C-1];吉林省教育厅项目(2016176)

摘　　要：纤维素酶的工业应用一直受成本高、酶活低的限制,而内切葡聚糖酶作为纤维素酶最主要的组成部分,提高内切葡聚糖酶的酶活力显得尤为重要。根据GenBank中收录的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)KCTC的模式菌株和解淀粉芽孢杆菌HZ79内切葡聚糖酶序列的相似性及orf分析设计特异性引物,以解淀粉芽孢杆菌的基因组DNA为模板,利用PCR反应克隆出长为1 500 bp的内切葡聚糖酶基因(egl基因),将目的基因与pMD19-T载体相连,转入大肠杆菌DH5α,阳性克隆经菌液PCR及双酶切验证,将验证正确的质粒送金唯智测序公司测序。比对结果表明:该克隆片段无碱基缺失,无移码突变,扩增序列准确、可靠。将测序正确的重组质粒转化BL21,进行大肠杆菌BL21(DE3)的原核表达,SDS-PAGE分析蛋白的大小为55 ku,等电点为8.12。为后续的内切葡聚糖酶基因突变体文库的建立,及egl酶酶活力及耐受性提高菌株的高通量筛选奠定前期基础。High cost and low enzyme activity are two factors that have always been restricting the in- dustrial application of cellulose, whereas incision-glucolase is the main part of cellulose enzyme, and to increase the activity of incision-glucolase is becoming more and more important. In this study, specific primers were designed according to starch Bacillus （Bacillus amyloliquefaciens） KCTC mod- el strains Bacillus HZ79 reconciliation starch incision-glucolase and orf sequence similarity analysis recorded by Gen Bank. With starch Bacillus genomic DNA as a template, 1 500 bp incision-glu- eolase genes （egl gene） were cloned by PCR reaction, the purpose gene was connected to pMD19- T carrier and then transformed into E. coli DH5 alpha. Positive clone was tested by microbial PCR and double enzyme, and then, the correct plasmid was sequenced by Jin Weizhi sequencing compa- ny. Comparison results showed that the cloned fragment had no base to miss, no frameshift muta- tions, and amplification sequences were accurate and reliable. Then the sequencing of recombinant plasmid was transformed into BL21, and expressed in E. coli BL21 （DE3）. SDS-PAGE analysis re- suits were as follows： the protein size： 55 ku; isoelectric point： 8.12. This will lay preliminary foundation for further incision-glucolase mutant library building, egl enzyme activity and high- throughput screening of tolerance-improved strains.

关 键 词：解淀粉芽孢杆菌 内切葡聚糖酶基因 原核表达 

分 类 号：Q786[生物学—分子生物学]