大肠杆菌MalQ基因的克隆与原核融合表达  

Cloning and Fusion Expression of Gene MalQ from E.coli

作　　者：王水兴[1] 郭勇[1] 许杨[2] 李燕萍[2] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]南昌大学中德联合研究院,江西南昌330047

出　　处：《华南理工大学学报（自然科学版）》2008年第4期122-126,共5页Journal of South China University of Technology(Natural Science Edition)

基　　金：广东省重点科技攻关项目(A301020201)

摘　　要：为了构建用于表达麦芽糖转糖基酶的基因工程菌,用PCR方法获得大肠杆菌(Escherichia coli)K12MalQ基因.将该基因插入原核表达载体pET-DsbA中,对质粒所含外源片段进行双向测序,并经局部相似性基本查询工具(BLAST)比较分析,发现其与GenBank中报道的大肠杆菌K12MalQ基因的同源性高达99%.重组质粒转化E.coliBL21(DE3)plysS,经异丙基硫代-β-D-半乳糖(IPTG)诱导后以融合蛋白形式表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示MalQ基因与DsbA表达的融合蛋白在目标位置相对分子质量约为103 000处有明显条带.经薄层层析分析粗酶液酶处理的麦芽三糖溶液,证实粗酶液具有麦芽糖转糖基活性.In order to construct a gene engineering bacterium expressing amylomaltase, gene MalQ was amplified from Escherichia coli K12 by PCR, and was cloned into pET-DsbA. Then, a two-direction sequencing was performed, and the results were analyzed by means of BLAST. There displayed high similarity （99%） of the obtained gene MalQ to the gene MalQ sequence of E. coli K12 reported in the GenBank. Moreover, the recombined plasmid pET-DsbA-MalQ was transformated into E. coli BL21 （DE3） plysS and was induced by IPTG. The fusion protein of gene MalQ and DsbA with a relative molecular mass of about 103000 was detected by means of sodium dodecyl sul- phate-polyacrylamide gel electrophoresis. 4-α-glucanotransferase activity of the crude enzyme was finally demonstrated by means of thin layer chromatography.

关 键 词：麦芽糖转糖基酶 基因重组 融合表达 大环糊精 基因克隆 

分 类 号：Q786[生物学—分子生物学]