地衣芽孢杆菌谷氨酸消旋酶基因克隆、原核表达及生物信息学分析  

Cloning, prokaryotic expression and bioinformatics analysis of glutamic acid racemase gene of Bacillus licheniformis

作　　者：邬旭龙 杨敏[1] 肖璐 张婧 杨苗 谢礼 安微 林华 WU Xulong;YANG Min;XIAO Lu;ZHANG Jing;YANG Miao;XIE Li;AN Wei;LIN Hua(The Pet Nutrition and Health Research Center,Chengdu Agricultural College,Chengdu 611130,China;Key Laboratory of Animal Genetics and Breeding of Sichuan Province,Sichuan Animal Science Academy,Chengdu 611130,China;Key Laboratory of Food Safety Testing of Sichuan Province,Technology Center of Chengdu Customs,Chengdu 610041,China)

机构地区：[1]成都农业科技职业学院宠物营养与健康研究中心,成都611130 [2]四川省畜牧科学研究院动物遗传育种四川省重点实验室,成都611130 [3]成都海关技术中心食品安全检测四川省重点实验室,成都610041

出　　处：《黑龙江畜牧兽医》2022年第8期114-118,138,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：成都市技术创新研发项目(2019-YF05-00119-SN);四川省中央引导地方科技发展专项(2020ZYD025);四川省重点研发项目(2020YFS0469);成都农业科技职业学院校级科研基金项目(20ZR101);海关总署科研项目(2019HK045)。

摘　　要：为了进一步分析谷氨酸消旋酶的结构和生物信息功能,试验以γ-PGA生产菌株地衣芽孢杆菌(Bacillus licheniformis)DY136为材料,PCR扩增其谷氨酸消旋酶基因(racE),纯化racE基因进行原核表达,并运用多种在线生物信息学分析软件对该基因编码蛋白质的理化性质、亲/疏水性、信号肽、结构域等进行分析。结果表明:PCR扩增racE基因条带大小为819 bp,对racE基因进行大肠杆菌原核表达,通过构建表达载体和优化表达条件,成功表达了大小为30.15 ku的重组融合蛋白。racE蛋白编码272个氨基酸,氨基酸序列与NCBI中公布的地衣芽孢孢杆菌racE蛋白(GenBank登录号为KAA0845975.1)氨基酸同源性为99.62%。racE蛋白理化性质稳定,是亲水蛋白,无跨膜结构域,不含有信号肽,α-螺旋是其主要的二级结构,主要定位于细胞质中(73.90%)。说明racE基因保守且相对稳定,在谷氨酸的合成中发挥重要作用。In order to further analyze the structure and bioinformatics function of glutamate racemase, the experiment used γ-PGA-producing strain Bacillus licheniformis DY136 as the material;the glutamic acid racemase gene racE was amplified by PCR, the target gene was purified for prokaryotic expression, and a variety of online bioinformatics analysis softwares were used to analyze its physicochemical properties, hydrophilicity/hydrophobicity, signal peptide, structural domain, etc. The results showed that the size of the PCR-amplified racE gene was 819 bp. Prokaryotic expression of the racE gene was carried out in E. coli;by constructing an expression vector and optimizing expression conditions, a recombinant fusion protein with a size of 30.15 ku was successfully expressed. racE protein encoding 272 amino acids. The amino acid sequence was 99.62% homologous to the Bacillus licheniformis racE protein published in NCBI(GenBank registration number KAA0845975.1). racE protein was stable in physicochemical properties, which was a hydrophilic protein without transmembrane domain and signal peptide. α-helix was its main secondary structure, which was mainly located in the cytoplasm(73.90%). The results suggested that the racE gene was conserved and relatively stable, and played an important role in the synthesis of glutamate.

关 键 词：地衣芽孢杆菌 谷氨酸消旋酶 racE基因 克隆 原核表达 生物信息学分析 

分 类 号：Q78[生物学—分子生物学] S852.616[农业科学—基础兽医学]