甘油激酶和3-磷酸甘油脱氢酶基因在大肠杆菌中的克隆表达  

Cloning and Overexpression of Glycerol Kinase and Glycerol-3-Phosphate Dehydrogenase Genes in E.coli

作　　者：袁超[1] 杨洋[1] 韩小容[1] 窦洁[1] 王慧[1] 周长林[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2014年第4期325-328,共4页Pharmaceutical Biotechnology

基　　金：中央高校基本科研业务费专项资金

摘　　要：莽草酸是抗流感有效药物"达菲"的前体物质。作者等之前的研究中,发现甘油作为碳源时重组大肠杆菌生物合成莽草酸的量最高,说明甘油代谢途径对莽草酸的生物合成有重要的影响,甘油激酶和3-磷酸甘油脱氢酶是大肠杆菌甘油代谢途径的关键酶,对它们进行克隆表达来研究甘油代谢途径对大肠杆菌生物合成莽草酸的影响。文章采用PCR方法从大肠杆菌DH5α基因组中扩增得到长度均为1.5 kb的甘油激酶基因(glpK)和3-磷酸甘油脱氢酶基因(glpD),并构建了重组表达载体pET-3b-glpD-glpK,转化E.coli BL21(DE3)后得到重组的E.coli BL21(DE3)-pET3b-glpD-glpK,并采用SDS-PAGE检测目的基因蛋白的表达。结果表明,glpK和glpD基因已成功构建到pET-3b-glpD-glpK质粒中,SDS-PAGE也检测到了目的基因的成功表达(55 k);同时,重组E.coli BL21(DE3)-pET3b-glpD-glpK的甘油消耗比出发菌株提高了16%,莽草酸积累量是出发菌株的3.5倍。Shikimic acid（ 3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid,SA） is an important chiral compound used as a key chemical building block for the antiviral drug Oseltamivir（ Tamiflu）,which is employed in the treatment and prophylaxis of both influenza A and influenza B viruses. In our previous study,SA accumulation in recombinant E. coli in glycerol cultures was higher than that in other cultures,suggesting that glycerol metabolic pathway was important for the biosynthesis of shikimic acid. Glycerol kinase and glycerol-3-phosphate dehydrogenase are key enzymes in glycerol metabolism of E. coli,which are important in the biosynthesis of shikimic acid. Gene glpD and glpK,1. 5 kb,were amplified from genomic DNA of E. coli DH5α,and were cloned into vector pET-3b to construct the expression vector pET-3b-glpK-glpD,which was transformed into E. coli BL21（DE3） to form an engineered strain of E. coli BL21（ DE3）-pET-3b-glpD-glpK. SDS-PAGE was used for protein expression analysis. The data showed that the recombinant vector pET-3b-glpD-glpK was constructed successfully and GlpD and GlpK were overexpressed at 55 ku in SDS-PAGE.Meanwhile,the glycerol consumption in recombinant E. coli BL21（ DE3）-pET3b-glpD-glpK was 16% increased over the parental strain and the shikimic acid accumulation in recombinant E. coli BL21（ DE3）-pET3b-glpD-glpK was 3.5-fold higher than that of the parental strain.

关 键 词：甘油激酶 3-磷酸甘油脱氢酶 甘油 莽草酸 大肠杆菌 克隆 表达 

分 类 号：Q55[生物学—生物化学]