肌酐水解酶基因工程菌的构建及功能研究  

The Construction of the Creatininase Gene Engineering Bacteria and Function Study

作　　者：杨波[1] 蒋芬[1] 朱艳[1] 杨成[2] 向铁勇 段绍斌[4] 蒋云生[4] 

机构地区：[1]南华大学附属第一医院肾内科,湖南衡阳421001 [2]长沙医学院临床系 [3]湖南省衡阳市第八中学生物教研室 [4]湘雅二医院肾内科

出　　处：《中南医学科学杂志》2016年第5期494-498,共5页Medical Science Journal of Central South China

基　　金：湖南省自然科学基金项目(No:13JJ3082)

摘　　要：目的构建肌酐水解酶的重组质粒转染至大肠杆菌中,研究其蛋白的表达及活性。方法根据Genbank上肌酐水解酶基因序列(D45424.1),进行生物合成,得到肌酐水解酶基因片段C,聚合酶链反应(PCR)扩增后,与质粒GV296连接,构建成重组质粒GV296-C,转染至大肠杆菌BL21(DE3),构建成基因工程菌GV296-C/BL21(DE3)。进行十二烷基硫酸钠—聚丙烯酰胺(SDS-PAGE)分析后,测定培养液肌酐浓度变化。结果成功构建重组质粒GV296-C,电泳显示目的片段约为0.783Kb,测序结果与D45424.1基因序列一致。重组质粒GV296-C转染至E.coli BL21(DE3)感受态细胞中,经SDS-PAGE分析酶蛋白的分子量约为29KD。结论成功构建工程菌GV296-C/BL21(DE3),诱导后高效表达肌酐水解酶,具有水解肌酐活性。Objective To construct recombinant plasmids of Creatininase,cloning into E. coli,and study its expression and enzyme activity. Methods According to the gene sequence of creatininase in Genbank（ D45424. 1）,it was biosynthesized to get the gene fragment of creatininase C,after the PCR amplified,and then was connected with GV296 to construct the recombinant plasmid GV296-C,and transformed to E.coli BL21（ DE3） to construct gene engineering strain GV296-C/BL21（ DE3）.After SDS-PAGE,the concentration of creatinine in the culture fluid was determined. Results The recombinant plasmid GV296-C was constructed successfully,enzyme fragment was about 0. 783 Kb as electrophoresed shown.The sequencing result was consistent with gene order（ D45424. 1）. The reconstructed plasmid GV296-C were transformed into E.col BL21（ DE3） competent cells.The protein of molecular weight was approximately 29 KD by SDS-PAGE.Conclusions The engineering strain GV296-C/BL21（ DE3） is constructed successfully,and the expression of the creatininase is induced to be efficient,which has the enzymes activity.

关 键 词：肌酐水解酶 基因工程菌 肌酐 慢性肾功能衰竭 

分 类 号：R692.5[医药卫生—泌尿科学]