| 杨波,蒋芬,朱艳,杨成,向铁勇,段绍斌,蒋云生.肌酐水解酶基因工程菌的构建及功能研究.[J].中南医学科学杂志.,2016,(5):494-498. |
| 肌酐水解酶基因工程菌的构建及功能研究 |
| The Construction of the Creatininase Gene EngineeringBacteria and Function Study |
| 投稿时间：2016-04-25 修订日期：2016-06-29 |
| DOI： |
| 中文关键词: 肌酐水解酶 基因工程菌 肌酐 慢性肾功能衰竭 |
| 英文关键词:creatininase gene engineering bacteria creatinine chronic renal failure |
| 基金项目: |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| Objective To construct recombinant plasmids of Creatininase,cloning into E.coli,and study its expression and enzyme activity.MethodsAccording 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.ResultsThe recombinant plasmid GV296-C was constructed successfully,enzyme fragment was about 0.783Kb 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 29KD by SDS-PAGE.ConclusionsThe 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. |
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