溶藻弧菌dtd基因的克隆及原核表达条件优化Cloning and Optimization of Prokaryotic Expression of D-Tyr-tRNATyr Deacylase(DTD) Gene from Vibrio alginolyticus
陈立明,庞欢瑛,简纪常,吴灶和
摘要(Abstract)：
根据NCBI数据库中已发表的溶藻弧菌(Vibrio alginolyticus)全基因组序列合成一对特异性引物,应用聚合酶链式反应(PCR技术)扩增溶藻弧菌HY9001菌株dtd基因,将其定向克隆到原核表达载体pET-32a构建重组表达质粒pET-DTD,并对其进行诱导温度、诱导时间、诱导IPTG浓度等条件的优化,最后探究其纯化时最佳咪唑洗脱浓度。结果表明:DTD蛋白成功表达,且其以包涵体的形式存在,在诱导温度37℃、IPTG浓度0.1 mmol/L条件下诱导5 h表达量最高,纯化最佳咪唑洗脱浓度为150 mmol/L。
关键词(KeyWords)： 溶藻弧菌;dtd;基因克隆;原核表达;条件优化
基金项目(Foundation): 国家科技支撑计划(2012BAD17B02);; 广东省自然科学基金(B12121)
作者(Author): 陈立明,庞欢瑛,简纪常,吴灶和
参考文献(References)：
- [1]梁海鹰.溶藻弧菌(Vibrio alginolyticus)HY9901株鞭毛蛋白基因的克隆与表达[D].广州:中国科学院南海海洋研究所,2010.
- [2]Austin B,Stobie M,Robertson P,et al.Vibrio alginolyticus:the cause of gill disease leading to progressive low-level mortalities among juvenile turbot,Scophthalmus maximus L in a Scott ish aquarium[J].J Fish Dis,1993,16(3):277-280.
- [3]Selvin J,Lipton A.Vibrio alginolyticus associated with white spot disease of Penaeus monodon[J].Dis Aquat Organ,2003,57(1):147-150.
- [4]Balcázar J L,Gallo-Bueno A,Planas M,et al.Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms[J].AntonLeeuw Int J G,2010,97(2):207-210.
- [5]陈清华.水产养殖业中抗生素使用的风险及其控制[J].水产科技情报,2009,36(2):67-72.
- [6]黄辉,毛芝娟,陈吉刚.哈维氏弧菌外膜蛋白OmpU的克隆、表达与免疫原性研究[J].华中农业大学学报,2010,29(3):346-350.
- [7]梁海鹰,夏立群,吴灶和,等.溶藻弧菌HY9901鞭毛蛋白flaB基因的克隆及原核表[J].水产学报,2010,34(1):139-146.
- [8]Yang H,Zheng G,Peng X,et al.D-Amino acids and D-Tyr-tRNA Tyr deacylase:stereospecificity of the translation machine revisited[J].FEBS letters,2003,552(2):95-98.
- [9]Soutourina J,Plateau P,Blanquet S.Metabolism of d-Aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae Cells[J].Journal of Biological Chemistry,2000,275(42):32535-32542.
- [10]Zheng G,Liu W,Gong Y,et al.Human D-Tyr-tRNATyr deacylase contributes to the resistance of the cell to D-amino acids[J].Biochem.J,2009,417:85-94.
- [11]Liu W,Liu C,Zhu J,et al.D-Tyr-tRNATyr Deacylase,a New Role in Alzheimer's-associated disease in SAMP8Mice[J].Chinese Medical Sciences Journal,2010,25(2):90-94.
- [12]Friedman M.Origin,Microbiology,Nutrition,and Pharmacology of D-Amino Acids[J].Chemistry&biodiversity,2010,7(6):1491-1530.
- [13]Erikson O,Hertzberg M,Nsholm T.A conditional marker gene allowing both positive and negative selection in plants[J].Nature biotechnology,2004,22(4):455-458.
- [14]Ruan B,Sll D.The bacterial YbaK protein is a Cys-tRNAPro and Cys-tRNACys deacylase[J].Journal of Biological Chemistry,2005,280(27)25887-25891.
- [15]Soutourina J,Plateau P,Blanquet S.Metabolism of d-Aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae Cells[J].Journal of Biological Chemistry,2000,275(42):32535-32542.
- [16]Leiman S A,May J M,Lebar M D,et al.D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis[J].Journal of bacteriology,2013,195(23):5391-5395.
- [17]Bhatt T K,Yogavel M,Wydau S,et al.Ligand-bound structures provide atomic snapshots for the catalytic mechanism of D-amino acid deacylase[J].Journal of biological chemistry,2010,285(8):5917-5930.
- [18]Yogavel M,Khan S,Bhatt T K,et al.Structure of D-tyrosyl-tRNATyr deacylase using home-source Cu K and moderate-quality iodide-SAD data:structural polymorphism and HEPES-bound enzyme states[J].Acta Crystallographica Section D:Biological Crystallography,2010,66(5):584-592.
- [19]庞欢瑛.溶藻弧菌毒力蛋白和保护性抗原的筛选[D].中国科学院研究生院,2010.
- [20]Carrio M M,Villaverde A.Construction and deconstruction of bacterial inclusion bodies[J].Journal of Biotechnology,2002,96(1):3-12.