QI Hai-ping,GANG jie,HU Wen-zhong,et al.Cloning of Douchi Fibrinolytic Enzyme Gene and Its Expression in Bacillus subtilis[J].Soybean Science,2012,31(01):159-161.[doi:10.3969/j.issn.1000-9841.2012.01.037]
豆豉纤溶酶基因的克隆及其在枯草杆菌中的表达
- Title:
- Cloning of Douchi Fibrinolytic Enzyme Gene and Its Expression in Bacillus subtilis
- 文章编号:
- 1000-9841（2012）01-0159-03
- 分类号:
- TS201.1
- 文献标志码:
- A
- 摘要:
- 以实验室筛选的UD8豆豉芽孢杆菌的总DNA为模板，应用PCR技术根据豆豉纤溶酶基因DNA序列(GeneBank AY720895.2)设计并合成1对引物，扩增出了豆豉纤溶酶基因,将该基因克隆到大肠杆菌-枯草芽孢杆菌穿梭质粒EC-BS8上，构建成表达质粒EC-BS8，再将其转化到枯草芽孢杆菌Bs6中。筛选表达菌株，实现了豆豉纤溶酶基因在枯草杆菌中高效表达。获得重组菌纤溶酶活力高达9 830 IU·mL-1，是出发菌株活力的2.9倍。经纯化SDS-PAGE鉴定重组豆豉纤溶酶相对分子质量为31.0 kDa。
- Abstract:
- With the total DNA extracted from Bacillaceae UD8 as template,one pair of primers were designed according to DNA sequence of Douchi fibrinolytic enzyme(DFE)gene(AY720895.2)on Genbank website,to amplify DFE gene by PCR.The PCR prodcuct was inserted into expression plasmid EC-BS8,and the plasmid was transformed into the host strain Bacillus subtilis. DFE gene was successfully expressed in Bacillus subtilis and the maximum Douchi fibrinolytic enzyme level reached 9 830 IU·mL-1,which was 2.9 fold higher of the initial strain.The cloned Douchi fibrinolytic enzyme purified molecular weight was 31.0 kDa with a single band from SDS-PAGE.
参考文献/References:
[1]Sumi H,Banba T,Kishimoto N.Strong pro-urokinase activators proved in Japanese soybean cheese natto[J].Journal of Japanese Society of Food Science and Technology,1996,43(10):1124-1127. [2]Urano T,Ihara H,Umemura K,et al.The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis?Cleaves and inactivates plasminogen activator inhibitor type I[J].The Journal of Biological Chemistry,2001,276:24690-24696. [3]刘宇峰，王金英，孙岸弢，等.豆豉纤溶酶保健功能食品的研制[J].大豆通报，2000，（2）：22-23.(Liu Y F,Wang J Y,Sun A T,et al.Development of Douchi fibrinolytic enzyme functional food[J].Soybean Bulletin,2000，（2）：22-23. [4]段智变，江汉湖，张书霞，等.纳豆激酶粗制液对家兔溶血栓作用及其机制研究[J].营养学报，2003，25（1）：46-51.(Duan Z B,Jiang H H,Zhang S X,et al.Studies on fibrinolytic function of Nattokinase and its mechanism[J].Acta Nutrimenta Sinica,2003,25（1）：46-51.) [5]吕美云，张新，陆云华.豆豉纤溶酶基因的克隆及其在毕赤酵母中的表达[J].安徽农业科学，2008，36(6)：2270-2271.(Lu M Y,Zhang X,Lu Y H.Cloning of Douchi Fibrinolytic enzyme gene and its expression in Pichia pastoris[J].Journal of Anhui Agricultural Sciences，2008，36(6)：2270-2271.) [6]刘成君,黄庆,赵莲,等.大肠杆菌-枯草杆菌穿梭质粒载体pSUGV4的构建[J].四川大学学报(自然科学版),2001,38(2):243-246.(Liu C J,Huang Q,Zhao L,et al.Construction of a Escherichia coli-bacillus subtilis shuttle vector PSUGV4[J].Journal of Sichuan University(Natural Science Edition),,2001,38(2):243-246.) [7]Astrup T,Mullertz S.The fibrin plate method for estimating fibrinolytic activity[J].Archives of biochemistry and biophysics,1952，40：346-351. [8]Sambrook J,Fritsch E F,Maniatis T..Molecular cloning:A laboratory manual[M].Cold Spring Harbor Laboratory Press,1989.齐海萍,钱和,王璋,等，淡豆豉纤溶酶的分离纯化[J].中国调味品,2005(11):21-27. [9]Qi H P,Qian H,Wang Z,et al.The purification of Douchi fibrinolytic enzyme from Dochi[J].China Condiment,2005(11):21-27.
相似文献/References:
[1]吴 兴,赵欢欢,张 锋,等.拟南芥AtGPAT9基因cDNA的克隆及其转化大豆的研究[J].大豆科学,2012,31(06):878.[doi:10.3969/j.issn.1000-9841.2012.06.004]
WU Xing,ZHAO Huan-huan,ZHANG Feng,et al.Cloning of AtGPAT9 cDNA from Arabidopsis and Its Transformation of Soybean[J].Soybean Science,2012,31(01):878.[doi:10.3969/j.issn.1000-9841.2012.06.004]
[2]赵欢欢,吴兴,张锋,等.拟南芥AtLACS9基因的克隆及其植物表达载体构建[J].大豆科学,2011,30(02):190.[doi:10.11861/j.issn.1000-9841.2011.02.0190]
ZHAO Huan-huan,WU Xing,ZHANG Feng,et al.Cloning and Plant Expression Vector Construction of AtLACS9 Gene from Arabidopsis Thaliana[J].Soybean Science,2011,30(01):190.[doi:10.11861/j.issn.1000-9841.2011.02.0190]
[3]胡英考,孟宪萍,李雅轩,等.大豆γ-生育酚甲基转移酶基因的克隆与表达分析[J].大豆科学,2011,30(02):198.[doi:10.11861/j.issn.1000-9841.2011.02.0198]
HU Ying-kao,MENG Xian-ping,LI Ya-xuan,et al.Cloning and Expression Analysis of Glycine max γ-tocopherol Methyltransferase Gene[J].Soybean Science,2011,30(01):198.[doi:10.11861/j.issn.1000-9841.2011.02.0198]
[4]王伟旗,侯文胜.大豆硫氧还蛋白基因的克隆与分析[J].大豆科学,2011,30(03):351.[doi:10.11861/j.issn.1000-9841.2011.03.0351]
WANG Wei-qi,HOU Wen-sheng.Clone and Analysis of Thioredoxin Gene from Soybean (Glycine max L.)[J].Soybean Science,2011,30(01):351.[doi:10.11861/j.issn.1000-9841.2011.03.0351]
[5]林苏霞,王晓梅,刘志刚,等.大豆主要过敏原GlymBd30K的抗原表位区基因的克隆表达、纯化及免疫原性鉴定[J].大豆科学,2010,29(02):186.[doi:10.11861/j.issn.1000-9841.2010.02.0186]
LIN Su-xia,WANG Xiao-mei,LIU Zhi-gang,et al.Cloning and Expression of the Antigenic Epitope of Gly m Bd 30K Protein from Soybean and Purification and Identification of Expressed Product[J].Soybean Science,2010,29(01):186.[doi:10.11861/j.issn.1000-9841.2010.02.0186]
[6]刘宁,刘若淼,马莹,等.中国大豆花叶病毒HC-Pro基因的克隆与序列分析[J].大豆科学,2010,29(04):549.[doi:10.11861/j.issn.1000-9841.2010.04.0549]
LIU Ning,LIU Ruo-miao,MA Ying,et al.Cloning and Sequence Analysis of HC-Pro Gene of Soybean Mosaic Virus[J].Soybean Science,2010,29(01):549.[doi:10.11861/j.issn.1000-9841.2010.04.0549]
[7]胡英考,李雅轩,蔡民华.大豆对羟苯丙酮酸双加氧酶基因的克隆与表达分析[J].大豆科学,2010,29(04):565.[doi:10.11861/j.issn.1000-9841.2010.04.0565]
HU Ying-kao,LI Ya-xuan,CAI Min-hua.Cloning and Expression Analysis of p-hydroxyphenylpyruvic Acid Dioxygenase Gene in Glycine max [J].Soybean Science,2010,29(01):565.[doi:10.11861/j.issn.1000-9841.2010.04.0565]
[8]白晶,张必弦,李新玲,等.C2H2型锌指蛋白基因的克隆与序列分析[J].大豆科学,2009,28(01):21.[doi:10.11861/j.issn.1000-9841.2009.01.0021]
BAI Jing,ZHANG Bi-xian,LI Xin-ling,et al.Cloning and Sequence Analysis of a C2H2 type Zinc Finger Protein Gene from Glycine soja[J].Soybean Science,2009,28(01):21.[doi:10.11861/j.issn.1000-9841.2009.01.0021]
[9]吴凯威,余晓,刘志刚,等.大豆泛变应原profilin基因的克隆、序列分析和表位预测[J].大豆科学,2007,26(01):1.[doi:10.3969/j.issn.1000-9841.2007.01.002]
WU Kai-wei,YU Xiao,LIU Zhi-gang,et al.CLONING， SEQUENCE ANALYSIS AND ANTIGEN EPITOPE PREDICTION OF PANALLERGEN PROFILIN IN SOYBEAN[J].Soybean Science,2007,26(01):1.[doi:10.3969/j.issn.1000-9841.2007.01.002]
[10]吴迪,刘斌,侯文胜,等.转座子标签法及其在大豆功能基因组研究中的应用前景[J].大豆科学,2007,26(02):254.[doi:10.3969/j.issn.1000-9841.2007.02.028]
WU Di,LIU Bin,HOU Wen-sheng,et al.TRANSPOSON TAGGING AND ITS POTENTIAL USE IN FUNCTIONAL GENOMICS STUDY OF SOYBEAN[J].Soybean Science,2007,26(01):254.[doi:10.3969/j.issn.1000-9841.2007.02.028]
备注/Memo
基金项目：辽宁省教育厅资助项目（2008135）。