利用Bio-BglBrick方法表达紫杉醇生物合成酶 |
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引用本文: | 金晶,王微,邵妤,陈章,凌焱,李玉霞,宋永波,刘刚,陈惠鹏.利用Bio-BglBrick方法表达紫杉醇生物合成酶[J].生物技术通讯,2014,25(5):616-620. |
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作者姓名: | 金晶 王微 邵妤 陈章 凌焱 李玉霞 宋永波 刘刚 陈惠鹏 |
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作者单位: | 1. 沈阳药科大学,辽宁沈阳110016;军事医学科学院生物工程研究所,北京100071 2. 军事医学科学院生物工程研究所,北京100071;吉林大学,吉林长春130012 3. 军事医学科学院生物工程研究所,北京100071;安徽大学,安徽合肥230601 4. 成都军区总医院呼吸科,四川成都,610083 5. 军事医学科学院生物工程研究所,北京,100071 6. 沈阳药科大学,辽宁沈阳,110016 |
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基金项目: | "十二五"重大新药创制国家科技重大专项"综合性新药研发技术大平台" |
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摘 要: | 目的:进行紫杉醇药物生物合成前五步催化酶二磷酸盐合酶(GGPPS)、紫杉二烯合酶(TS)、紫杉二烯5α羟化酶(THY5α)、紫杉二烯5α-O-乙酰转移酶(TAT)和紫杉烷10β羟化酶(TDH)基因在大肠杆菌异源生物合成途径的组建及串联,实现单个表达及串联表达,并试图通过连续生物催化获得紫杉醇中间体紫衫二烯。方法:依据合成生物学中Brick基因组装方法,通过对载体pET30a的酶切位点进行定向改造,设计独特的BglⅡ/BamHⅠ和XbaⅠ/SpeⅠ串联表达盒,在大肠杆菌BM Rosetta(DE3)中表达产物。结果:设计多基因表达盒,实现紫杉醇药物生物合成前五步催化酶的单个表达,GGPPS和TS以及THY5α、TAT和TDH的串联表达。结论:利用BglBrick/BioBrick基因组装方法,可以实现紫杉醇生物合成催化酶的快速组装及后续表达。
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关 键 词: | 紫杉醇 生物合成 异源表达 基因组装 |
Expression of Taxol Biosynthetic Enzymes via Bio-BglBrick Method |
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Institution: | JIN Jing, WANG Wei, SHAO Yu, CHEN Zhang, LING Yan, LI Yu-Xia, SONG Yong-Bo, LIU Gang, CHEN Hui-Peng( 1. Shenyaug Pharmaceutical University, Shenyang 110016; 2. Beijing Institute of Biotechnology, Beijing 100071; 3. Jilin University, Changchun 130012; 4. College of Life Science, Anhui University, Hefei 230601; 5. General Hospital of Chengdu Military Region, Chengdu 610083; China) |
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Abstract: | Objective: Genes encoding five sequential pathway steps of taxol biosythesis, which are geranylgeranyl diphosphate synthase (GGPPS), taxadiene synthase (TS), taxadiene- 5α- hydroxylase (THY5α), taxadiene- 5α- O- aeetyl-transferase(TAT) and taxane-10β-hydroxylase(TDH), were installed in a single E.coli host. Then through biosynthesis, the host can produce an intermediate taxadiene. Methods: Brick gene assembly method is a flexible standard for biological part assembly, through it we mutated the vector pET30a, designing Bgl Ⅱ/BamH Ⅰ and Xba ⅠI/Spe Ⅰ expression cassette. Results: The Bgl Ⅱ/BamH Ⅰ and Xba Ⅰ/Spe Ⅰ muhi-gene expression cassette was designed successfully. The five genes were achieved single genes expressin individually and multi-genes expression si- multaneously, GGPPS and TS, THY5α, TAT and TDH. Conclusion: The use of BglBrick method can help fulfill the rapid assembly of. taxol biosynthesis enzymes and sequential expression. |
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Keywords: | paclitaxel biosynthesis heterologous expression gene assembly |
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