重组甘氨酸脱羧酶基因工程菌生产L-5-甲基四氢叶酸  

Recombinant glycine decarboxylase engineered bacteria for production of L-5-methyl-tetrahydrofolate

作　　者：韩林[1] 许激扬[1] 卞筱泓[1] 邵飞[1] 吴东儿 朱虹[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国新药杂志》2013年第20期2379-2382,2409,共5页Chinese Journal of New Drugs

基　　金：中央高校基本科研业务费专项资金(JKQ2011046)

摘　　要：目的:针对叶酸代谢通路中的甘氨酸脱氢酶(GDC)构建高效表达GDC的重组菌E.coli BL21(pET22b-gcvP)并检测L-5-甲基四氢叶酸(L-5-MTHF)的增加量。方法:采用PCR法获得GDC基因gcvP,分别在其两端加上HindⅢ和XhoⅠ酶切位点,经HindⅢ和XhoⅠ双酶切后,连接到经同样双酶切的表达载体pET22b中,经限制性内切酶酶切和测序验证正确后,转化至E.coli BL21中。乳糖诱导重组蛋白表达,进行SDS-PAGE电泳,HPLC检测L-5-MTHF产量。结果:经双酶切与测序鉴定证实原核重组载体pET22b-gcvP构建成功,表达产物相对分子质量约为104×103,与GDC大小相符。与原始菌相比,重组菌的L-5-MTHF的产量增加了23.1%。结论:重组甘氨酸脱羧酶基因工程菌能够提高L-5-MTHF产量。Objective： To construct recombinant strain of E. coli BL21 （pET22b-gcvP） which efficiently expresses glycine decarboxylase （GDC） in folate metabolism pathway, and detect the increment of 5-L-methyl-tetrahydrofolate. Methods： The gene gevP encoding GDC was amplified from the E. coli BL21 by using PCR technique. The 5＇end of the primes was designed with Hind Ⅲ and Xho Ⅰ restriction sites respectively. The fragment was connected to expression vector pET22b which was digested by restriction enzymes Hind Ⅲ and Xho I. The target gene was confirmed by double-enzyme cleavage and DNA sequencing. Then recombinant vector was transformed into E. coli BL21. The recombinant protein expression was induced by lactose and verified by SDS-PAGE. The L-5- methyl-tetrahydrofolate production was detected by HPLC. Results： Double-enzyme cleavage and DNA sequencing confirmed that the recombinant vector pET22b-gcvP was successfully constructed. SDS-PAGE analysis showed that the expression product was about 104 ~ 103 , which matched GDC size. Compared with the original bacteria, the L- 5-methyl-tetrahydrofolate production by recombinant strain increases by 23.1%. Conclusion： Glycine decarboxylase engineered bacteria can improve L-5-MTHF production.

关 键 词：L-5-甲基四氢叶酸 甘氨酸脱羧酶 克隆 原核表达 双酶切 

分 类 号：R977.22[医药卫生—药品]