基因工程法生产L-5-甲基四氢叶酸的研究  

Study on the genetic engineering for L-5-methylenetetrahydrofolate production

作　　者：刘娅梅[1] 卞筱泓[1] 许激扬[1] 李兰娥[1] 张映桥[1] 许建良[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《中国生化药物杂志》2012年第3期244-247,共4页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的在E.coli BL21(DE3)中表达5,10-亚甲基四氢叶酸还原酶(MTHFR),并检测MTHFR的表达情况及L-5-甲基四氢叶酸的增加量。方法采用PCR法获得MTHFR基因(metF)全长DNA,分别在其5'端和3'端加上BamHⅠ和SacⅠ酶切位点。经BamHⅠ和SacⅠ双酶切后,连接到经同样双酶切的表达载体pET-28a(+)中,经限制性酶切、菌落PCR和测序验证正确后,转化至E.coli BL21(DE3),经IPTG诱导重组蛋白表达,SDS-PAGE检测MTHFR表达量,HPLC检测L-5-甲基四氢叶酸量。结果经酶切与测序鉴定证实原核重组载体pET28a-MTHFR构建成功,表达产物相对分子质量约为33 000,与MTHFR大小相符,工程菌的L-5-甲基四氢叶酸的产量显著增加。结论通过将MTHFR的基因导入L-5-甲基四氢叶酸产生菌能够提高L-5-甲基四氢叶酸产量。Purpose The 5,10-methylenetetrahydrofolate reductase（MTHFR） was expressed in E.coli BL21（DE3）,and the expression of MTHFR and the increment of 5-methylenetetrahydrofolate were detected.Methods The metF gene encoding MTHFR was amplied by PCR and fused to the expression vector pET-28a（＋） which was transfected into E.coli BL21（DE3）.The recombinant MTHFR was expressed following IPTG induction and verified by SDS-PAGE.The amount of L-5-methyltetrahydrofolate was detected by HPLC.Results The results of digestion and sequencing indicated that the recombinant expression vector pET28a-metF was successfully constructed.SDS-PAGE analysis showed that the target product was about 33 kD which was consistent with the anticipation,and the amount of 5-methylenetetrahydrofolate also significantly increased.Conclusion L-5-methyltetrahydrofolate producing strains containing expression vector pET28a-metf can improve the L-5-methyltetrahydrofolate production.

关 键 词：L-5-甲基四氢叶酸 原核表达 双酶切 

分 类 号：Q78[生物学—分子生物学]