基因工程法生产L-5-甲基四氢叶酸的研究

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。

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

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.