中国人肠道产甲酸草酸杆菌甲酰辅酶A转移酶基因的分离、克隆和鉴定

Molecular Cloning,Identification and Expression of frc,the Formyl-CoA Transferase Gene of Oxalobacter Formigenes

目的克隆产甲酸草酸杆菌甲酰辅酶A转移酶基因(frc),检测其在真核细胞中的表达。方法用聚合酶链式反应技术从产甲酸草酸杆菌基因组DNA中扩增frc基因片段并插入真核表达载体获取重组质粒pEGFP-frc,通过限制性内切酶酶切电泳和测序鉴定重组质粒。重组质粒转染人胚肾293细胞,检测frc基因在mRNA和蛋白水平上的表达。被转染细胞在含草酸培养液中培养,检测0～72h培养液中草酸浓度的变化。结果frc基因的真核表达载体构建成功。转染293细胞后24～72h,可观察到明亮的绿色荧光,在mRNA和蛋白水平上可检测到frc基因在真核细胞中的表达。转染pEGFP-frc细胞12～72h培养液中草酸浓度明显低于转染空载体的细胞(P<0.05)。结论中国人肠道产甲酸草酸杆菌中可分离出frc基因;frc基因可在真核细胞293细胞中表达并使293细胞获得代谢草酸的能力。

Objective To study the molecular cloning, identification and expression of frc, the formyl-CoA transferase gene of Oxalobacter formigenes from the intestines of Chinese people. Methods The fragment of frc gene was amplified by polymerase chain reaction from the genomic DNA of Oxalobacter formigenes and then cloned into eukaryotic expression vector pEGFP-C1. The recombinant plasmid was identified by restriction fragment electrophoresis and sequencing. Human embryo kidney 293 cells were transfected with the recombinant plasmid and the expression of mRNA and fusion protein was detected. The transfected cells were cultured in oxalate-containing medium and the concentration of oxalate was measured in 0-72 h. Resuits The recombinant eukaryotic expression vector was constructed successfully. Relucent green fluorescence emerged from the 293 cells 24-72 h after transfection. The frc mRNA and fusion protein were detected from the transfected cells. The concentration of oxalate in the medium of pEGFP-frc transfected cells was lower than that of pEGFP-C1 transfected cells in 12-72 h （P〈0.05）. Conclusion The frc gene can be cloned from the Oxalobacter formigenes in the intestines of Chinese people and expressed in eukaryotic cells.