【摘要】 目的:探讨中国人肠道产甲酸草酸杆菌甲酰辅酶A转移酶基因(frc)的分离、克隆和鉴定。方法:提取中国人肠道产甲酸草酸杆菌的基因组DNA,利用PCR技术扩增frc基因片段并克隆入真核表达载体pEGFP-C1,重组质粒命名为pEGFP-frc,通过限制性内切酶酶切电泳和测序鉴定插入片段。将pEGFP-frc通过脂质体转染293细胞,通过逆转录PCR(RT-PCR)和蛋白印迹(Western blot)分别从mRNA和蛋白水平检测frc基因在真核细胞中的表达。结果:中国人肠道产甲酸草酸杆菌frc基因全长1 287 bp,存在53个碱基和4个氨基酸残基的变异,与GenBank中的frc基因的碱基序列的同源性为95.88%,氨基酸残基序列的同源性为99.07%。转染293细胞后24~72 h,可观察到明亮的绿色荧光,RT-PCR和Western blot分别从mRNA和蛋白水平上检测到frc基因在真核细胞中的表达。结论:中国人肠道产甲酸草酸杆菌中可以分离出frc基因;中国人肠道产甲酸草酸杆菌frc基因存在一定的变异;frc基因可在真核细胞293细胞中表达。更多还原

【Abstract】 Objective:To study the cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people.Methods:The genomic DNA of Oxalobacter formigenes was extracted from the intestines of Chinese people.The fragment of frc gene was amplified by polymerase chain reaction and was linked with eukaryotic expression vector pEGFP-C1.The recombinant plasmid was named as pEGFP-frc and was identified by restriction-enzyme cutting and sequencing.Human embryo kidney 293 cells were transfected with the recombinated plasmid pEGFP-frc by LipofectamineTM 2000.RT-PCR were performed to detect the mRNA express of frc.Western blot were performed to detect the fusion protein express of FCoAT-EGFP.Results:The total length of frc gene from Oxalobacter formigenes in the intestines of Chinese people was 1287bp,and there were mutation of 53 nucleotides and 4 amino-acid residue.The homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%.293 cells transfected with the recombinated plasmid express relucent green fluorescence.The mRNA of frc gene was detected by RT-PCR.The fusion protein express of FCoAT-EGFP was detected by Western blot.Conclusions:The frc gene can be cloned from the Oxalobacter formigenes in the intestines of Chinese people and there are some mutation with the frc gene.The mutation frc gene can express in eucaryotic cell.更多还原