Study On The Molecular Cloning And Eukaryotic Expression Of Formyl-CoA Transferase And Oxalyl-CoA Decarboxylase Of Oxalobacter Formigenes: Implications For Gene Therapy Of Hyperoxaluria

Objective: Oxalate is a highly toxic natural byproduct of normal cellular metabolism. Abnormally synthesis major in liver or hyperabsorption in gut of oxalate can lead to the accumulation of oxalate in vivo and result in disorder of oxalate metabolism, hyperoxaluria. To study the molecular cloning, identification and expression of frc, the formyl-CoA transferase (frc) gene of Oxalobacter formigenes from the intestines of Chinese people was subcloned to a eukaryotic expression vector and the expression of frc gene was identified.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 was performed to detect the mRNA express of frc. Western blot were performed to detect the fusion protein express of FCoAT-EGFP. The transfected cells were cultured in oxalate-contained medium and the concentration of oxalate was measured at 0~72 h.Results: The total length of frc gene from Oxalobacter formigenes in the intestines of Chinese people was 1287 bp, 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%. The recombinant eukaryotic expression vector was constructed successfully. Relucent green fluorescence emerged from HEK293 cells 24~72 h after transfection. The frc mRNA and fusion protein were detected from the cell transfected. The concentration of oxalate in the medium of pEGFP-frc transfected cell was lower than that of pEGFP-C1 transfected cell at 12~72h (P<0.05).Conclusion: 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. Objective: Oxalate is a highly toxic natural byproduct of normal cellular metabolism. Abnormally synthesis major in liver or hyperabsorption in gut of oxalate can lead to the accumulation of oxalate in vivo and result in disorder of oxalate metabolism, hyperoxaluria. To study the cloning and identification of oxc gene from Oxalobacter formigenes in the intestines of Chinese people, the oxalyl-CoA decarboxylase (oxc) gene of Oxalobacter formigenes was subcloned to a eukaryotic expression vector and the expression of oxc gene was identified.Methods: The genomic DNA of Oxalobacter formigenes was extracted from the intestines of Chinese people. The fragment of oxc gene was amplified by polymerase chain reaction and was linked with eukaryotic expression vector pEGFP-C1. The fragment of oxc gene was identified by restriction-enzyme cutting and sequencing. Human embryo kidney 293 cells were transfected with the recombinated plasmid by LipofectamineTM 2000. RT-PCR were performed to detect the mRNA express of oxc. Western blot were performed to detect the fusion protein express of OCoAD-EGFP.Results: The total length of oxc gene from Oxalobacter formigenes in the intestines of Chinese people was 1707bp, and there were mutation of 109 nucleotides and 16 amino-acid residue. The homology of nucleotides and amino-acid residue with the sequence in GenBank was 93.61% and 97.18%. 293 cells transfected with the recombinated plasmid expressed relucent green fluorescence. The mRNA of oxc gene was detected by RT-PCR with a 347bp amplified product. The fusion protein express of OCoAD-EGFP was detected by Western blot with a 90kD protein.Conclusion: The oxc gene can be cloned from the Oxalobacter formigenes in the intestines of Chinese people and there are some mutation with the oxc gene. The mutation oxc gene can express in eucaryotic cell.