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Cloning and identification of frc gene from Oxalobacter frmigenes
Debo Kong1, Zhiqiang Chen, Zhangqun Ye
1Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. kongdebo@gmail.com
Abstract:
The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chinese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.
Insights
Researchers cloned the Oxalobacter formigenes frc gene from Chinese individuals. This gene, crucial for oxalate metabolism, was successfully expressed in human cells, demonstrating its functional activity.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Oxalobacter formigenes is a gut bacterium important for oxalate homeostasis.
- Understanding the genetic basis of its function, like the frc gene, is crucial for metabolic research.
Purpose of the Study:
- To clone and characterize the frc gene from Oxalobacter formigenes in the Chinese population.
- To investigate the expression and functional activity of the cloned frc gene in eukaryotic cells.
Main Methods:
- Genomic DNA extraction and PCR amplification of the frc gene.
- Cloning into pEGFP-C1 vector, creating a recombinant plasmid (pEGFP-frc).
- Transfection of human embryonic kidney 293 cells, followed by RT-PCR and Western blotting.
Main Results:
- The frc gene fragment was successfully cloned and sequenced, measuring 1287 bp.
- High homology (95.88% nucleotide, 99.07% amino acid) was observed with existing GenBank sequences.
- Transfected 293 cells exhibited green fluorescence, and frc mRNA and the FCoAT-EGFP fusion protein were detected.
Conclusions:
- The frc gene from Oxalobacter formigenes of Chinese origin is successfully expressed in eukaryotic 293 cells.
- The expressed frc gene retains its enzymatic activity in the mammalian cell environment.
