The cloning and identification offrc gene from Oxalobacterformigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacterformigenes was extracted, frc gene fragment was amplified by polym...The cloning and identification offrc gene from Oxalobacterformigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacterformigenes was extracted, frc gene fragment was amplified by polymerase chain reaction （PCR） and linked with pEGFP-CI. 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 fro 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 thatfrc gene cloned from the Oxalobacterformigenes in the intestines of Chinese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.展开更多
基金This project was supported by a grant from National Natural Sciences Foundation of China (No. 30371423)
文摘The cloning and identification offrc gene from Oxalobacterformigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacterformigenes was extracted, frc gene fragment was amplified by polymerase chain reaction （PCR） and linked with pEGFP-CI. 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 fro 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 thatfrc gene cloned from the Oxalobacterformigenes in the intestines of Chinese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.
