Cloning and identification of frc gene from Oxalobacter frmigenes.
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.
Kong
Debo
D
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. kongdebo@gmail.com
Chen
Zhiqiang
Z
Ye
Zhangqun
Z
Yang
Weimin
W
Yao
Linfang
L
Guo
Hui
H
Liu
Guanlin
G
Zeng
Lingqi
L
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D015256:Coenzyme A-Transferases / Q000235:genetics* / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D020624:Oxalobacter formigenes / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D001426:Bacterial Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015256:Coenzyme A-Transferases; C064813:formyl-coenzyme A transferase