Construction and identification of recombinant lentiviral expressing vectors formyl-CoA transferase gene and Oxalyl-CoA decarboxylase gene of oxalobacter formigenes from Chinese feces

Abstract

Objective To clone the formyl-CoA transferase gene (FRC),and Oxalyl-CoA decarboxylase gene ( OXC),in oxalobacter formigenes from Chinese feces (OxCF),and construct the recombinant lentiviral expressing vectors pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED,providing a basis for further study on gene therapy for hyperoxalurias.Methods Total RNA was isolated from OxCF,and the FRC and OXC cDNA was amplified by polymerase chain reaction (PCR) and then ligated with pMD18T simple vectors after retrieve and purification.The ligation products were transformed into competent DH5α.The positive recombinant clones were selected and identified by a complementation,restriction endonuclease digestion.The cloning vector and the lentiviral vectors pLenti6.3/v5 DEST-IRES-EGFP and pLenti6.3/v5 DEST-IRES-DsRED first digested with BamHⅠ were ligated and transformed respectivly.The enzyme and PCR analyses were performed to confirm the recombinant vector,and then DNA sequence were analysis.The titer of constucted lentivirus vectors were tested.Results Two fragments of 1287 bp and 1707 bp were obtained by PCR respectivly.The enzyme and PCR analyses revealed that the correct FRC and OXC cDNA was cloned.The sequence of pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED was identical to that of cloned cDNA respectivly.HEK293T cells had green and red fluorescence 24 h after transfection.The titers of two types of virus were 1.15 × 108 TU/ml and 9.75 × 107 TU/ml respectively.Conclusion FRC and OXC are cloned correctly and the recombinant lentiviral vectors pLenti6.3-FRC-IRES-EGFP and pLenti6.3-OXC-IRES-DsRED are constructed successfully. Key words: Oxalobacter formigenes; Lentiviral vector; Hyperoxalurias