Cloning Of Full-length CDNA,Bioinformatic Analysis And Prokaryotic Expression Of CYP2J Of Bactrian Camel

The cytochrome P450 2J enzyme plays an important role in metabolism of endogenous substances in body and exogenous compounds and has always been the focus of pharmacology and toxicology research.The main purpose of this study was to clone full-length c DNA sequence of CYP2J of Bactrian camel,to further predict the structures,properties and functions of CYP2J protein encoded by open reading frame of CYP2J gene and to successfully express this protein by prokaryotic expression system.Firstly,specific primers were designed according to the known fragment of CYP2J gene of Bactrian camel,RACE technique was used to clone Bactrian camel 3'and 5'sequence and splice 3'and 5'sequence fragments by DNAMAN software to obtain the full-length cDNA of CYP2J of Bactrian camel.Subsequently,Structure,properties and evolutionary relationship of CYP2J protein were analyzed by a variety of bioinformatics software such as DNAMAN,MEGA 6 and Prot Param etc.Finally,a recombinant plasmid pET21a-CYP2J containing the coding sequence of CYP2J was constructed by using pET-21a?+?as a fused expression vector and was induced to expressed in Escherichia coli BL21.The expression and purification of CYP2J protein was analyzed by SDS-PAGE and Western Blot.The results showed the full-length cDNA of CYP2J was obtained.It was 1770 bp in length containing a 5'-untranslated region?5'-UTR?of 8bp and a 3'-untranslated region?3?-UTR?of 253 bp and the ORF of 1509 bp which encodes 502 amino acid residues.Bionformatic analysis showed predicted molecular weight,isoeletric point value,predicted molecular formula of CYP2J protein was 57.98 kD,8.45 and C₂₆₄₅H₄₀₉₉N₇₁₃O₇₂₆S₁₅,respectively.The protein was predicted to be a stable hydrophilic protein,did not have a signal peptide but had a transmembrane segment?aa residues 12to 35?,was anchored to endoplasmic reticulum,and the secondary structure of CYP2J was mainly composed of alpha helix and random coil.Further phylogenic analysis displayed that the protein had the closest genetic relationship with those CYP2J of sheep and cattle,all of them had one FxxGxRxCxG/A which was a consensus heme-binding motif in Cytochrome P450 family.Prokaryotic expression results showed that efficient expression of CYP2J protein could be realized after induced with 0.5 mM IPTG in E.coil BL21 for 6 h at 37?.In conclusion,the complete c DNA sequence of Bactrian camel CYP2J gene was successfully obtained,the encoding protein sequences of ORF were analyzed by bioinformatics and the prokaryotic expression of its encoding protein was achieved in this study,which laid the good foundation for the antibody preparation of CYP2J gene of Bactrian camel and expression and distribution of CYP2J of Bactrian camel in protein level.