Detection Of CYP2J Gene Expression In Bactrian Camel Using RT-qPCR And The Construction Of Expression Vector In Vitro

In order to detect the CYP2J gene expression and distribution in different tissues of Bactrian camel and to provide possible clues for revealing the correlation of Bactrian camel’s biological properties of resistance to salt-sensitivity hypertension and CYP2J genes, the Bactrian camel CYP2J genes were detected in mRNA level by using Real-Time Quantitative PCR method in this research, and its in vitro expression vector was built for further studies of CYP2J gene expression in protein levels.Primers design accorded to the predict sequence of bactrian camel CYP2J logined in the GenBank（XM₀06187584）. ACTB was seclected as house-keeping gene. SYBR Green I method was used in Real Time PCR amplication, and the 2-ΔΔCT relative quantitative method was used in data processing. All methods mentioned above were used in detection of bactrian camel CYP2J expression and distribution in eight kinds of tissues:liver, heart, spleen, lung, kidney, intestine, blood vessels, pancreas and sheep livers at gene level. The results indicated that the heart is the main tissue where CYP2J express. And the level of CYP2J expression in camel hearts was significantly higher than sheep livers, and kidney. Except for hearts, levels of CYP2J expression in livers, small intestines and pancreas was reduced in turn. As for the spleens, lungs and blood vessels, It’s very low.In this study, RT-PCR was applied to the amplification of the 1509 bp complete CDs area of bactrian camel CYP2J mRNA which was used in TA-cloning after gel-recycling purification. After which, the CYP2J gene was connected with pMD18-T vector and transferred into E.coli DH5α cells. Cultivation for multiplication after the verification of double enzymes digest with HindⅢ and KpnⅠ, and plasmid PCR. The recombined vector pMD1-T-CYP2J and was digested by double enzymes mentioned above. And CYP2J was connected with pET-32a digested by the same enzymes. The new recombined vector was transferred into DH5α cells for the identification of positive clones. Double enzymes digest with HindⅢ and Kpnl and plasmid PCR were used to the verification of successful recombined pET-32a-CYP2J vectors. The recombined plasmids pMD18-T-CYP2J and pET-a-32-CYP2J double enzyme digest electrophoresis diagram showed specific purpose bands at about 1500 bp represent CYP2J and the 2700 bp and 5900 bp bands respectively represent two plasmids; Electrophoresis diagrams of PCR amplification products with the templates of 2 kinds of recombined plasmids are demonstrated at about 1500 bp specific bands of CYP2J. And the similarity of comparation between sequencing results and Genbank is 99%. The results above proved that the bactrian camel CYP2J prokaryotic expression vector named pET-32a-CYP2J had been successfully built.Accordingly, this study successfully detected the expression and distribution of bactrian camel CYP2J within the 8 main types tissues at the RNA level, and successfully constructed CYP2J’s prokaryotic expression vector.All results above had laid the foundation for the subsequent studies at protein level.