Gene cloning,tissue expression and preparation of monoclonal antibody of Lateolabrax japonicus PPARγ

Peroxisome proliferator-activated receptors(PPARs)are a family of ligand-activated nuclear transcription factors that play pivotal roles in lipid and energy homeostasis.A cDNA of 1 688 bp encoding PPARγ was isolated from liver total RNA of Japanese Seaperch(Lateolabrax japonicus)using RT-PCR by degenerate primers based on sequence of other animals published on GenBank.The obtained 1 688 bp Seaperch PPARγ included 1 569 bp open reading frame encoding a protein of 522 amino acid residues with a theoretical pI of 6.06 and molecular weight of 59.02 ku.The blast analysis indicated that the deduced amino acid sequence of Seaperch PPARγ shared the highest identity of 93.1% with Dicentrarchus labrax,92.3% with Sparus aurata and 61.8% with Homo sapiens. RT-PCR analysis showed that the Seaperch PPARγ was ubiquitously expressed in 9 tissues tested,with the highest expression in liver,gill and lipid tissues,followed by spleen,brain,intestine and kidney,the lowest in heart and muscle.The cDNA sequence of PPARγ open reading frame was cloned into a prokaryotic expression vector pET-28a(+).The recombinant plasmid pET-28a-PPARγ1569 was transfected into Escherichia coli.BL21(DE3)pLysS and the PPARγ expression was induced at 30 ℃ by addition of 1 mmol/L isopropyl-β-d-thiogalactopyranoside.After 4 h induction with IPTG,the expressed recombinant protein with an apparent molecular mass of 66 ku was found by SDS-PAGE and confirmed by Western-blotting using an antibody specific to 6-His tag.The expressed PPARγ protein was insoluble and present in the inclusion bodies.These inclusion bodies were solubilized by 6 mol/L guanidine hydrochloride and purified on a Ni²⁺ affinity column(Ni²⁺ His-binding column).After purification,the recombinant PPARγ was used to immunize mice and the specific polyclonal antibody was obtained.Indirect ELISA(enzyme linked immunosorbent assay)was established to test the titer of the polyclonal antibody,the result was positive and the titer reached 16 000.Our research serves as a basis for further research into fish PPARγ’s biological characterization and function.