Abstract:
Objective The prokaryotic expression of peroxisome proliferator-activated receptor δ (PPARδ) in Nile tilapia was determined for the preparation of a polyclonal antibody against the receptor protein.
Method The bioinformatics analysis on PPARδ was conducted to arrive at a primer designed for amplification. The amplified products were subcloned into expression vector pET-B2m to construct recombinant plasmid for transformation into E. coli B21 with IPTG induction. The expressed protein was identified by SDS-PAGE and purified with a Ni-NTA prior to immunization on Japanese white rabbits. A polyclonal antibody with its titer determined by ELISA and specificity analyzed by the western blotting was obtained.
Result The prokaryotic expression vector pET-B2m-PPARδ was successfully constructed. The recombinant protein with molecular weight of 90 KD was expressed in both inclusion body and soluble proteins. The titer of the rabbit anti-PPARδ polyclonal antibody was 1 ∶ 2 048 000. The western blot confirmed that the antibody reacted specifically with PPARδ protein.
Conclusion This study successfully expressed and purified the PPARδ recombinant protein of Nile tilapia, and subsequently, obtained the polyclonal antibodies with high specificity and efficacy that could be used for further research on PPARδ in Nile tilapia.
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