ABSTRACT
The objective of this study was to investigate the effects of recombinant adiponectin on chicken liver cells. The full-length chicken adiponectin gene was amplified by PCR and cloned into the vector pET-32a, followed by the transformation of the vector into Escherichia coli BL21. SDS-PAGE was used to detect and analyze the purity of the expressed recombinant protein. Induction was performed with 1 mM IPTG at 30 °C for 3 h, and the recombinant thioredoxin–adiponectin fusion protein was purified using Ni-NTA affinity chromatography. Chicken adiponectin was successfully expressed and purified in a bacterial system. In addition, the chicken recombinant adiponectin demonstrated that it ameliorates palmitic acid- and oleic acid-induced adipogenesis, in which an increase in β-oxidation and a decrease in lipogenesis-related genes may be involved. In summary, chicken recombinant adiponectin enhances fatty acid metabolism in LMH cells.
chicken; fatty acid metabolism; recombinant protein
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The chicken adiponectin cDNA contains 735 bp, which translates into a protein that is 244 amino acids in length.
A) Expression of chicken recombinant adiponectin from pET32a-adiponectin in E. coli. Samples were eluted using buffers containing 50 mM imidazole (lane 1), 125 mM imidazole (lane 2), 250 mM imidazole (lane 3), and 500 mM imidazole (lane 4). B) The expression profile of recombinant adiponectin via non-reducing SDS-PAGE. Samples were eluted by buffers containing 125 mM imidazole (lane 1).
A) Representative images of Oil Red O staining; B) Quantification of Oil Red O staining.Con - control; OAPA - oleic acid and palmitic acid; Ad10 - chicken recombinant adiponectin, 10 μg/mL; Ad20 - chicken recombinant adiponectin, 20 μg/mL.Data were expressed as relative mean ± SEM (n = 5 in each group).Different letters indicate statistical differences.
A) SREBP1c; B) FAS; C) ACC; D) CPT1α; E) PPARα.Con - control; OAPA - oleic acid and palmitic acid; Ad10 - chicken recombinant adiponectin, 10 μg/mL; Ad20 - chicken recombinant adiponectin, 20 μg/mL.SREBP1c - sterol regulatory element-binding transcription factor 1c; FAS - fatty acid synthase; ACC - acetyl-CoA carboxylase; CPT1α - carnitine palmitoyltransferase 1α; PPARα - peroxisome proliferator-activated receptor α.The mRNA were determined using real-time PCR and normalized to β-actin.Data were expressed as the relative mean ± SEM (n = 5 in each group).Different letters indicate statistical differences.