Secreted Expression Of Recombinated NPY Gene Of Dairy Cow In Pichia Pastoris And Biological Activities Detection

A feature of energy metabolization in perinatal cows is energy negative balance, resulting from energy intake decreasing but demand increasing. On one hand, energy negative balance will inevitablely lead to adipose mobilization, and the lipolysis promotes gluconeogenesis in the liver, in order to alleviate the energy shortage; on the other hand, a large amount of the non-esterified fatty acid (NEFA) will enter into the liver, most of which will oxidize to provide energe through the Krebs cycle and part of which will generate ketone bodies. Therefore, when the rate of NEFA intake exceeds its oxidation rate in liver, it will definitely enhance the generation of ketone bodies, causing or worsening Ketosis. The previous research in our lab indicated that neuropeptide Y (NPY) could improve feed intake and alleviate energy negative balance in perinatal cows; NPY could positively regulated the expression and activity of the key enzymes related to liver gluconeogenesis during the pathogenetic progresss of Ketosis.Total RNA was extracted from hypothalamus tissue of Holstein newborn calf, and RT-PCR was employed to clone the NPY mature peptide gene. Then the gene was added a FLAG tag to its N-terminal, and the tagged gene was inserted into a pPICZaA to construct the plasmid pPICZaA-FLAG-NPY. The plasmid pPICZaA-FLAG-NPY was transformed into Pichia pastoris strain GS115 by electroporation. The transformants were screened for multicopy recombinants using Zeocin, and induced with methanol. The expressed products were analyzed by Tricine-SDS-PAGE and Western blotting. Tricine-SDS-PAGE showed that Pichia pastoris cells integrating the plasmid pPICZaA-FLAG-NPY produced a high level of FLAG-NPY and the relative molecular mass of expressed protein was about 4.5kDa. The results of Western blotting immunogenicity showed that FLAG-NPY had strong antigen immune reactions with the M2 antibody against the FLAG tag. Those results indicated that FLAG-NPY protein was successfully expressed in Pichia pastoris. Primary bovine hepatocytes was cultured in vitro, and then real time fluorescence quantitative PCR and biochemical kits was applied to measure the effect of NPY on liver gluconeogenesis key enzyme by adding different concentrations of recombinant FLAG-NPY (0,50,100,200,500,1000 ng/ml). The result showed that the recombinant FLAG-NPY had stimulative effect on both the transcription level and the activity of PEPCK and PC.To sum up, the present study successfully cloned the FLAG-NPY fusion gene and successfully constructed eukaryotic expression plasmid pPICZaA-FLAG-NPY. The fusion gene was expressed in Pichia pastoris, and its biological activities were also investigated. The research established the experimental foundation for further research and provided a theoretical basis for the prevention and control of Ketosis in dairy cows.