High Activity Expression And Functional Characteristics Of Neuropeptide Y In Larimichthys Crocea (Large Yellow Croaker)

As one of the important marine economic species in China,large yellow croaker?Larimichthys crocea?has suffered serious damage to its natural resources due to over fishing throughout the year,and its fishery resources need to be supplemented.Therefore,it is of great practical significance to study the regulation mechanism related to the growth of Pseudosciaena crocea.Based on the mammalian cell expression system,the high activity expression technology of neuropeptide Y?NPY?of Pseudosciaena crocea was developed,and the activity of the expression product was evaluated;the fusion expression vector of FLAG-LcNPY2R/7R?FLAG-Larimichthys crocea Neuropeptide Y2/Y7 Receptor?,LcNPY2R/7R-EGFP?Larimichthys crocea Neuropeptide Y2/Y7 Receptor-EGFP?was successfully constructed,The signal transduction mechanism mediated by Lc NPY2R/7R was studied.The eukaryotic expression system of large yellow croaker NPY was successfully constructed by inserting the fragment of Lc NPY mature peptide,LcNPY 18-36 truncate,and the combination of hexahistidine linked mature peptide and hexahistidine linked 18-36truncate into pcDNA3.1?+?eukaryotic expression vector.The synthesis peptide NPY was used as a control,and the activity of the peptide expressed in HEK293T cells was analyzed by Western blotting and internalization.The results showed that the expression product of pcDNA3.1-NPY recombinant expression vector had bio-activity,and the highest concentration of the expressed peptide could reach the level of 100 nM of the synthesized peptide LcNPY;four eukaryotic expression vectors of NPY could induce the endocytosis of LcNPY2R,which indicated that the constructed eukaryotic expression system produced peptides with high activity.The results show that LcNPY2R/7R can be activated by LcNPY,and LcNPY2R/7R can inhibit the cAMP accumulation caused by forskolin after being activated by LcNPY,which indicates that the activation of LcNPY2R/7R can couple G_?i protein;in addition,LcNPY2R/7R can cause significant calcium flow signal after being activated,and the G_?qq inhibitor FR900359 can inhibit the Ca²⁺flow caused by LcNPY2R/7R,and the data of calcium chelator indicate that the intracellular and extracellular Ca²⁺is involved in intracellular signaling,indicating that both receptors are activated to couple G_?i and G_?qq proteins simultaneously.The results also showed that LcNPY2R/7R could significantly mediate ERK1/2 phosphorylation after activation,and showed the dependence of Lc NPY ligand concentration and time gradient.The activation level was regulated by both intracellular and extracellular calcium ions.Endocytosis is an important way to desensitize GPCR.The endocytosis of LcNPY2R/7R is gradient dependent on the treatment time and concentration of LcNPY,and can be inhibited by Y2 receptor inhibitorsIn conclusion,this study successfully expressed the bioactive LcNPY through the construction of eukaryotic expression system by mammalian cell line,and the expression products are expected to form low-cost bioactive peptide biological products through further production process optimization.It is applied to the feeding and growth of large yellow croaker,and provides good technical support for the protection and breeding of germplasm resources of large yellow croaker.In this paper,we successfully constructed a high activity expression system of large yellow croaker NPY,and evaluated the high activity characteristics of the product through the verification of receptor activation characteristics;The signal transduction mechanism of NPY and its receptor in large yellow croaker was expanded.The change of cAMP and Ca²⁺,ERK1/2 phosphorylation and endocytosis mediated by LcNPY2R and LcNPY7R were discussed.It will support the further comprehensive study on the signal transduction pathway and physiological function of neuropeptide receptor of large yellow croaker.