Isolation,Purification And Immunomodulatory Activity Of Polypeptides From Bee Pupae

Bee pupae is the larvae and pupae of bees,which are rich in nutrients,including protein,fat and trace elements.Bee pupa protein has biological activities such as anti-oxidation,immune regulation and anti-tumor.However,at present,only simple processes were used in bee pupae products in China,most of the resources of bee pupa were not effectively exploited,resulting in waste of bee pupa resources.Only a little had been reported on the preparation,purification and biological activities of bee pupae polypeptides.The preliminary research on the bee pupa polypeptide had been conducted in our research group,and the results indicated that the purified bee pupa polypeptide component had strong antioxidant activities with values for further study.Based on the preliminary work,bee pupa polypeptide was prepared by enzymatically hydrolyzing with alkaline protease from bee pupa protein,and purified by DEAE-Sepharose Fast Flow ion chromatography and Sephadex G-25 gel column chromatography.Two polypeptides purification components BPP-21 and BPP-22 were obtained and characterized preliminarily.The immunoregulatory activity and mechanism of the purified bee pupae polypeptide components in vitro were studied by mouse macrophage cell line RAW264.7.A mouse model of immunosuppression induced by cyclophosphamide was used to study the immunomodulatory activity of the purified components of bee pupa polypeptide in vivo.The main research contents are listed below:(1)Preparation,purification and preliminary characterization of bee pupa polypeptide were conducted.Alkaline protease was used to hydrolyze bee pupa protein to obtain bee pupa polypeptide by enzymatic hydrolysis,and four purified components(BPP-1,BPP-2,BPP-3 and BPP-4)of bee pupa polypeptide were obtained after purifying by DEAE-Sepharose Fast Flow ion chromatography column,the yields of four purified components were 2.3%,6.8%,3.5% and 3.2%,respectively.Sephadex G-25 gel column was used to further purify the higher yield component(BPP-2),and two purified components BPP-21 and BPP-22 were obtained with yields of 38.43% and 45.62%,respectively.Tricine-SDS-PAGE gel electrophoresis analysisshowed that BPP-21 and BPP-22 were homogeneous with molecular weights around1000 Da.Amino acid analysis showed that BPP-21 and BPP-22 contained 16 and 14 amino acids,among which including 7 and 6 essential amino acids,respectively.The results indicated that among the essential amino acids,Lysine was highest in BPP-21 and BPP-22,and had the high nutritional value.(2)The immunomodulatory effects and mechanisms of the purified bee pupae polypeptide components BPP-21 and BPP-22 were studied in RAW264.7macrophages in vitro.The results showed that BPP-21 and BPP-22 had no significant inhibitory activity on RAW264.7 cells within the concentrations ranged from 12.5 to200 μg/mL.BPP-21 and BPP-22 significantly stimulated IL-2、TNF-α、IFN-γ and NO productions and increased the mRNA expressions of IL-2、TNF-α、IFN-γ and iNOS.BPP-21 and BPP-22 could stimulate macrophages to produce reactive oxygen species(ROS),increased the activity of superoxide dismutase(SOD)in macrophages in varying degrees,and had no obvious oxidative damage to cells.Studies on immunomodulatory mechanisms indicated that BPP-21 and BPP-22 could up-regulate the phosphorylation levels of extracellular regulatory protein kinase(ERK)and p38,promoting the expression of nuclear transcription factors EIK-1,CREB and MEF2,and thus enhancing the immune activity of RAW264.7 cells.(3)The immunosuppressive mouse model induced by cyclophosphamide(CTX)was used to study the immunomodulatory effects of the purified bee pupae polypeptide component BPP-22(25,50 and 100 mg/kg)on immunosuppressed mice.The results showed that the high-dose group of BPP-22 could significantly relieve the weight loss trend of mice caused by CTX and increase the growth rate of mouse body weight.The middle and high-dose groups could significantly improve the spleen and thymus index of immunosuppressed mice,significantly improve the phagocytic function of mononuclear-macrophages of immunosuppressed mice,and promote the secretion of cytokines(IL-2,IFN-γ)and serum immunoglobulin(IgA,IgM and IgG)in immunosuppressed mice.In the high-dose group,BPP-22 could significantly increase the ear swelling,the number of white blood cells,lymphocytes and red blood cells in immunosuppressed mice.These results suggested that BPP-22 could enhancethe immune activity of CTX-induced immunosuppressed mice.