| Royal jelly is the secretion from hypopharyngeal gland, palate gland and brain gland of5-15day workers. It not only controls caste determination, becoming worker or queen in development for honeybee larvae, but also has nutritional functions for human. Recently, the proteins and peptides from royal jelly have being attracted more attention, especially after Kimura’s new discovery that the main royal jelly protein1(MRJP-1) is the key factor of caste differentiation in honeybee. According to the domestic and foreign relevant reports, this thesis studied on the extraction, nutritional functions and application of MRJPs and MRJP-1. The main results were as follows:1) Extraction and separation of MRJPs and MRJP-1MRJPs powder was prepared with fresh royal jelly by centrifugation, dialysis and lyophilization. The weight of MRJPs was about7.34%of fresh royal jelly. SDS-PAGE analysis of MRJPs showed4protein bands, which might be77-87kDa MRJP-5,60-70kDa MRJP-3,57kDa MRJP-1and49kDa MRJP-2, successively, according to relevant reports.MRJP-1was prepared with fresh royal jelly by ultracentrifugation for two times. The content of MRJP-1was about3.21%of fresh royal jelly, and part of MRJP-1still remained in the supernatant, according to SDS-PAGE analysis. Therefore, the content of MRJP-1in royal jelly should be more than3.21%. At the same time, MRJP-1only showed1protein band with57kDa in SDS-PAGE profile.2) Preparation of specific polyclonal antibody of MRJP-1The specific polyclone antibody of MRJP-1was prepared with designed peptides as antigen. Firstly, the specific peptide from MRJP-1amino acid sequences which was differed from those of other MRJP members (MRJP-2-MRJP-9) was selected and synthesized chemically. Secondly, rabbits were immunized with MRJP-1peptide and their serum was collected. Thirdly, rabbit serum was purified by affinity chromatography and specific polyclonal antibody of MRJP-1was obtained (named R2).The Western blot analysis showed that R2possessed high specificity for MRJP1from MRJPs. Elisa analysis showed that R2has a higher antibody titer with1:20000for MRJP-1. As a contrast, the antibody of MRJP-1was prepared with MRJP-1expressed in the recombinant Escherichia coli.(named Rl) as antigen. Rl could recognize all MRJP members. Elisa analysis showed that Rl has titer with1:10000which is significantly lower than that of R2.3) Determination of MRJP-1content in royal jelly by ElisaThe content of MRJP-1in royal jelly was determined by Elisa. MRJP-1prepared previously was selected as antigen, and R2was selected as primary antibody. Optical density was measured at450nm and630nm. Firstly, the standard curve between optical density and concentration of MRJP-1was established. Afterwards, MRJPs was used as antigen, and optical density was measured by the same method. The results showed royal jelly contained3.85%of MRJP-1. As a contrast, the content measured with R2as primary antibody was higher than the content measured with Rl as primary antibody.As a quantitative analysis of the antigen-antibody, Elisa could reach a high sensitivity, due to its high catalytic efficiency of the enzyme. Moreover, the operation of this method was simple, convenient and low cost. It could be applied to identify the quality of royal jelly products by measuring the content of MRJP-1.4) Research on the prolification activity for culturing cells of MRJPs and MRJP-1The effect of MRJPs and MRJP-1in the absence of serum on cell growth was evaluated by cell morphology and density. According to relevant reports and condition of our laboratory, spc-A-1cells. sw480cells, sw620cells,293T cells, Hepli4cells, HCT-116cells and A2780cells were selected. The investigated results showed that MRJPs and MRJP-1could replace FBS to cells partly, but its activity was less effective than that of FBS. The cell survival rates of spc-A-1. sw480, sw620,293T and Hepli4cultured with MRJPs and MRJP-1were higher than that of control, but lower than that of FBS, significantly. The prolification activity for cell culture of MRJPs and MRJP-1for cells differed were different. For example, the cell survival rates of HCT-116and A2780cultured with MRJPs, MRJP-1and control had no significant difference. There was no significant different between MRJPs and MRJP-1, which suggested that MRJP-1was the key factor in MRJPs in terms of cell culture.This thesis provided the new knowledge firstly for detecting the quality of royal jelly with special antibody, and replacing partially FBS with MRJP-land MRJPs as cell growth factors to culture five cell strains at home and abroad. |
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