A Method For Purifying Major Royal Jelly Protein 1 Polyclonal Antibody And Primary Structure Characterization Of The Antibody

The main component that exerts an immunomodulatory function in royal jelly is protein,particularly major royal jelly protein 1?MRJP1?,which is a major glycoprotein.MRJP1 is considered to be the dominant protein in royal jelly allergy,and people who are allergic to royal jelly may have acute asthma,contact dermatitis,allergic reactions,and severe deaths.Studies have found that many of the physiological functions of glycoproteins are accomplished with the participation of glycan chains,and even the role of the glycan chain is more direct,more prominent,and affects the conformation of glycoproteins.In order to preserve the role of the glycan chain,this study is of great significance in the preparation of polyclonal antibodies using natural MRJP1 without excising the glycan chains.The main purpose of this thesis is to find a effective method to purify the specific polyclonal antibody IgG,and establish a method for analyzing the primary structure of the antibody by computer search and manualsearch.The experimental results provide basic data for the subsequent application of MRJP1 polyclonal antibody in therapeutic drugs and diagnostic reagents in vitro.The main findings are as follows:?1?Extraction of MRJP1 and preparation of rabbit polyclonal antibody:Three different solutions of MRJPs were obtained by ultracentrifugation.MRJP2 and MRJP3 were mainly present in UMRJPs.Three main proteins were observed in MMRJPs.MRJP1 was mainly in LMRJPs,accounting for about 92.95%.The immunization experiment of New Zealand rabbit was carried out using the isolated MRJP1 as an antigen.A serum with a titer of 1:56,000 was obtained.After purification of serum by protein A affinity chromatography,the 50 kDa heavy chain and the 25 kDa light chain were shown on the denaturing electrophoresis gel.Under non-denaturing conditions,there was only one band and the molecular weight was about 150kDa,which was in accordance with the antibody conditions.The IgG from MRJP1 polyclonal antibody was successfully obtained in the experiment.?2?Isoelectric focusing electrophoresis separates the polyclonal antibody and selects the best specific component:Ten different components were distinguished by isoelectric focusing electrophoresis,and then the ten different polyclonal antibodies were eluted by electroelution.The results of SDS-PAGE and Native-PAGE indicate that the electroelution method can successfully obtain different multi-resistance components from isoelectric dispensing.The ELISA method was used to verify the OD₄₅₀50 values of the ten components for different substrates?MRJP1,MRJP2 and MRJP3?.It was found that the response of Component 5 to MRJP1 was strong and the response to MRJP2 and MRJP3 was weak,so Component 5 was considered to be more specific for MRJP1.The results of the ELISA method were also verified by the Western Blot assay.Subsequent experiments will select Component 5.?3?The primary structure characterization of the antibody of Component 5:Double enzymes enzyme the antibody andfragment of the antibody is determined by LC-MS/MS.The analytical method of antibody characterization based on the Peaks Studio software and manual calculation is established.Comprehensive analysis can obtain the sequence of the heavy chain:LSLEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAPGEGLEYIGFIE SNRTTYYANWVD/N?+0.98?GRFTISG/K/S/QTSTTVDLKITSPTTED/N?+0.98?TATYFCA RGYKRAFDPWGP/EKFYGYDYWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTL GCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVA HPATNTKVDKTVAPSTCSKPTCPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEP VTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKT VAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTW YINNEQVRTARPPLREQQFNSTIRVVSTLPITHQDWLRGKEFKCKVHNKALPAPIEKTI SKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTT PAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK,a total of 542 amino acids.Comprehensive analysis can obtain the sequence of the light chain:ADL/NVVMTQTPASVSEPVGGTVTLKCQASQSLSSY/AALAWYQQKPGQPPKLLIYA/S/GASTLASGVP/SSRFEGSGSGTE/Q?+0.98?FTLTLSDVQECDAATYYCQAGYNSVDTN NV/NSVNTNNV/TWTNNNNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCV ANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYT CKVTQGTTSVVQSFNRGDC,a total of 216 amino acids.The resulting sequence did not find a consistent record on the Uniprot website and it belonged to the newly discovered sequence.