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Intercommunity Epitopes Study of Most Economy Fish Serum Immunoglobulin

Author: ZhengHui
Tutor: LinTianLong
School: Fujian Agriculture and Forestry University
Course: Preventive Veterinary Medicine
Keywords: monoclonal antibodies monoclonal antibody purification epitope phagepeptide library immune
CLC: S941
Type: Master's thesis
Year: 2012
Downloads: 46
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Abstract


Antigen-specific immune response induced by the basic structural and functional units thoseare epitopes.Epitope-depth study will help us understand the structural and functionalcharacteristics of the protein antigen,it has an important role in the epitope vaccine design andimmunodetection.We had annlyzed26kinds of common economic fish serumimmunoglobulin in system such as Epinephelus awoara,Siniperca chuatsi,Pseudosciaenacrocea and so on.And filtered out the broad recognition spectrum of monoclonal antibodies.Inthis study, conducted in-depth study on this basis,we use phage display peptide library todetermine the monoclonal antibodies who have broad recognition abiliy for spectrum in orderto identify epitopes.It will lay a good foundation in the research and development for thebroad recognition spectrum of fish identified immunoglobulindetection reagents.First,recovering and subculturing the Monoclonal antibody cell lines of Epinephelusawoara,Siniperca chuatsi.then Inducing the mice to produce ascites.Ascites using differentmethods to purify,titer and measure its concentration.The ascites step-by-step salting out withsaturated ammonium sulfate,Further purification by IgM purification columns.Finding thepurity is significantly improved by SDS-PAGE after the further purification.The ascites ofSiniperca chuatsi only use protein A sepharose cl-4B affinity chromatography to purify.thetitre is0.982×10-8before purified,it increased by10times after purified.The results bySDS-PAGE showed that the unrelated protein is significantly reduced and the protein achieveshigh purity, which can conform with the requirements of the follow-up experiments.Secondly,panning the phage display peptide library using the purified ascites.After threerounds of panning,positive phage has effective enrichment.ELISA results show that after eachround of panning OD values than the one before has a significant increase.picking6-7Plaquesfrom The phage tablet of the three rounds to amplificate the positive phage.DNA wasextracted by using M13single-stranded phage DNA Extraction kit,then sequenced.sixsamples measured have the same sequence that is CTT CCT CTG CCT ATT AAGAAT.suggesting that their encoded amino acid sequence is LPLPIKN.Three of the sevensamples of Epinephelus awoara have the same sequence that is AGT TCT ACT ATT ATGGAG CGT,suggesting that their encoded amino acid sequence is SSTIMER.According to theChou-Fasman method,the common epitopes of the two monoclonal antibodies areconformational.The Subsequent competition experiments showed Phage display analogepitope can be identificated by monoclonal antibody.It is really the serum antibodycompetition epitope.Finally,Testing the monoclonal antibody of Epinephelus awoara is specific.Using BSAto immune Cyprinus carpio.collecting The venous blood of the tail after immunedrespectively at10,2030days.The ELISA assay results show that the immunohistochemicalfish can produce specific antibodies.Proving that the monoclonal antibody of Epinephelusawoara reacts with the serum immunoglobulin of Cyprinus carpio. the monoclonal antibodycan detect how the serum antibody level changes.In summary,the results of this study has important reference value for the development ofthe broad recognition spectrum of fish immunoglobulin detection reagents.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Fish Diseases
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