Expression And Purification Of Bovine C-Reactive Protein And Preparation Of Single Chain Antibody

C-reactive protein(CRP),one of the acute phase proteins,is widely distributed in vertebrates.Two different pentapeptide proteins were isolated from bovine serum by calcium-dependent affinity chromatography on high pyruvate agarose gel.One of the proteins cross-reacted with some rabbit anti-human CRP antisera,so it was named bovine CRP.CRP has "anti-inflammatory" and "pro-inflammatory" function showing its pleiotropy.CRP levels increase sharpl y with injury,infection and inflammation and decrease rapidl y with remission.The phage display technology is to insert the required gene fragments into the phages of filamentous phage coat protein III or filamentous phage coat Ⅷ,and display the antibody molecules on the surface of the phage through the processing and packaging of the phage itself.Then the specific antibodies were screened from the phage antibody library by the specific binding of antigens and antibodies.The specific physiological function of bovine CRP as an indicator of inflammation in bovine diseases,combined with phage display technology,can be used to obtain specific antibodies with high antigen binding capacity,which has a high potential for future diagnostic and therapeutic applications.In this project,we immunized Balb/c mice by expressing recombinant bovine CRP protein,obtained lymphocytes by grinding the spleen of mice,extracted total RNA and reverse transcribed into c DNA,performed three rounds of amplification,and then screened the CRP mouse-derived single chain antibody by phage display technology,through the above method,we pre-expressed a high concentration of bovine prokaryotic CRP protein,immunized mice and obtained high serum potency We also constructed a phage antibody library with a library capacity of 1.3×108,and finally screened two positive clones with different gene sequences and high binding activity to bovine prokaryotic CRP protein to induce their expression of murine-derived single-chain antibodies.The aim of this project is to provide a reliable basis for the diagnosis and treatment of bovine diseases with the obtained single-chain antibodies.Since the utilization value of bovine CRP is less studied at home and abroad,this experiment also provides a broad application prospect and is of great significance for the study of bovine CRP.