Preparation Of Monoclonal Antibodies Against Bursin And Identification Of Heptapeptide Mimotopes Of Bursin From Random Phage Display Library

Bursin, a tripeptide hormone that not only induces early B-cell differentiation, butalso promoted the generation of antibody and corresponding T-cell-differentiating wasisolated from extracts of bursa of Fabricius and was found to have the struciurelysyl-histidyl-gly-cine amide. But because of limited quantity available from poultrybursa of fabricius extraction and high cost of chemical synthesis, it is difficult thatbursin associated products widely used in livestock and poultry production. In thisstudy, we will produce high affinity bursin monoclonal antibody （MAb） and screenbursin mimic epitope from phage heptapeptide library to use as a substitute ofimmunological enhancement for the purpose of stock farming.1. Preparation of artificial antigenThere were several candidates amino group could be coupled with carrier proteinsto produce antibody. Although most of these antibodies could be used to constructimmunological detection methods, the biological activity of phage mimic epitopescreened by the mAb which were produced by different coupling methods may havesignificant difference. We applied two different antigen to couple the different aminoacid residues of bursin with carrier proteins.（1）The amino group on-Lys of bursin was conjugated to bovine serum albumin（BSA） or chicken ovalbumin （OVA） by EDC and SMCC method to produceBSA-KHG-NH₂or OVA-KHG-NH₂and BSA-S-KHG-NH₂respectively. We useBSA-KHG-NH₂immunized BALB/c mice to produce antibody. The results show thatthe antibody titer are up to1:102400and the multibody reaction with OVA-KHG-NH₂can be competitively inhibited by bursin and GKHG-NH₂but not KHGK peptide.Indicate the immuned mice have prduced specific andibodies against Bursin.Thestandard curve of indirect competitive ELISA assay constructed by the linearity rangeis between15ng/mL and1000ng/mL,50%competitive inhibition concentration ofbursin is292ng/mL.（2）The amino group on-Gly of bursin was directional conjugated to bovineserum albumin （BSA） or chicken ovalbumin （OVA） by EDC. Immunized BALB/cmice by used KHG-NH-BSA.Although the immunized mice have produced higherantibody titers, the multibody reaction with KHG-NH-OVA or KHG-NH-8MAP can’tbe competitively inhibited by bursin and GKHG-NH₂、KHGK peptide. It wasspeculated that the Immunized mice didn’t produced high titer specific antibodyagainst Bursin.2. The preparation of Bursin MAb（1）We screened18hybridoma cells that can stable secret anti-bursin antibodyfrom five mices immunized with BSA-KHG-NH₂after four times hybridization, theywere each named4A11、7B2、2D2、2A6、3E10、5D8、5F2、5F8、6D4、8C11、 11C4、11E9、12A5、12G10、14A9、15C10、15H5and17B2. The screened18strainsMab have directiones combined with antigen, strongly suggested that these monoclonalantibodies can’t bound to antigen if the structural epitope of bursin with the amino group on-Gly；Binding assay indicate that all the18MAbs have significant difference combinedwith Bursin.50%competitively inhibited by Bursin（IC50）test between in20.3～122.3ng/mL;Cross reactivity of18anti-Bursin Mab with GKHG-NH₂are between in22.8～387.6%,cross reactivity with KHGK are lower than4.3%.Their affinity testbetween in3.95×10⁶².39×10⁷, their ascites titer from1.28×10⁵to5.12×10⁵。Select2D2and11C4from18strains MAb for screened the mimic epitope from the phageheptapeptid.We constructed a indirect competitive ELISA method based on2D2mAband the detection limit line arity range is between7.8～250ng/mL, and50%competitive inhibitied concentration of bursin is25.6ng/mL, the regression equation isy=134.76-22.131×Ln（x） and the correlation index R²=0.9987. We tested the bursincontent of six bursa fabricius samples and the result revealed that per gramme tissuecontained50.59、78.53、89.25、47.70、92.54and94.39ng bursin respectively.（2） Immunized5BALB/c mice with KHG-NH-BSAand hybridized twice,andhave four times hybridization,but none hybridoma cell can be screened.3. Screening the mimic epitope and research of it’s biological activityTreat2D2MAb as ligand, screened the mimic epitope of bursin from the phageheptapeptide library by four times solid phase affinity elutriation method, finally wegot27positive phage clone. According to the assay of phage and bursin competitivebinding with2D2MAb, we found that phage No.40has the highest biological activity（IC50=60.1ng）,and it suit for the immune activity.We treated hybridoma cell2C6strain（P35MAb of anti-Swpv）with differentconcentration of bursin, GKHG-NH₂, KHGK, KHG and five phages with differentbiological activity. Our results indicate that5μg/mL of bursin led to a increase of35.4%antibody secretion, another peptides haven’t significant effect,and treated with1.5×10¹⁶pfu/mL of phage No.40result in antibody concentration increasing with26.6%but other peptides and phage do not have significant effect on antibodysecretion of hybridoma cell P35.Taken together, our data indicated that the positivephage clone has the analogous function of bursin and to be an efficient alternative ofbursin to display the immune enhancement activity.