Studies On The Phage Displayed Mimotopes Of A Protective Monoclonal Antibody (SSj14) Against Schistosoma Japonicum

Objective: To obtain peptide mimicking epitopes with monoclonal antibody ssj14 against Schistosoma japonicum using phage display technique and study their immuno-protection mechanism.Methods: We screened a phage random 12 peptide library using a protective monoclonal antibody ssj14-mediated biopanning and detected many positive phage clones with direct ELISA. Then 33 positive phage clones were randomly selected for their insert sequencing and 11 different clones sequences were obtained. The competitive ELISA assay and western-blotting were performed to analyse the above positive phage clones for their ssj14-mediated binding activity. According to the results, we further chosed three optimal candidate clones and the mixed positive clones(1x10~11 pfu/mice for each clone) with FCA adjuvant to immune BALB/c mices and tested the immuno- protective effects as well as the levels of IL-12 in vivo after challenge with Schistosoma japonicum cercaria. Synthesized peptide HNNSLPFFKLAT was evaluated for its potential immuno-protection of worms reduction and eggs reduction in immunized mice challeged with Schistosoma japonicum cercaria. The peptide conjugated with BSA immune BALB/c mices with 40ug of peptide mixed with FCA adjuvant or FCA plus of IL-12 (lug/mice) adjuvant. SEA-coated ELISA and synthesize peptide-coatedsed ELISA were estabilished to detect the positive and negative sera, then compared the results between the two method. Results: After 3 round of selection for biopanning, specific ssj14-binding phage clones were enriched efficiently; positive rate for the specifity in screened clones was 90%(30/33), and 11 of 33 clones were chosed for sequencing. According to the sequencing results, we got the following 11 sequences: HNNSLPFFKLAT ,AHRHPISFLSTL , HIKHVTPRSNLS , YQSQSFSTSLFD , NFMESLPRLGMH, TSKTLTDLFSYA , TMSNPITSLISV , EAVNRWLPVREL , QTIARHHLRPTV , KHYDPFHHRMPQ , AWTPWHVHKPSG, named P1-P11 respectively. The competitive ELISA assay and western-blotting showed the highest inhibition rate between SEA and antibody ssjl4 reached 67.03%. The positive clones of phage were all strongly recognized by the ssj 14 Mab in the western-blotting. All the results showed that the positive phage clones are immunogenic. The positive clones could induce significant reduction of adult worms in mice, the reduction rate induced by P11, P1, P2 and mixed group were 13. 84%, 23. 48%, 22. 69%, 52. 83%, respectively, as well as does in liver eggs per gram(LEPG) 47.23%, 48.56%, 34.17%, 65.47%, in eggs per gram 28.89%, 44.89%, 51.32%. Compared to the TBS control group, they could increase the level of IL-12 after the third immunization. The IL-12 level of Pll,PI,P2 and mixed group reach 20.43pg/ml, 25.75 pg/ml, 22.02 pg/ml, 30.34 pg/ml respectively and much higher than the control group.Two groups of peptide using either FCA adjuvant or FCA plus IL-12 adjuvant got 13.6%and 20.0% LEPG. The SEA-coated ELISA tested and distinguished all the positive and negative sera, compared to the peptide-coated ELISA which has 97.5 % specifity (39/40) .The two methods did not show significant difference.This study may provide the promising method looking for candidate vaccine of Schistosoma japonicum.