Screening Specific B-cell Epitopes Of Trichomonas Vaginalis By Phage Random Peptide Library

Backgrounds and ObjectivesTrichomonas vaginalis (T. vaginalis), is a kind of flagellate which can parasites in the urogenital tract of human, Trichomonas vaginalis causes Trichomonosis, the most common sexually transmitted infection in humans, According to the WHO, the number of people suffering from trichomonosis in the world per year is approximately180million, this infection also predisposes individuals to infection of HIV and Mycoplasma. Currently, the rapid diagnoses for Trichomonosis is limited to the sensitivity and specificity, the research and development for T. vaginalis vaccine is lagging behind. In this study, we expect to biopanning specific B-cell epitope of T. vaginalis from the Ph.D.-7with the methods and techniques for molecular biology, immunology and bioinformatics, then detected the immunogenicity, sensitivity and specificity of the biopanning results, the purpose of this study is to provide a theoretical basis for the rapid diagnosis and the vaccine for T. vaginalis.Materials and Methods1. T. vaginalis clinical isolates (collected from the clinical laboratory of the Fifth Affiliated Hospital of Zhengzhou University) were inoculated into TYM medium and maintained as axenic cultures, then make the whole-parasite antigen and cryopreserved T. vaginalis.2. Immunized rabbits (Purchased from Experimental Animal Center of Henan Province) fortnightly by subcutaneous the whole-parasite antigen, detected the antibody titer by ELISA after the third subcutaneous, collected, separated and purified the serum when the antibody titer meet the requirements, then detected the immunogenicity by Western-Blot and IFA (immunofluorescence assay).3. Use the purified rabbit anti-T. vaginalis IgG for three rounds biopanning by the Ph.D.-7(Purchased from NEB inc, USA), detected the biopanning results by phage ELISA and sequenced the positive phage clones, sequence alignment for the sequencing result with the known sequence of T. vaginalis’s protein form GenBank, then make the bioinformatics analysis from Protscale server.4. Immunized BALB/c mice (Purchased from Experimental Animal Center of Henan Province) weekly by subcutaneous the selection positive phage clones, establish the positive control, negative control and solvent control at the same time. Collected and separated serum after the third subcutaneous, then detected the immunogenicity by Western-Blot and ELISA and detected the parasites location of the positive phage display peptides by IFA.5. Established the T. vaginalis mouse model by subcutaneous infection, observed the abscess formation and the number of live parasites in pus daily. Use the infection serum to detected the sensitivity and specificity of the positive phage clones by ELISA.6. Detected the cytokine level of IL-4and IFN-γ from the mice which were immunized with the positive epitope peptides by ELISA.Results1. T. vaginalis reached the highest growth density at about48h and its logarithmic phase is between36～48h, T. vaginalis reached pure culture after three generations. The SDS-PAGE result showed that the protein bands distribution of the whole-parasite antigen is between10-150kDa and the most densely bands are between30-70kDa.2. ELISA results showed that the whole-parasite antigen can cause a strong immunoreaction after immunized rabbits, the titer of antibody is greater than1:106. Western-Blot result showed that a total of15protein of the whole-parasite antigen can recognized by the serum that is three bands more than the result of mouse serum. The immunofluorescence result showed that the immunoreaction between the whole-parasite antigen and the serum can lead to red fluorescence.3. We obtained14positive phage clones after three rounds biopanning. There were no similarity between P1-P14and the known sequence of T. vaginalis’s protein form GenBank on the primary structure. The results of phage ELISA and the bioinformatics analysis showed that the immunogenicity of P3, P5and P11are stronger than the others.4. ELISA results showed that P3, P5and P11can cause strong immunoreactions after immunized BALB/c mise, differences are statistically (P<0.05). Western-Blot result showed that the serum of phage clone P11can specifically recognized a protein band (42kDa) of the whole-parasite antigen. The immunofluorescence result showed that the immunoreactions between P3, P5and T. vaginalis were weaker than P11because of the green fluorescence were weak in P3and P5but P11had a bright green fluorescence on the membrance of T. vaginalis.5. Subcutaneous live parasites to mice lead to abscess formation and live parasites can be detected in the pus within10d. ELISA results showed that P3, P5and P11have high specificity (P<0.05) and their sensitivity were1μg/ml,2μg/ml,3μg/ml respectively.6. The cytokine ELISA results showed that at one week after the last immunization, the cytokine level of IL-4and IFN-y from P3、P5and P11groups were higher than M13and PBS groups (P<0.05), the cytokine level of IL-4was significantly elevated, there was no significant differen of cecytokine level between P3、P5and P11(P>0.05) Conclusions1. The whole-parasite antigen of T. vaginalis has a good immunogenicity and its serum has a high antibody titer.2. There were14simulation B-cell epitopes had biopanning by the ues of the Ph.D.-7. Among these simulation epitopes, P3、P5and P11have good immunogenicity, sensitivity and specificity, the results suggested that these simulation epitopes may have potential applications in the aspect of specific diagnostic reagents.3. The immunized from P3、P5and P11to mice could induce humoral immunity and cellular immunity and humoral immunity is dominant.