Screening And Identification Of The Mimic Epitope Of The Adhesion Protein Of Mycoplasma Genitalium And The Observation Of Immunological Competence Of MAP

BackgroundMycoplasma genitalium (M. genitalium) is a newly defined pathogen of causessexually transmitted diseases. M. genitalium has been well described as a pathogenwith acute and chronic nongonococcal urethritis (NGU), and may cause genital tractdiseases in women, such as bacterial vaginosis cervicitis, pelvic inflammatory disease(PID), endometritis and infertility. And M. genitalium can cause conjunctivitis andpneumonia for newborn by the mother`s genital tract infection during childbirth. M.genitalium is one of the main pathogens cause opportunistic infection for AIDSpatients, and a synergistic factor of human immunodeficiency virus (HIV), thus it isknown as the AIDS-associated mycoplasma.So far, there is still not available vaccine to meet the clinical needs. Thus clinicaldoctors are badly in need of safe, effective vaccines to prevent M. genitalium infection.M. genitalium adhesion protein (MgPa) is the major adhesion of M. genitalium andit`s C-terminal part (amino acid1248～1364) is the most immunogenic region. ThusMgPa plays an important role in the diagnosis and prevention of M. genitalium. Thisstudy aim to screen the mimic epitopes of MgPa by phage display random peptidelibrary, and prepare the eight branches MAP containing the mimic epitopes, and studythe immunogenicity of MAP.ObjectivesTo express and purify the recombinant adhesion protein of M. genitalium (rMgPa)containing the dominant epitope (1075~1364aa), and prepare and purify the rabbit anti-rMgPa polyclonal antibody (pAb). A12-mer phage display peptide library wasscreened by using the purified pAb for target molecular in order to obtain theantigenic mimic epitopes of MgPa, and thus facilitate the understanding of theantigenic structure of MgPa. The multiple antigen peptides (MAP) containing themimic epitope were prepared, and the humoral and cellular immune response levelswere analyzed in MAP-immunized BALB/c mice, in order to provide the theoreticalbasis for the development of the safe, effective multi-epitope-baseded markervaccines to prevent M. genitalium infection.Methods（1）The recombinant prokaryotic expression plasmid was transformed into E.co1iRosettaTM2(DE3) to express the adhesion protein of M.genitalium. Therecombinant protein (rMgPa) was identified by ELISA, SDS-PAGE andWestern blot, and then purified by Ni-NAT immunoaffinity chromatography.The concentration of MgPa was analyzed by bicinchoninic acid method.（2） New Zealand Whites rabbits were immunized with the purified MgPa togenerate the corresponding polyclonal antibody. Polyclonal antibody wasinitially purified by saturated ammonium sulfate, and then affinitychromatography with CNBr-activated Sepharose4B coupled with recombinantprotein. The immune sera were characterized by ELISA and Western blotanalysis.（3）The purified pAb was used to target molecular to screen the immunodominantmimic epitopes of MgPa by using a random12-peptide phage display library.Phage clone were randomly selected and then the single chain DNA wereextracted and purified, DNA sequence analysis and computer-basedbioinformatics analysis were performed to define the consensus amino acidresidues of the mimotopes by MIMOX. The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed byELISA, competitive ELISA and Western blot analysis.（4）The eight branches MAP containing the screened mimic epitopes of MgPa wereprepared using poly-lysine as the core matrix. The purity of MAP was analyzedby reverse phase high performance liquid chromatography (RP-HPLC), andthen the molecular weights of MAP were characterizated by MassSpectrometry.（5）A total of30female BALB/c mice were randomly divided into5groups: GroupPBS, Group WP, Group AS, Group KH and Group mixed MAP. The miceswere inoculated intramuscularly with100g of MAP or100L of PBS attwo-week interval for four times. The sera of mice were collected and stored at-20℃in the day before every immunization or the day before execution. Themice spleen lymphocytes were separated for preparing the spleens cellssuspensions.（6）The specific IgG antibody and the subtype of IgG antibody in serum of theimmunized mice and. IFN-γ or IL-4levels in the cultured supernatant of spleenlymphocytes were detected by indirected ELISA. The proliferation responsesof the spleen lymphocyte were detected using MTT assay and delegated bystimulation index (SI).Results（1）The results of SDS-PAGE showed the recombinant protein containing6×His,with molecular weight of about37kD, was successfully expressed in E.co1iRosettaTM2(DE3). And the target protein in bacteria was mainly in the form ofinclusion bodies, the target protein with high purity were obtained by Ni-NATaffinity chromatography. The protein concentration reaches1160μg/mL byBCA method.（2）The corresponding polyclonal antibody were obtained by immunizing New Zealand rabbits with the purified and refolded rMgPa. The results of ELISAdemonstrated that the titer of pAb in serum is1∶25600. The results ofWestern blot showed that the polyclonal antibody has high specificity.SDS-PAGE analysis proved that the pAb in sera has high purity and themolecular weight of light chain of the prepared pAb was about25kD, and thatof heavy chain was approximately50kD, therefore, the molecular weight ofpAb about150kD.（3）After four rounds of biopanning to phage display random12peptide library, theyield ratios of the first and fourth round biopanning were1.45×10-6and9.25×10-4, respectively, which demonstrated the specific phages weresignificant enriched. The single-stranded DNA were successfully extractedfrom74phage clones and sequenced. The exogenous inserts from74phageclones distinguished45peptides sequences that can be divided into three groupsaccording to the different amino acid sequence. The results of bioinformaticsanalysis by MIMOX and comparative analysis for the45different peptidesequences revealed three different consistent core sequence wereP-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L andK-S-L-S-R-X-D-X-I.（4）Amongst45peptides, a total of36peptides were ELISA positive and theabsorbance values of23phage clones were higher than1.5,which demonstratedthe high reactivities with pAb. Competitive binding assay showed that thespecific binding between these phages and pAb could be partially inhibited bydifferent concentration of rMgPa, and the inhibitory effect was correspondinglyincreased when the concentration of rMgPa increased.23phage clones that theabsorbance values were higher than1.5could specifically bind with pAb.（5）The purity of three MAP were higher than90%by reverse phase highperformance liquid chromatography analysis. The results of MS analysis showed the molecular weight of MAP was basically consistent with theexpected theoretical molecular weight, which three MAP containing the mimicepitopes were successfully prepared.（6）The A450value of IgG antibody in the last immunized sera of group WP, AS,KHand mixed group were respectively0.935±0.028、0.640±0.022、0.841±0.103and1.326±0.025, which were significantly higher than that of PBS group(0.118±0.023)(p<0.01). And the A450value from mixed group was obviouslyhigher than those of group WP, AS and KH (p<0.05).（7）The titers of specific IgG antibody were1∶2560、1∶640,1∶2560, and1∶5120for group WP, AS, KH and mixed group, respectively. The IgG2a antibodywas mainly antibody in the mice sera of Group WP, AS, KH and mixed group,and the corresponding ratios IgG2a/IgG1were respectively1.835±0.125,1.708±0.180,1.690±0.202and2.095±0.179, which were significantly higherthan that of PBS group (0.967±0.142，p<0.01).（8）The contents of IL-4in the supernatant of the cultured spleen lymphocyte ofGroup WP, AS, KH and mixed group were respectively55.588±2.407,42.996±1.935,54.754±2.270and81.598±2.649pg/mL, which were higher thanthat of Group PBS（16.673±1.662）(p<0.01). And the IL-4level of mixed grouphas significantly difference from those of group WP, AS and KH (p＜0.01).（9）The stimulation indexs (SI) of the spleen lymphocyte from Group WP, AS, KHand mixed group were higher than that of Group PBS (1.107±0.032，p<0.01).And the SI of the mixed group was higher than those of other groups (p<0.01).（10）The contents of IFN-γ in the supernatant of the cultured spleen lymphocyte ofGroup WP, AS, KH and mixed group were significantly higher than that ofGroup PBS (31.476±1.717),(p<0.01). Meanwhile, the contents of IFN-γ fromthe mixed group was obviously higher than that those of other groups (p<0.01). Conclusion（1）The rMgPa with molecular weight of about37kD was successfully expressed inE.co1i RosettaTM2(DE3) and purified by by Ni-NAT affinity chromatography.（2）The rabbit anti-rMgPa pAb that had high titer and specificity was successfullyprepared and purified by affinity chromatography.（3）The mimic epitopes of MgPa were successfully screened and identified by phagedisplay peptide library and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent theimmunodominant mimic epitopes of MgPa.（4）The three corresponding MAP (WP, AS and KH) containing the three mimicepitopes were successfully prepared, identificated and purified.（5）The WP, AS and KH could induce strong specific cellular immune and humoralimmune response. And the competence of mixed group was stronger than thatof the Group WP, AS and KH.