Observation on the immunological competence of multiple antigen peptides based on the mimic epitope of the adhesion protein of Mycoplasma genitalium / 中华微生物学和免疫学杂志

Objective To provide experimental evidence for the development of multi-epitope-baseded marker vaccines through investigating the humoral and cellular immune responses in BALB/c mice induced by the multiple antigen peptides (MAPs) with the mimic epitope.Methods Three types of MAPs in eight branched forms containing the mimic epitope of Mycoplasma genitalium adhesion protein (MgPa) were prepared using poly-lysine as the core matrix.The purity of MAPs was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).The molecular weights of MAPs were characterized by Mass Spectrometry.The BALB/c mice were immunized intramuscularly for four times with single or mixed MAPs.The specific IgG antibody and the subtype of IgG antibody in serum of the immunized mice were detected by indirect ELISA.The proliferative responses of the spleen lymphocytes were detected using MTT assay.The ELISA were used to detect IFN-γ and IL-4 levels in the cultured supematant of spleen lymphocytes.Results The three types of MAPs containing the mimic epitopes were successfully prepared with high purity.They,could stimulate mice to produce specific IgG antibodies,of which,the major antibody isotype was Th1 immune response-associated IgG2a.Compared with the single MAP immunization group,the mixed-MAPs immunized mice produced more IgG,IgG1 and IgG2a antibody (P＜0.05).Furthermore,these MAPs could enhance the specific proliferation of spleen lymphocytes in immunized mice and induce the production of IFN-γ and IL-4.The levels of IFN-γand IL-4 in mixed-MAPs group were significantly higher than those of the single MAPs group (P＜0.01).Conclusion The three types of MAPs could induce strong specific cellular and humoral immune responses.The immunological competence of the mixed-MAPs was stronger than those of the single MAP.

Screening and identification of antigenic mimics of the adhesion protein of Mycoplasma genitalium by phage display peptide library / 中华微生物学和免疫学杂志

ObjectiveTo screen a 12-mer phage display peptide library by the polyclonal antibody (pAb) against the recombinant adhesion protein of Mycoplasma genitalium (rMgPa) in order to obtain the antigenic mimic epitopes of MgPa.MethodsThe purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library.Seventy-four recombinant phage clones were randomly selected,and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX.The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA,competitive ELISA and Western blot analysis.Results After four rounds of biopanning,a significant enrichment of phages was achieved,the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences.Amongst 45 peptides,36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa.Immunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M.genitalium,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.Results of bioinformatics analysis by MIMOX demonstrated that S,A,F for cluster 1,A,K,I,T and L for cluster 2,K,S,L,R,D and I for cluster 3,may be the key consensus amino acid residues in the aligned mimotopes,respectively.ConclusionAntigenic mimics on MgPa were successfully identified 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 the immunodominant mimic epitopes of MgPa.And S,A,F K,I,T,L,R and D may be the key amino acid residues for the epitopes of MgPa.

Biopanning of HCV antigen epitopes from a random 12-peptide library by anti-hepatitis C virus polyclonal antibody / 中南大学学报(医学版)

Objective To analyze the epitopes of anti-hepatitis C virus(HCV)antibodies by peptide library biopanning. Methods Phage random peptide library of 12 amino acids was immunoscreened with purified IgG from the sera of hepatitis C patients. Positive clones which were obtained after 3 rounds of biopanning were detected by ELISA and 4 of them were sequenced. Results After 3 rounds of screening, the radio of output to input increased from (4.6×10~(-4))% to (5.3×10~(-2))%, meaning the enrichment was effective. At the third round of screening, all the selected clones proved to specifically react with the sera for immunoscreening. Four positive phage clones were sequenced, which shared a very conservative sequence and was named as C1. Its inserting sequence in the coat protein III was deduced to be GSMSPYVRWYTP, and the positive rate of C1 reacted with 20 cases of HCV patients was 85%.Conclusion The antigen-mimic peptide C1 is successfully screened from 12 random phage peptide library and it has some diagnostic value.

Screening of mimic epitope for MUC1 based on prediction of the antigenic epitope / 重庆医科大学学报

Objective:To predict the B cell epitope for MUC1 antigene and screen the mimic epitope of MUC1 from random phage dispay peptide library. Methods:In order to predict the B cell epitope for MUC1 antigen,the secondary structure and antigenicity was analysed with various methods. The purified Ma695 Ab was used to screen in phage random 12 peptide library. The positive clones were identified by sandwich ELISA and competitive inhibiton assay. Results:17 distinct antigenic epitope regions in MUC1 were identified by computation. 14 positive clones were acquired after 3 rounds of screening. Amino acid sequences deduced from DNA sequences showed four different sequences:KHYDPFHHRMPQ,QADTARSVALAG,VPSKPDLHVRSI and MTPIHYWNHNRV. The inhibitory assay showed that the 4 mimic epitope peptides displaying on the phage surface could effectively inhibit the combination of antibody with antigen and the inhibitory rates of each mimic epitope were 50% higher than that in the controls. Conclusion: Prediction of the B cell epitope for MUC1 can provide a basic clues for studies on structure and function of MUC1. The results indicated that KHYDPFHHRMPQ,QADTARSVALAG,VPSKPDLHVRSI and MTPIHYWNHNRV are the mimotopes which could mimic the epitope of MUC1.

Selection and prokaryotic expression of MUC1 mimic epitope / 第三军医大学学报

Objective To screen MUC1 antigen mimic epitope by phage display peptide library technology and to construct a recombinant plasmid expressing MUC1 antigen mimic epitope.Methods MUC1 mimic epitope was screened by Biopanning,DNA sequencing and amino acid sequence comparison.The gene was constructed into PET-31b(+) expression vector and expressed in Escherichia coli BL21(DE3) after transformation and induction by IPTG.The complete protein of the host bacteria was extracted for SDS-PAGE.The recombinant protein was purified by affinity chromatography on a Ni~(2+)-sepharose column and detected by Western blotting.Results The selected MUC1 mimic epitope could specifically combine with MUC1 monoclonal antibody.The recombinant expression vector PET-31b(+)-MUC1 was constructed successfully after the fusion protein was induced with IPTG.A specific protein band was shown on SDS-PAGE profile and detected by Western blotting.Conclusion An MUC1 mimic epitope was screened out.The epitope fusion protein was successfully expressed for the development of tumor vaccines targeting MUC1.