Screening and identification of the polypeptides specifically binding to the adhesion protein of Myco-plasma genitalium / 中华微生物学和免疫学杂志

Objective To screen and identify the polypeptides specifically binding to the adhesion protein of Mycoplasma genitalium(MgPa) by using the Ph. D.-12TM phage display peptide library for further understanding the biological function and the possible pathogenic mechanism of the MgPa. Methods The Ph. D.-12TM phage display peptide library was used for 3 rounds of biopanning with the purified recombinant MgPa ( rMgPa) as the given target. The phages were collected for amplification after biopanning. The single strand DNA of phage clones were extracted and purified by using the sodium iodide method for further se-quencing. ELISA, competitive binding assay and dot immunobinding assay were performed to analyze the specific binding of positive phages to rMgPa. Results A significant enrichment of phages was achieved after 3 rounds of biopanning. Eleven different phage exogenous sequences (P1-P11) were detected among the 38 phages randomly selected from the agar. Two core sequences were deduced according to the repeating times of amino acids among the 11 polypeptide sequences, which were V-H-W-D-F-R-Q-W-W-Q-P-S and D-W-S-S-W-V-H/Y-R-D-P-Q-T/S. Ten out of the 11 representative phages ( P1-P10 ) specifically combined with the rMgPa. Conclusion Two polypeptides specifically binding to rMgPa were successfully screened out, which provided the tool for further investigation on the biological function of MgPa and the pathogenic mecha-nism of Mycoplasma genitalium.

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.

Expression and immunological characterization of the major epitope of P1 adhesin protein of Mycoplasma pneumoniae and its clinical application research / 中华微生物学和免疫学杂志

Objective To study the application of P1 adhesin protein epitopes in diagnosis of Mycoplasma pneumoniae(Mp) infected patient. Methods The major epitope(P1-534) of P1 adhesin protein were predicted by ProPred and ANTIGENIC according to its primary structure. The high value fragment was cloned into a constructed recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21(DE3) and the fusion protein was identified by Western blot. BALB/c mice were immunized with purified protein to test its immunogenicity. Then the purified protein was used as antigen to test the serum of Mp infected patient by ELISA, and compared with the Mp whole cell antigen. Results The P1-534 protein was successfully expressed and purified. ELISA data showed that P1-534 protein could elicit high levels of IgG in immunized mice, the sensitivity and specificity of P1-534 were determined to be 85.00% and 97.67%, while the Mp whole cell antigen were 72.50% and 74.42%. Conclusion The results conformed that the recombinant epitope has certain immunogenicity,and its sensitivity and specificity are better than Mp whole cell antigen. P1-534 protein can be used as an antigen for immunodiagnosis of Mp infection.