| [Objective]The mimic epitopes of Mycobacterium tuberculosis were screened from a phage display random peptide library using the purified mixed tuberculosis-positive serum as a target.The application value of phages and polypeptides in the serodiagnosis of tuberculosis were detected.[Methods]?1?The purification of mixed TB-positive serum was performed by saturated ammonium sulfate.The concentration of purified mixed serum was determined by the BCA protein assay kit.?2?The preliminary purified TB-positive serum was used as a target for 3 rounds of biopanning to screen the mimic epitopes of M.tuberculosis from phage display random 12 peptide library.Screened phages were collected and amplified.The single-stranded DNAs of the selected M13 phages were extracted using sodium iodide method forDNAsequencing.Protein-proteinBlastanalysis?http://blast.ncbi.nlm.nih.gov/Blast?was carried out to analyze the homologies between the two 12-mer peptides and M.tuberculosis antigen.?3?ELISA and dot immunobinding assay were used to verify binding affinityto positive phages with mixed tuberculosis-positive serum.?4?Indirect ELISA was measured to detectreactivities ofcore phages with TB-positive sera,TB-negative sera,M.pneumonia-positive sera andC.pneumonia-positive sera,andthe application value was identified in the serodiagnosis of tuberculosis.?5?12-mer peptides were synthesized usingsolid phase method and purified usingreverse phase highperformance liquid chromatography?RP-HPLC?.Indirect ELISA was used to detectreactivities of12-mer peptides with TB-positive sera,TB-negativesera,M.pneumonia-positiveseraandC.pneumonia-positive sera,andthe application value was identified in the serodiagnosis of tuberculosis.[Results]?1?TB positive serum protein with high puritywas obtained using saturated ammonium sulfate,and its concentration reaches1500 g/mL by the BCA protein assay kit.?2?3 rounds of biopanning were performed using the purified TB-positive serum was as target molecule for phage display random12 library.The yield of the first round to the third round of panning was 5.0×10-9,1.4×10-7 and 2.3×10-6,respectively,which demonstrated phage clones with specific binding activity were enriched significantly.The single-stranded DNAs of screened 37phages were extracted and sequenced,then the corresponding exogenous amino acid sequences were deduced based on the reverse complement sequence of the DNA sequence.37 polypeptides could be roughly divided into two groups due to the difference of the amino acids sequence,S-V-S-V-G-M-K-P-S-P-R-P?CS1?and T-M-G-F-T-A-P-R-F-P-H-Y?CS2?.CS1 and CS2 may be the mimic epitope of these antigens fromM.Tuberculosis based on the result of BLAST.?3?The results of ELISA demonstrated that the representative phages could bind to tuberculosis-positive sera,and dot immunobinding assay showed thatthere were clear spots for all representative phage clones.These results indicated that the positive phage clones could combine with TB-positive serum specifically.?4?The indirect ELISA assay demonstrated that2 core phagescould combine specificallywith serumfrom tuberculosis patient.Core phage1 and 2 achieved sensitivities of 78.57%and 72.62%,and specificities were 95.31%and 93.75%,respectively.?5?The purity of the two synthetic peptides is more than 99%,and themolecularweightsofthesyntheticpeptidesare basicallyconsistent with those predicted by Compute PI/MW tool.The indirect ELISA assay demonstrated that two synthetical12-mer peptidescould combine with serumfrom tuberculosis patient specifically.CS1 and CS2 achieved sensitivities of 89.41%and85.88%,and specificities were 90.63%and 87.50%,respectively.[Conclusion]?1?Two mimotopes of Mtb were successfully screened.?2?The corresponding two core phagesthat couldcombine specificallywithserumfrom tuberculosis patient showed higher sensitivity and specificity,and had the potential for the serodiagnosis of TB.?3?The corresponding two 12-mer peptides can combine specificallywith serumfrom tuberculosis patient showed the higher sensitivity and specificity,and had the potential for the serodiagnosis of TB. |
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