[Screening of the binding peptides of Mycobacterium tuberculosis H(37)Rv].

OBJECTIVE

To screen the Mycobacterium tuberculosis H(37)Rv binding peptide using phage-displayed random peptide libraries, and to analyze the binding capacity of the peptide with Mycobacterium tuberculosis.

METHODS

Inactive Mycobacterium tuberculosis H(37)Rv was used for screening of the binding peptide from the Ph.D.-7 peptide library, and Mycobacterium smegmatis was used for reverse screening during the 2(nd) to 4(th) rounds of screening. After 4 rounds of screening, single phages were randomly selected for DNA sequencing. The selected clones were tested by indirect enzyme linked immunosorbent assay (ELISA). The peptide of positive clone, which showed the highest affinity, was synthesized in vitro with fluorescent markers. The specific combination of the peptide with 16 mycobacterium standard strains and 3 other microbes (Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) were observed by fluorescence microscopy.

RESULTS

After 4 rounds of biopanning, remarkable enrichment of the phages that specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 5 sequences were obtained. Five phages with different sequences were detected using indirect ELISA and all of them were found to be positive clones. Phage 8 showed the highest affinity with target molecule. The peptide of phage H8 was synthesized in vitro with fluorescent markers, and it was confirmed that the peptide could bind with H(37)Rv and other 15 mycobacterium including Mycobacterium smegmatis, but not with 3 other microbes.

CONCLUSIONS

By using phage-displayed random peptide libraries, we obtained the binding peptide of H(37)Rv. It was shown that the peptide could bind with Mycobacterium tuberculosis specifically, which provided a new way for the detection of Mycobacterium tuberculosis in vitro.