title: [Screening and identification of C7C peptide ligands of Mycobacterium tuberculosis].
abstract: OBJECTIVE
  To obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library, and preliminarily identify the binding capacity of the peptide to Mycobacterium.
  
  
  METHODS
  Inactive Mycobacterium tuberculosis reference strain H37Rv was used as the target molecule to screen the Ph. D.-C7C peptide library, and Mycobacterium bovis, BCG was used for reverse screening. After 4 rounds of affinity screening, single phages eluted by H37Rv and BCG were selected for DNA sequencing. ELISA was used to detect the binding affinities of different single phage clones. The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers. Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides, comparing with linear binding peptides obtained before.
  
  
  RESULTS
  After 4 rounds of biopanning, phages that could bind with target molecules were remarkable enriched. 16 common sequences were obtained by sequencing analysis of single phages. With ELISA, phage SB1, SB5, SB8 and SB26 all showed higher affinity with H37Rv and BCG, the ratio to negative control of which were ≥ 2.1, but could not bind to the 3 nonmycobacteria, which were identified as the positive clones. Based on the results of flow cytometer detection, the affinities to H37Rv of 4 cyclic peptides SB1, SB5, SB24, SB26 were (73.2 ± 6.3)%, (63.2 ± 5.3)%, (32.9 ± 3.1)%, (89.4 ± 7.0)%, and to BCG were (65.6 ± 6.1)%, (48.6 ± 4.5)%, (10.3 ± 1.8)%, (86.6 ± 7.9)%, separately, which were all higher than H8 ((4.0 ± 1.0)%, (5.5 ± 1.2)%) . From the results of fluorescence microscopy observation, all of the fluorescent labeled cyclic peptides SB1, SB5, SB24, SB26 could bind to H37Rv and showed higher fluorescence intensities, which also had certain affinities to other 18 mycobacteria, but the fluorescence intensities were lower than H37Rv, and didn't bind to 3 non-mycobacteria.
  
  
  CONCLUSIONS
  Based on the replacement of linear 7 peptide library with C7C peptide library, new ligands of Mycobacterium tuberculosis were achieved successfully, which showed significantly higher binding affinities to mycobacteria.
authors.affiliation: Shanghai Pulmonary Hospital Affiliated to Tongji University, Shanghai Key Laboratory of Tuberculosis, Shanghai 200433, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Hui
authors.initials: H
authors.lastname: Ma
authors.forename: Xiaochen
authors.initials: X
authors.lastname: Huang
authors.forename: Junmei
authors.initials: J
authors.lastname: Lu
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Email: shhuzy@163.com.
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Hu
mesh_terms: D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D008024:Ligands; D009169:Mycobacterium tuberculosis; D019151:Peptide Library; D010455:Peptides
chemical_list: D008024:Ligands; D019151:Peptide Library; D010455:Peptides