Ph d 12tm phage display peptide library

The Ph.D.-12^TM Phage Display Peptide Library (New England Biolabs, MA, USA) was screened using CDR3δ peptides as follows: a 96-well plate was coated overnight with 150 μL/well of coating buffer (containing 10 μg of CDR3δ peptide) at 4°C and then blocked overnight with 0.1 M NaHCO₃ (pH 8.6) containing 5 mg/mL bovine serum albumin at 4°C. The primary library solution was added to the wells (10 μL per well containing 10¹¹ colony-forming units) and then shaken gently at room temperature for 30 min. After thoroughly washing with Tris-buffered saline (TBS) containing 0.1% Tween 20, the CDR3δ peptide-binding phages were eluted in acidic buffer [0.2 M glycine-HCl (pH 2.2)], followed by immediate neutralization with 1 M Tris-HCl (pH 9.1) or by competitive elution with a high concentration of CDR3δ peptide (100 μg/mL). The concentration of Tween 20 in the washing buffer was 0.1% in the first round and 0.5% in the subsequent two rounds of screening.

The Ph.D.-12^TM Phage Display Peptide Library and E. coli ER2738 were purchased from New England Biolabs (Beverly, MA, USA). The Eca109 cell line and human esophageal cancer tissue sections were obtained from the tissue library of the First Hospital of Jilin University (Changchun, China). Anti-M13 mouse monoclonal antibody was purchased from Amersham Biosciences (Piscataway, NJ, USA). Horseradish peroxidase (HRP)-labeled goat anti-mouse antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-mouse antibody, proteose peptone, and yeast extract were purchased from Shanghai Rui Qi Biological Technology Co., Ltd (Shanghai, China).

The Ph.D-12^TM Phage Display Peptide Library (New England Biolabs, Cambridge, MA, USA) was used to perform in vivo phage display in our rat model of bacterial OM. In this library the pIII protein displays linear 12mer peptides with a diversity of 2.9 × 10⁹ unique peptide sequences, at about 100 copies per sequence, in 10 μL of supplied phage. Tetracycline resistant E. coli ER2738 host bacteria were used for filamentous phage infection to allow for the blue/white screening using IPTG/X-gal plates for all the titration steps. The M13KE wild-type (WT) “empty” phage was used as a control for the determination of unspecific TM binding, ME internalization and phage transport. The WT phage is the M13KE phage in which the pIII gene does not code for a random linear 12mer peptide sequence.