An Avidin-Biotin Conjugation Technique for Presenting Target Antigens on Mycobacterium bovis BCG

Published: May 31, 2024

Abstract

Source: Liao, T. A., et al., Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System. J. Vis. Exp. (2018).

The video demonstrates a technique for loading antigens on Mycobacterium bovis BCG to improve its immunogenic properties. The method uses the avidin-biotin system to coat the bacterial surface with exogenous antigens.

Protocol

1. Biotinylation of BCG Cell Surface

  1. Grow BCG in Middlebrook 7H9 broth with 10% OADC (oleic acid, albumin and dextrose solution) and 0.05% Tween 80 at 37 °C on a shaker platform at 50 rpm until OD = 0.5-1.
    NOTE: BCG is a biosafety level 2 pathogen and all experiments concerning BCG should be done in a biosafety cabinet with proper personal protective equipment.
  2. Wash 109 BCG 3 times with 500 µL of ice-cold endotoxin free PBS plus 0.1% Tween80 (PBST) (pH 8.0). Suspend BCG pellet in sterile endotoxin free PBS.
  3. Immediately before use, prepare 1 mL of 10 mM Sulfo-NHS SS biotin in sterile filtered water.
    1. Incubate bacteria with 1 mL of 0.5 mM of Sulfo-NHS SS biotin at room temperature for 30 min.
  4. Wash labeled bacteria 3 times with 500 µL of iced cold PBST to remove non-reacted biotinylation reagent.
    1. Re-suspend pellet in 1 mL of PBST.

2. Binding of Monomeric Avidin-Fusion Protein to Biotinylated BCG Surface

  1. Mix 5 x 108 biotinylated BCG with Avi-protein (10 μg/mL final in PBS-T) for 1 h at room temperature on shaker platform.
  2. Wash bacteria 3 times with 500 µL of iced cold PBST.

Divulgaciones

The authors have nothing to disclose.

Materials

Endotoxin-free RPMI 1640 StemCell Technologies 36750
Sulfo-NHS SS biotin Thermo Fisher 21328
Middlebrook 7H9 broth BD Diagnostic Systems 271310
OADC BD Diagnostic Systems B11886
Tween 80 Sigma-Aldrich P1379

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Citar este artículo
An Avidin-Biotin Conjugation Technique for Presenting Target Antigens on Mycobacterium bovis BCG. J. Vis. Exp. (Pending Publication), e22273, doi: (2024).

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