an avidin-biotin conjugation technique for presenting target antigens on mycobacterium bovis bcg

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

Work in a biosafety cabinet using personal protective equipment because BCG is a level II pathogen.
To biotinylate the surface of BCG cells, first, grow the cells on a shaker platform to the proper density. Collect about 1 billion cells, and wash them three times using 500 microliters of ice-cold endotoxin-free PBST. Then, spin down the cells, and suspend them in sterile endotoxin-free PBS. Next, prepare fresh 10-millimolar Sulfo-NHS SS biotin in sterile filtered water. Then, incubate bacteria in 1 milliliter of broth containing 0.5 millimolar of Sulfo-NHS SS biotin. Perform the incubation at room temperature for 30 minutes.
Next, wash the now labeled bacteria three times with 500 microliters of ice-cold PBST, to remove the unreacted reagent. Then, resuspend the pellet in 1 milliliter of PBST.
Mix 500 million biotinylated BCGs with avidin fusion protein, for a final concentration of 10 micrograms of protein per milliliter. Let this reaction go for an hour at room temperature on a shaker. Next, wash the bacteria three times with 500 microliters of ice-cold PBST.

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1. Biotinylation of BCG Cell Surface
<ol>
	<li>Grow BCG in Middlebrook 7H9 broth with 10% OADC (oleic acid, albumin and dextrose solution) and 0.05% Tween 80 at 37 &#176;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.</li>
	<li>Wash 109 BCG 3 times with 500 &#181;L of ice-cold endotoxin free PBS plus 0.1% Tween80 (PBST) (pH 8.0). Suspend BCG pellet in sterile endotoxin free PBS.</li>
	<li>Immediately before use, prepare 1 mL of 10 mM Sulfo-NHS SS biotin in sterile filtered water.&#8203;
	<ol>
		<li>Incubate bacteria with 1 mL of 0.5 mM of Sulfo-NHS SS biotin at room temperature for 30 min.</li>
	</ol>
	</li>
	<li>Wash labeled bacteria 3 times with 500 &#181;L of iced cold PBST to remove non-reacted biotinylation reagent.
	<ol>
		<li>Re-suspend pellet in 1 mL of PBST.</li>
	</ol>
	</li>
</ol>
2. Binding of Monomeric Avidin-Fusion Protein to Biotinylated BCG Surface
<ol>
	<li>Mix 5 x 108 biotinylated BCG with Avi-protein (10 &#956;g/mL final in PBS-T) for 1 h at room temperature on shaker platform.</li>
	<li>Wash bacteria 3 times with 500 &#181;L of iced cold PBST.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Endotoxin-free RPMI 1640</td>
			<td>StemCell Technologies</td>
			<td>36750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS SS biotin</td>
			<td>Thermo Fisher</td>
			<td>21328</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 broth</td>
			<td>BD Diagnostic Systems</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>OADC</td>
			<td>BD Diagnostic Systems</td>
			<td>B11886</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Liao, T. A., et al., <a href="https://app.jove.com/v/56421">Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System.</a> 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.

1. Biotinylation of BCG Cell Surface

	Grow BCG in Middlebrook 7H9 broth with 10% OADC (oleic acid, albumin and dextrose solution) and 0.05% Tween 80 at ...

Begin with a suspension of Mycobacterium bovis BCG, treated with a non-ionic surfactant, to prevent bacterial clumping.
Next, add a biotinylation reagent and incubate.
This biotinylation reagent, a complex of biotin complexed to a functional linker, facilitates binding biotin molecules to bacterial surface proteins, labeling bacteria.
Post-incubation, centrifuge the biotin-labeled bacterial suspension to remove unreacted biotinylation reagent.
Resuspend the bacteria in a buffer. Add a solution of avidin-fusion protein, a recombinant protein comprising a target antigen domain fused to monomeric avidin.
During incubation, avidin binds to biotin, facilitating bacterial surface modification with target antigens.
Centrifuge the suspension to remove unbound fusion proteins.
Resuspend the antigen-coated bacteria in a buffer containing a non-ionic surfactant. The antigen coating on the bacteria can enhance its property to trigger immune responses.

20 Views. تقنية اقتران أفيدين والبيوتين لتقديم المستضدات المستهدفة على المتفطرة بوفيس BCG

Video: تقنية اقتران أفيدين والبيوتين لتقديم المستضدات المستهدفة على المتفطرة بوفيس BCG - Experiment

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

تحديد التعبير عن سطح الخلية ومعدل إندوسيتيك من البروتينات في الثقافات أستروسيت الابتدائية باستخدام بيوتينيلاتيون

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

JoVE Journal

Neuroscience

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

البكتيريا الببتيد العرض لاختيار الانزيمات بيوتينيلاتينج رواية

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...

Immunology and Infection

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

تحديد مناعة اللقاح باستخدام الخلايا المتغصنة المشتقة من وحيدات الأبقار

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

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

Transcript

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