Name: expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system
Uploaded: 2018-01-31T13:00:00
Description: Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

Vi takker Dr. R Stokes for BCG Pasteur stamme og A. Talal for teknisk support. Vi takker også GenScript for hjælp med gen syntese.

Alle dyrene blev opretholdt i henhold til protokoller godkendt af Animal Care og brug udvalg på University of British Columbia. Forsøg blev godkendt af Animal Care og brug udvalg og udført efter den canadiske Rådet om dyrs pleje retningslinjer. Forsikring om dyrs velfærd antallet er A11-0247.
1. generation af monomere begærlighed Fusion proteiner udtrykker plasmider
<ol><li>Sub klon monomere begærlighed sekvens12 i pDEST17 plasmid mellem de "CTC" og "GAA" webst...

<ol>
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Vi rapporteret i denne undersøgelse en ikke-genetiske metode til hurtig og effektiv visning af eksogene proteiner på BCG overflade tilføje enten specifikke antigener eller specifikke funktionelle egenskaber forventes at effektivt forbedre den bakterie immunogenicitet. Vi viste, at BCG cellens overflade kunne være let biotinylated til øjeblikkelig overflade dekoration med begærlighed fusion proteiner. Den samlede procedure kan udføres inden for 2 timer, mens genetiske transformation og udvalg af positive kloner kr?...

Forskellige strategier er blevet foreslået at erstatte den nuværende TB vaccine BCG, herunder protein adjuverende systemer, viral vektoriserede teknologier, svækkede levende M.tb stammer og genetisk modificerede stammer, BCG, enten for at indføre gener over udtrykker BCG antigener, der ikke er tilstrækkeligt til udtryk under infektion1 eller Mtb-specifikke antigener ikke forekommer i BCG2. Genteknologi, står dog over for mange hindringer herunder den usikre niveau af sikkerhed, den tidskrævende proces og den lave effektivitet af udtryk vektorer4,5. Med hensyn til forbedring af BCG, en alternativ tilgang er nødvendig for at forbedre immunogenicitet uden behov for usikre genetiske vekslen.
I denne undersøgelse introducerer vi en roman strategi for visning af rekombinante proteiner af interesse på BCG cellens overflade, der er baseret på den velkendte høj-affinitet begærlighed interaktion med biotin. Denne tilgang giver mulighed for hurtig og reproducerbare fastgørelse af rekombinant begærlighed fusion proteiner på overfladen af biotinylated BCG, der letter bred manipulationer af BCG at opnå maksimal forbedring af dets effektivitet samtidig opretholde sin fremragende sikkerhed optage, observeret i løbet af årtier af brug.
Avidin affinitet for biotin er meget høj (Kd = 10−15 M) og når dannet, biotin-begærlighed komplekset er meget stabil og kan kun afbrydes under denatureringen betingelser6. Dog for denne type af interaktion til at tjene som en gene overførsel metode alternativ, er langsigtede men reversible visning af rekombinante proteiner nødvendige. Derfor indførte vi her en lav affinitet monomere begærlighed (Kd = 10−7 M), fører til reversibel udgivelsen af protein fra overfladen dekoreret BCG engang indtagne inde antigen præsentere celler. For at give en proof of concept, vi har testet denne metode bruger et monomere begærlighed kimære protein svarende til et surrogat antigen stammer fra ægalbumin (æg)7,8. Resultaterne viste, at BCG cellens overflade kan er være dekoreret nemt og hurtigt med monomere begærlighed fusion proteiner og at denne binding til BCG overflade er stabil og reproducerbar uden påviselige ændringer i bakteriel vækst og overlevelse. Også, vi fandt at BCG dekoreret med monomere begærlighed sammensmeltet med æg (AviOVA) kan fremkalde en svarer til induceret af BCG immunrespons genetisk udtrykker den samme antigen både in vitro- og in vivo. Denne teknologi af reversibel visning af proteiner af interesse på bakteriel overfladen er derfor en effektiv erstatning for traditionelle gen overførsel metoder og kan skabe en platform for bred manipulationer af BCG og yderligere programmer i vaccine udvikling.

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	<tbody>
		<tr height="21">
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			<td style="width:229px;">StemCell Technologies&#160;</td>
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			<td>Thermo Fisher&#160;</td>
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			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S3762</td>
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			<td height="26" style="height:26px;">Phycoerythrin (PE)-conjugated I-Ab-OVA323-339 tetramer&#160;</td>
			<td>MBL International&#160;</td>
			<td>TS-M710-1</td>
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			<td height="21" style="height:21px;">AF647 rat anti-mouse I-A/I-E</td>
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			<td height="21" style="height:21px;">PeCy7 rat anti-mouse CD4</td>
			<td>BD Bioscience&#160;</td>
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			<td height="21" style="height:21px;">PE rat anti-mouse CD8 Ab</td>
			<td>BD Bioscience&#160;</td>
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			<td height="24" style="height:24px;">AF 647 rat anti-mouse H-2kb&#160;</td>
			<td>BD Bioscience&#160;</td>
			<td align="right" style="width:119px;">562832</td>
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			<td style="width:229px;">Thermo Fisher</td>
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			<td height="21" style="height:21px;width:531px;">AF 647 rat anti-mouse CD4</td>
			<td style="width:229px;">BD Bioscience&#160;</td>
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			<td height="21" style="height:21px;">Ultra-small gold-conjugated goat anti-rabbit IgG&#160;</td>
			<td>Electron Microscopy Sciences&#160;</td>
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			<td>BD Diagnostic Systems</td>
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			<td height="21" style="height:21px;width:531px;">OADC</td>
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			<td>Invitrogen</td>
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			<td>GenScript</td>
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			<td>Invitrogen</td>
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			<td>Invitrogen</td>
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			<td height="21" style="height:21px;width:531px;">pDONR221 plasmid</td>
			<td style="width:229px;">Invitrogen</td>
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			<td height="21" style="height:21px;">Ni-NTA columns&#160;</td>
			<td>Qiagen</td>
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			<td>Thermo Fisher&#160;</td>
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			<td height="21" style="height:21px;">Flurosave</td>
			<td>Calbiochem-Novabiochem</td>
			<td align="right" style="width:119px;">345789</td>
			<td></td>
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			<td height="21" style="height:21px;">Axioplan II epifluorescence microscope</td>
			<td>Carl Zeiss Inc</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CCD digital camera&#160;</td>
			<td>Retiga EX, QImaging</td>
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			<td></td>
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			<td height="21" style="height:21px;">Tecnai G2 200kV electron microscope&#160;</td>
			<td>FEI Company</td>
			<td style="width:119px;">G2 200Kv&#160;</td>
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			<td height="21" style="height:21px;">70&#956;m Falcon cell strainer&#160;</td>
			<td style="width:229px;">Thermo Fisher&#160;</td>
			<td align="right" style="width:119px;">87712</td>
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			<td height="21" style="height:21px;">EasyStep mouse biotin positive selection kit&#160;</td>
			<td>StemCell</td>
			<td align="right" style="width:119px;">18556</td>
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			<td height="21" style="height:21px;width:531px;">biotin-Ter119/Ertyroid cells antibody</td>
			<td style="width:229px;">BioLegend</td>
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			<td height="21" style="height:21px;">Brefeldin A</td>
			<td>BD Pharmingen</td>
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expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system

Tuberkulose (TB) er en alvorlig infektionssygdom og kun tilgængelig vaccine M. bovis bacillus Calmette Guérin (BCG) er sikker og effektiv til beskyttelse mod børns svær TB meningitis og nogle former for formidlet TB, men undlader at beskytte mod pulmonal TB, som er den mest udbredte form for sygdommen. Lovende strategier til at forbedre BCG i øjeblikket stole enten på sin transformation med gener kodning immundominant M. tuberkulose (Mtb)-specifikke antigener og/eller komplementering med gener kodning Co-faktorer, som ville stimulere antigen præsentere celler. Store begrænsninger for disse strategier omfatter lav effektivitet, lave stabilitet og udtryk vektorer usikker sikkerhedsniveau. I denne undersøgelse præsenterer vi en alternativ tilgang til forbedring af vaccine, som består af BCG komplementering med eksogene proteiner af interesse på overfladen af bakterier, i stedet for transformation med plasmider kodning tilsvarende gener. Først, proteiner af interesse er udtrykt i fusion med monomere begærlighed i standard E. coli udtryk systemer og derefter bruges til at dekorere overfladen af biotinylated BCG. Dyreforsøg ved hjælp af BCG overflade dekoreret med surrogat ægalbumin antigen viser, at den modificerede bakterien er fuldt immunogen og i stand til at inducere specifikke T-celle svar. Alt støtte data præsenteres her kraftigt en roman og effektiv metode for omformningen den nuværende BCG-vaccine, der erstatter den møjsommelige konventionelle tilgang af komplementering med eksogene nukleinsyrer.

Med de generelle procedurer beskrevet ovenfor, blev det undersøgt, gennemførligheden af BCG overflade biotinylation og dekoration med surrogat antigen æg. Immunogenicitet af den modificerede BCG blev derefter testet in vivo. Bakteriel overfladen var let mærket med biotin til hurtig visning af begærlighed kimære antigener uden påviselige ændringer i bakteriel fænotyper. Den resulterende modificerede BCG er effektivt indtages af antigen præsentation celler og kan fremkald...

Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

En ny teknik til hurtig antigen visning på en bakteriel overflade præsenteres, som indebærer overflade biotinylation efterfulgt af udsættelse for proteiner af interesse i fusion med monomere begærlighed. Lastning BCG med udvalgte antigener med held forbedrer sin immunogenicitet, tyder på, at overflade dekoration kan erstatte traditionelle genetiske metoder.

Udtryk af eksogene antigener i Mycobacterium bovis BCG Vaccine via ikke-genetiske overflade dekoration med begærlighed-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Research

JoVE Journal

Immunology and Infection

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Dyrkning Mikroglia fra Neonatal og Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Fremstilling af rekombinant arenavirus for Vaccine Development i FDA-godkendte Vero celler

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Ekspression af funktionelle Rekombinant hæmagglutinin og neuraminidase Proteiner fra Novel H7N9 influenza virus ved hjælp baculovirusekspressionssystemet

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In vitro analyse af MyD88-medieret Cellular immunrespons til West Nile Virus mutantstamme Infektion

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃƒÅ½Ã‚ÂµRI

Udvikling af en<em&gt; In vitro</em&gt; Modelsystem til at studere interaktionen af<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE med sin højaffinitetsreceptoren FceRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-GuÃƒÆ’Ã†'Ãƒâ€šÃ‚Â©rin

En CFSE-baserede Assay til at studere den Migration af murine Skin dendritiske celler i drænende lymfeknuder Under Infektion med<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analyse af lymfocyt Ekstravasation Ved anvendelse af en<em&gt; In vitro</em&gt; Model af humant blod-hjernebarrieren

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Rensning af membran til kabinen for endoplasmatiske Reticulum-associerede nedbrydning af eksogene antigener i Cross-præsentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberkulose i mus med Reporter enzym fluorescens

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...