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 belastningen og A. Talal kundestøtte. Vi har også takke GenScript for hjelp med Gen syntese.

Alle dyrene ble opprettholdt i henhold til protokoller godkjent av Animal Care og bruk komiteer ved University of British Columbia. Eksperimenter ble godkjent av Animal Care og bruk komiteer og utført i henhold til kanadiske Rådet for dyr omsorg retningslinjer. Velferd er dyr forsikring A11-0247.
1. generasjon monomerisk Avidin Fusion proteiner uttrykke plasmider
<ol><li>Sub klone monomerisk avidin sekvens12 i pDEST17 plasmider mellom "CTC" og "GAA" områder som til...

<ol>
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Vi rapporterte i denne studien en ikke-genetiske tilnærming for rask og effektiv visning av eksogene proteiner på BCG overflaten til bestemte antigener eller bestemte funksjonelle egenskaper forventes å effektivt forbedre den bakterien immunogenisitet. Vi viste at celleoverflaten BCG kan være lett biotinylated for øyeblikkelig landtransport dekorasjon med avidin fusion proteiner. Totalt prosedyren kan utføres innen 2 timer, mens genetisk transformasjon og utvalg av positiv kloner krever 2 til 3 måneder av tidsfors...

Ulike strategier har vært foreslått å erstatte gjeldende TB vaksinen BCG, inkludert protein adjuvant systemer, viral vectored teknologier, dempes live M.tb stammer og genmodifiserte BCG stammer, enten for å innføre gener over uttrykke BCG antigener som ikke er tilstrekkelig uttrykt under infeksjon1 eller Mtb-spesifikke antigener ikke finnes i BCG2. Genteknologi, men står overfor mange barrierer inkludert usikker sikkerhet, den tidkrevende prosessen og lav effektivitet av uttrykket vektorer4,5. Når det gjelder å forbedre BCG, er alternativ metode nødvendig for å forbedre immunogenisitet uten behov for usikker genetisk alternations.
I denne studien introduserer vi en ny strategi for visning av rekombinante proteinene rundt på BCG cellens overflate som er basert på den velkjente høy affinity avidin interaksjonen med biotin. Denne fremgangsmåten kan rask og reproduserbar vedlegg av rekombinant avidin fusion protein på overflaten av biotinylated BCG, som muliggjør bred manipulasjoner av BCG å oppnå maksimal forbedring av effekt samtidig opprettholde sin utmerkede sikkerhet registrere, observert over tiår med bruk.
Avidin affinitet for biotin er ekstremt høy (Kd = 10−15 M) og når dannet, biotin-avidin komplekset er meget stabil og kan bare bli forstyrret under denaturing forhold6. Men for denne type samhandling som et gen overføring metoden alternativ, er langsiktig men reversibel visning av rekombinante proteinene nødvendig. Derfor introduserte vi her en lav affinitet monomerisk avidin (Kd = 10−7 M) som fører til reversibel utgivelsen av protein fra overflaten dekorert BCG når inntatt i antigen presentere celler. For å gi et bevis på konseptet, testet vi denne metoden bruker en monomerisk avidin chimeric protein tilsvarer en surrogat antigen avledet fra ovalbumin (OVA)7,8. Resultatene viste at celleoverflaten BCG kan være enkelt og raskt dekorert med monomerisk avidin fusion proteiner og at denne bindingen til BCG overflaten er stabil og reproduserbar uten synlig endringer i bakterievekst og overlevelse. Også vi fant at BCG dekorert med monomerisk avidin smeltet sammen med OVA (AviOVA) kan indusere en immunrespons lik som indusert av BCG genetisk uttrykke samme antigen både i vitro og i vivo. Denne teknologien av reversibel proteiner av interesse på bakteriell overflaten er derfor en effektiv erstatning av tradisjonelle genet overføring og gir en plattform for bred manipulasjoner av BCG og videre søknader i vaksine utvikling.

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			<td>Carl Zeiss Inc</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 alvorlige infeksjonssykdommer og kun tilgjengelig vaksine M. bovis bacillus Calmette-Guérin (BCG) er trygg og effektiv beskyttelse mot barn alvorlig TB hjernehinnebetennelse og noen former for spres TB, men ikke klarer å beskytte mot lunge TB, som er den mest utbredte sykdommen. Lovende strategier for å forbedre BCG nå stole på transformasjonen med gener som koder immunodominant M. tuberkulose (Mtb)-spesifikke antigener og/eller complementation med gener co-faktorer som vil stimulere antigen-koding presentere celler. Store begrensninger på disse inkluderer lav effektivitet, lav stabilitet og usikker nivået av sikkerhet for uttrykket vektorer. I denne studien presenterer vi en alternativ tilnærming til vaksine forbedring, som består av BCG complementation med ytre proteiner rundt på overflaten av bakterier, i stedet for transformasjon med Plasmidene koding tilsvarende gener. Først proteiner av interesse uttrykt i fusion med monomerisk avidin i standard E. coli uttrykk systemer og deretter brukes til å dekorere overflaten av biotinylated BCG. Dyreforsøk bruker BCG overflate dekorert med surrogat ovalbumin antigen viser at endrede bakterien er fullt immunogenic og kan indusere bestemt T celle svar. Dataene som presenteres her sterkt støtte helt, en roman og effektiv metode for omforme den gjeldende BCG-vaksinen som erstatter arbeidskrevende konvensjonell tilnærming til complementation med ytre nukleinsyrer.

Med den generelle prosedyrer beskrevet ovenfor, ble muligheten for BCG overflate biotinylation og dekor med surrogat antigen OVA undersøkt. Immunogenisitet av den endrede BCG var testet i vivo. Bakteriell overflaten var enkelt merket med biotin for rask visning av avidin chimeric antigener uten synlig endringer i bakteriell fenotyper. Den resulterende endret BCG er effektivt ingested av antigen presentasjon celler og kan indusere en egg-spesifikk immunrespons i mus.
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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 teknikk for rask antigen visning på en bakteriell overflate presenteres, som innebærer overflate biotinylation etterfulgt av eksponering for proteiner interesse sammensmelting med monomerisk avidin. Lasting BCG valgte antigener vellykket forbedrer sin immunogenisitet, antyder at landtransport dekorasjon kan erstatte tradisjonelle genetisk tilnærminger.

Uttrykk for eksogene antigener i Mycobacterium bovis BCG vaksinen via ikke-genetiske overflaten dekorasjon med Avidin-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

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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

Expression of Functional rekombinant hemagglutinin og neuraminidase Proteiner fra Novel H7N9 Influensa virus Bruke Baculovirus Expression System

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

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In Vitro Analyse av MyD88-mediert cellulær immunrespons til West Nile Virus Mutant Strain Infeksjon

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

Utvikling av en<em&gt; In vitro</em&gt; Modellsystem for å studere samspillet mellom<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE med sin high-affinitetsreseptor 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 basert analyse for å studere migrasjon av murine Skin dendrittiske celler i Tømming lymfeknuter Under infeksjon 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

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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

Rensing av membran kupé for endoplasmatiske retikulum-assosiert nedbrytning av eksogene antigener i Cross-presentasjon

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 tuberculosis 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 ...