Name: expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel h7n9 influenza virus using the baculovirus expression system
Uploaded: 2013-11-06T23:00:00
Description: Watch this Scientific Journal Video about Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System at J

We would like to thank Patrick C. Wilson for monoclonal antibody CR9114. We also thank Jennifer Debeauchamp and Richard Webby for the original A/Anhui/1/13 plasmids. Partial support for this work was provided by the National Institute for Allergy and Infectious Diseases-funded Program Project Grant AI097092-01A1 and CEIRS (Centers of Excellence for Influenza Research and Surveillance, HHSN26620070010C). FK was supported by an Erwin Schr&ouml;dinger fellowship (J 3232) from the Austrian Science Fund (FWF).

1.. Fremstilling af rekombinant baculovirus <ol><li> Kloning i baculovirus transfer-plasmider <ol><li> Clone H7 HA og N9 NA ektodomænet (eller enhver anden HA eller NA gen) ind modificerede pFastBac transferplasmider (se indledningen). Vektorer til HA indeholdende et C-terminalt trimerisation domæne og et hexahistidin-tag, samt NA indeholdende et N-terminalt tetrameriseringsdomæne domæne og hexahistidin-tag, er blevet beskrevet 1,10,11,22 (figur 1) og kan rekvireres fra forfatterne. <br...

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Although expression of influenza virus HA and NA proteins in insect cells using the baculovirus expression system is generally straight forward, there are several important points to consider. It is important, as pointed out in the introduction, that the ectodomains of HA and NA are expressed in fusion with trimerization or tetramerization domains, respectively. These domains ensure correct folding of the molecules usually assisted by the transmembrane domain, which is not present if only the ectodomain is expressed. For...

Som en første reaktion på den seneste fremkomsten af ​​romanen H7N9 influenzavirus stamme i Kina, vi købte plasmider, der bærer de genomiske sekvenser for hæmagglutinin (HA) og neuraminidase (NA) gener af de to første isolater. Ved hjælp af disse plasmider var vi i stand til hurtigt at konstruere baculovirale ekspressionsvektorer til produktion af rekombinant HA og NA i insektceller. Dette ekspressionssystem er veletableret i vores laboratorium og har vist sig afgørende for mange af vores igangværende forskningsprojekter 1-8. Insektceller er i stand til at folde korrekt og posttranslationelt ændre komplekse proteiner. Disse modifikationer indbefatter N-bundet glycosylering, hvilket er vigtigt for mange virale glycoproteiner. Som sådan er det ikke overraskende, at baculovirussystemet helt er etableret for at udtrykke influenzavirus overfladeantigener. I virkeligheden har mange af HA og NA krystalstrukturer blevet løst ved hjælp af insektcellelinier udtrykte proteiner 9-11. En anden fordel vedsystem ligger i dens pålidelige højt proteinindhold udbytter på op til 30 mg HA / L cellekultur (baseret på vores erfaring med mere end 50 forskellige HA-proteiner) - en konsekvens af virus-infektion drevet udtryk fra den stærke polyhedrinpromotor. Fjernelse af de transmembrane og endodomain af HA og NA-proteiner giver mulighed for ekspression af sekreterbare opløselige versioner af proteiner, og dermed i høj grad letter rensningsprocessen. Desuden er disse ektodomæner hexahistidin mærkede og derfor kan oprenses ved hjælp af affinitetskromatografi under anvendelse af Ni2 +-harpiks. De transmembrane domæner af HA og NA proteiner bidrager til homotrimer og homotetramer dannelse, hhv. For at bevare disse oligomerizations, vi tilføje en C-terminal trimerisering domæne til HA-og en N-terminal tetrameriseringsdomæne domæne til NA-konstruktionerne (figur 1). Vi har påvist, at en sådan trimerisation domæne stabiliserer funktionel, konserved konformationelle epitoper på stilk-domæne af HA 1.. Den korrekte tetrameriseringsdomæne af NA vil også kunne bidrage til at korrigere foldning og NA-funktion 10. Sekvenser og Baculo-transfervektorer huser trimerisation eller tetrameriseringsdomæne domæner kan rekvireres fra forfatterne eller fra andre laboratorier i området, 9,10,12. Et andet vigtigt problem for ekspression af secernerede proteiner i insektceller er valget af den rigtige cellelinie. Mens Spodoptera frugiperda afledte Sf9 celler understøtter baculovirus replikation meget godt og er generelt bruges til virus redning og formering, har de begrænset kapacitet til at udskille store mængder protein. Trichoplusia ni afledt BTI-TN-5B1-4-celler (almindeligvis kendt som High Five) har en højere udskillelse kapacitet og er cellelinien valg til ekspression i denne protokol 13,14. Endvidere er det nyttigt at reducere eller endda fjerne kalvefosterserum (FBS) fra ekspressionssystemer kulturer. Vi bruger derfor medier med reduceret (3%) FBS indhold til dyrkning af de virus, der arbejder aktier, og vi udfører protein udtryk i serum frie medier. Rekombinante HA og NA-proteiner anvendes til mange immunologiske teknikker. Måske den mest almindelige er i ELISA-assays til måling af serum konvertering ved vaccination hos mennesker eller dyr 4,6,7,15. Har, og kontorer er også brugt i ELISPOT-analyser, da både stimulerende molekyler og detektionsreagenser for antistofsecernerende celler 16. Deres anvendelse som molekylære lokkemad tillader specifikt sorterer plasmablasts eller andre typer af B-celler, som derefter kan anvendes til isolering (terapeutiske) monoklonale antistoffer (mAb&#39;er). Rekombinante HA og NA molekyler er yderligere anvendt i karakteriseringen af disse mAb&#39;erne 8. Andre eksempler studere pH-stabilitet eller receptor specificitet har udtrykt af forskellige virusisolater, eller kvantitativt at vurdere specifikke NA enzymatiskeaktivitet eller resistens over for inhibitorer. Endvidere rekombinant, oprenset HA kan bruges til at standardisere HA indholdet af inaktiverede influenzavirus vacciner. Vigtigere er det, rHA (og til en vis grad NA) vacciner bliver i øjeblikket testet i dyremodeller og humane kliniske forsøg med en vaccine kandidat bliver godkendt af FDA i 2013 2,17-19. Fordelen ved disse nye vacciner er, at på grund af den rekombinante karakter af disse proteiner kunne arbejdskrævende proces at generere høje vækst reassorterede virus undgås. Måske endnu mere relevant er det, at deres HA udbytte er høj og reproducerbar fra stamme til stamme. Også, da disse vacciner er produceret i et æg-fri-system, de ikke indeholder protein forurenende stoffer, der er problematiske for personer med æg allergi. Her valgte vi at udtrykke HA og NA-proteiner fra to isolater af romanen kinesiske H7N9 influenzavirus, A / Anhui/1/13 og A/Shanghai/1/13 20,21. Disse er rettidige eksempler, men den beskrevne protokol kan anvendes til ekspression af influenza A-og B-HA eller NA-proteiner og kan tilpasses til at udtrykke andre secernerede virale eller cellulære proteiner. Trimere eller tetramere transmembrane proteiner kan klones ind ekspressionskassetterne anvendes til HA og NA, hhv. Men mest opløselige udskilte cellulære proteiner, som interferoner for eksempel ikke brug for et ekstra domæne til stabilisering og alene kan udtrykkes med en C-terminal hexahistidinmærke.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus expression system</td>
			<td>Invitrogen</td>
			<td>10359-016</td>
			<td>Follow manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TNM-FH insect medium</td>
			<td>Gemini Bioproducts</td>
			<td>600-311</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone SFX-Insect</td>
			<td>Thermo Fisher</td>
			<td>SH30278.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellfectin II Reagent</td>
			<td>Invitrogen</td>
			<td>P/N58760</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F68</td>
			<td>Sigma</td>
			<td>1000702664</td>
			<td></td>
		</tr>
		<tr>
			<td>SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>1018240</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra Centrigual filters Ultracel 30K</td>
			<td>Millipore</td>
			<td>UFC902024</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Columns (5 ml)</td>
			<td>Qiagen</td>
			<td>34964</td>
			<td></td>
		</tr>
		<tr>
			<td>Max efficiency DH10Bac bacteria</td>
			<td>Invitrogen</td>
			<td>10361-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink HiPure Plasmid Filter Midiprep Kit</td>
			<td>Invitrogen</td>
			<td>K210015</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399-100G</td>
			<td>for elution and wash buffers</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>ATCC</td>
			<td><a href="http://www.atcc.org/Products/All/CRL-1711.aspx" target="_blank">CRL-1711</a></td>
			<td></td>
		</tr>
		<tr>
			<td>High Five cells</td>
			<td>Invitrogen</td>
			<td>B85502</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of functional recombinant hemagglutinin and neuraminidase proteins from the novel h7n9 influenza virus using the baculovirus expression system

Baculovirusekspressionssystemet er et kraftfuldt værktøj til ekspression af rekombinante proteiner. Her bruger vi den til at producere foldede og glykosyleret korrekt versioner af influenza A-virus overfladeglycoproteiner - hæmagglutinin (HA) og neuraminidase (NA). Som et eksempel, vi valgte HA-og NA-proteiner udtrykt ved romanen H7N9-virus, der for nylig dukkede op i Kina. Men protokol kan let tilpasses til HA og NA-proteiner udtrykt ved enhver anden influenza A og B-virusstammer. Rekombinant HA (rHA) og NA (RNA) proteiner er vigtige reagenser til immunologiske assays, såsom ELISPOT og ELISA, og de er også i bred anvendelse for standardisering vaccine, antostofopdagelse, isolering og karakterisering. Endvidere kan rekombinante NA-molekyler anvendes til at screene for inhibitorer med små molekyler og er nyttige til karakterisering af den enzymatiske funktion af NA, samt dens følsomhed over for antivirale lægemidler. Rekombinante HA-proteiner er også being testet som eksperimentelle vacciner i dyremodeller, og en vaccine baseret på rekombinant HA for nylig blev godkendt af FDA til anvendelse i mennesker. Den metode, vi beskriver her til at producere disse molekyler er ligetil og kan lette forskning i laboratorier for influenza, da det giver mulighed for produktion af store mængder af proteiner hurtigt og til en lav pris. Selv her har vi fokus på influenzavirus overfladeglycoproteiner, kan denne metode også bruges til at fremstille andre virale og cellulære overfladeproteiner.

HA-molekylerne fra A/Shanghai/1/13 og A/Anhui/1/13 udtrykt godt med udbytter på ca 20 mg / L kultur. NA molekyler af begge stammer udviste moderat udtryk niveauer fra 0,2 mg / L kultur for A/Shanghai/1/13 til 0,7 mg / L for A/Anhui/1/13. Alle fire præparater protein havde meget lidt at ingen urenheder (figur 8A og B) med, har værende af højere renhed end nationale kontorer, som kan forklares med udtrykket niveau. A/Shanghai/1/13 HA blev yderligere analyseret ved hjælp af ELISA med ...

Watch this Scientific Journal Video about Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System at J

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

Her beskriver vi en måde at udtrykke foldede og funktionelle korrekt influenzavirus overfladeantigener afledt fra romanen kinesiske H7N9 virus i insektceller. Teknikken kan tilpasses til at udtrykke ektodomæner eventuelle virale eller cellulære overfladeproteiner.

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 ...

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Emballagen HIV-eller FIV-baserede Lentivector ekspressionskonstruktioner og transduktion af VSV-G-pseudotypet virale partikler

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Affinitetsrensning af Influenzavirus ribonukleoprotein komplekser fra kromatin af inficerede celler

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

Differentiering funktionelle roller af genekspression fra immun og ikke-immun celler i Mouse Colitis af knoglemarvstransplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Redning af rekombinant Newcastle Disease-virus fra cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

Skalerbar High Throughput Selection Fra fagpræsenterede Syntetiske antistofbiblioteker

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Co-immunoudfældning af musen Mx1 Protein med influenza A-virus nukleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Nasal Wipes for influenza A-virus Detection og Isolering fra svin

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Fluorescensbaseret neuraminidase-inhiberingsassay i vurdering af den følsomhed over influenzavira til neuraminidaseinhibitoren Class of Antivirale

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...