Name: making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry
Uploaded: 2018-05-27T14:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

J.J.G. verdikleri destek için Ulusal Bilim Vakfı (DMR-1654405) ve kanser önleme Araştırma Enstitüsü of Texas (CPRIT) (RP170752s) kabul eder.

1. hazırlık
<ol><li>Lysogeny suyu (LB) agar yapmak ve tabak12dökün.</li>
	<li>
bl21(de3) wtQβ kat protein içeren bir pET28 plazmid ile dönüşümü.
	<ol>
<li>E. coli BL21(DE3) yetkili hücrelerde bir buz banyosu çözülme. Hücre microcentrifuge tüp içinde yer 50 μL.</li>
		<li>Plazmid 2 μL bir tüpün içine ekleyin ve hafifçe tüpü hafifçe vur. O zaman 30 dk için buz üzerinde kuluçkaya.</li>
		<li>Isı şok hücreler için 45 s tam olarak 42 ° C'de olduğu bir su banyosunda Sonra hemen Isı-şok buz banyosunda tüpü yerleştirin ve 5 min için kuluçkaya.</li>
		<li>950 μL herhangi bir antibiyotik içeren LB medya ekleyin.</li>
		<li>37 ° C'de 60 dk için 200 devirde kültür sallamak</li>
		<li>100 μL LB agar plakaları kültürünün (sefaloridin) plaka ve plaka gecede 37 ° C'de kuluçkaya Gerektiğinde beyaz kolonileri seçin.</li>
	</ol>
</li>
	<li>
Süper en iyi suyu (SOB) medya olun.
	<ol>
<li>Otoklav iki 2 L Erlenmeyer şişeler superdry bir döngüsü şaşkın.</li>
		<li>Aseptik ortamında, tartmak ve tryptone 20.0 g ekleyin, 5.0 g maya ekstresi, susuz magnezyum sülfat, 2,469 g 0,584 g sodyum klorür ve 0,186 g potasyum klorür her şişesi için.</li>
		<li>Birim için 1 L Ultrasaf Su ile her şişesi ve basınçlı kap sıvı döngüsü getirmek.</li>
		<li>Bir kez SOB medya ısıyla sonra oda sıcaklığında ulaştı, sefaloridin (100 mg/mL) 1 mL medya her litre için ekleyin ve 4 ° C'de depolayın</li>
	</ol>
</li>
	<li>
0.1 M potasyum fosfat tampon (pH 7,00) olun.
	<ol>
<li>1 M potasyum Yem mono çözüm 68.045 g potasyum 500 mL Ultrasaf Su Yem mono çözülerek olun.</li>
		<li>1 M potasyum dibasic çözüm 87.09 g potasyum Ultrasaf Su 500 mL dibasic çözülerek olun.</li>
		<li>38,5 mL potasyum Yem mono çözüm ve potasyum dibasic 61,5 mL 1 L şişe ekleyin.</li>
		<li>PH gerekirse 7,00 için ayarlamak ve 1 L. bir birime getirmek</li>
	</ol>
</li>
	<li>
5-%40 0.1 M potasyum fosfat tampon (pH 7,00) sükroz degradeler olun.
	<ol>
<li>50 mL santrifüj tüplerde 0.1 M potasyum fosfat tampon (pH 7,00) çözünmüş % 5-40 (% 5'lik artışlarla artan) sükroz ile çözümleri hazırlayın.</li>
		<li>3.3 mL % 5 sükroz çözeltisi alt kısmındaki uzun iğne kullanarak 38 mL yuvarlak alt Polikarbonat tüp kasası ve beş diğer tüpler için bu işlemi yineleyin.</li>
		<li>Dikkatle 3.3 mL % 10 sükroz çözeltisi tüpün dibinde mevduat ve degrade rahatsız değil gibi iğne dikkatli bir şekilde çıkarın. Diğer beş tüpler için yineleyin. 
		 </li>
	</ol>
</li>
	<li>3.3 mL katmanları sükroz çözümleri, rahatsız etmeyin degradenin dikkatli olurken her tüpün içinde % 40'a %15 artan yatırmak devam edin.</li>
	<li>Tamamlandığında, folyo degradelerle üst kısımları kapak ve-80 ° C'de depolayın</li>
</ol>2. Qβ ifadesidir
<ol><li>1:1 çamaşır suyu/etanol ile tezgah alanı silin.</li>
	<li>İki 3 mL starter kültür aseptik ortamında aseptik bir ortamda E. coli BL21(DE3) tek kolonileri SOB medya 3 mL içine ekleyerek getirin.</li>
	<li>Bir shaker bir 37 ° C ve %0 bağıl nem (rH) odada 250 rpm'de üzerinde bir gecede büyümek.</li>
	<li>
SOB medya Starter kültürü aşılamak:
	<ol>
<li>Her iki 3 mL starter kültürler shaker alın ve aseptik bir ortamda, her starter kültür iki birinin içine dökün 2 L Erlenmeyer şişeler taze SOB medya her 1 litre ile şaşkın.</li>
		<li>Bir shaker bir 37 ° C ve % 0 rH odada 250 rpm'de inoculated medya yerleştirin.</li>
	</ol>
</li>
	<li>OD600 0,9-1.0 ulaşıncaya kadar bir shaker bir 37 ° C ve % 0 rH odada 250 rpm'de bakteri büyümek.</li>
	<li>1 mL 1 M izopropil β-D-1 thiogalactopyranoside (IPTG) protein ifade ikna etmek için P1000 pipet kullanarak ekleyin.</li>
	<li>Medya bir 37 ° C ve % 0 rH odada 250 rpm'de shaker üzerinde gece bırakın.</li>
	<li>Medya shaker ertesi sabah ve santrifüj 1000 ml'lik şişe de 20,621 × g 4 ° C'de 1 h için hücreleri hasat için kullanarak kaldırın.</li>
	<li>
Süpernatant atın ve hücre Pelet toplamak.
	<ol>
<li>Süpernatant yaklaşık 5 mL bakterileri öldürmek için çamaşır suyu ile bir şişe içine dökün. Bu atık.</li>
		<li>Santrifüj şişe altındaki hücre Pelet scrape ve Pelet 50 mL santrifüj tüpüne koymak için bir spatula kullanın.</li>
	</ol>
</li>
</ol>3. Qβ arınma
<ol><li>Hücre Pelet ~ 20-30 ile resuspend mL 0.1 M potasyum fosfat tampon (pH 7,00).</li>
	<li>Resuspension yok parçalar olduğundan emin olun ve bir microfluidizer işlemci üreticisinin protokolüne göre kullanarak hücreleri parçalayıcı ( Tablo malzemelerigörmek). Parçacıklar verimini artırmak için en az iki kez hücreleri parçalayıcı.</li>
	<li>Lysate 20,621 x g de 250 mL santrifüj şişelerde 4 ° C'de 1 saat için santrifüj kapasitesi</li>
	<li>Pelet atın ve süpernatant ml hacmi ölçmek. Bu değeri 0.265 tarafından çarpar ve bu miktarın g amonyum sülfat süpernatant için.</li>
	<li>En az 1 h protein çökelti 200 devirde heyecan tabakta için 4 ° C'de karıştırın.</li>
	<li>20,621 x g 4 ° C'de 1 h için 250 mL şişe santrifüj kapasitesi</li>
	<li>Süpernatant atın ve Pelet yaklaşık 10 mL 0.1 M potasyum fosfat tampon (pH 7,00) ile resuspend.</li>
	<li>1:1 kloroform/n-butanol eşit miktarda ham örnek için vortexing tarafından bir kaç saniyeliğine ekleyip karıştırın.</li>
	<li>4 ° C'de 30 dakika 20,621 × g de 38 mL tüpler santrifüj kapasitesi</li>
	<li>Pasteur pipet kullanarak üst sulu katmanı yeniden elde etmek. Sulu ve organik katmanı arasında kurdu tabaka gibi jel almak değil dikkatli olun.</li>
	<li>Altı % 5-40 önceden yapılmış sükroz degradeler çözülme ve 2 mL her ekstresinin yükleyin.</li>
	<li>Ultracentrifuge 99,582 x g ücretsiz yavaşlama ile 4 ° C'de 16 h için de.</li>
	<li>Bir ışık yayan diyot (LED) ışık her tüpün altında Parlatıcı ve bir mavi bant görünür olmalıdır. Bu parçacıklar uzun iğne şırınga ile yeniden elde etmek.</li>
	<li>Ultrapellet 370,541 x g 4 ° C'de 2,5 h için parçacıklar</li>
	<li>Süpernatant atın ve 0.1 M potasyum fosfat tampon (pH 7,00) ile arıtılmış parçacıkların şeffaf Pelet resuspend.</li>
</ol>4. miktar ve ürün onayı
<ol><li>Bradford tahlil ürün13ölçmek için kullanın.</li>
	<li>Polyacrylamide jel elektroforez (SDS-sayfa) ürün14onaylamak için çalışma azaltılması ve sigara azaltılması Sodyum Lauryl Sülfat. Not: SDS-sayfa azaltarak kat protein molekül ağırlığı onaylamak için kullanılır; Sigara azaltılması-SDS-sayfa daha yüksek sipariş yapısı emin olmaktır.</li>
</ol>5. Qβ DB bileşikler conjugating
<ol><li>
Disulfides Qβ üzerinde azaltmak.
	<ol>
<li>0.0020 gram dissolve tris(2-carboxyethyl) fosfamin (TCEP) 1 mL 100 x hisse senedi çözüm yapmak Ultrasaf Su içine. Not: azaltma önce taze TCEP hazırlayın.</li>
		<li>Qβ (5 mg/mL) 200 µL microcentrifuge tüp içine ekleyin.</li>
		<li>100 x TCEP hisse senedi çözüm 20 µL ekleyerek izleyin.</li>
		<li>1s için oda sıcaklığında kuluçkaya.</li>
	</ol>
</li>
	<li>
Azaltılmış disülfür dibromomaleimide Polietilen glikol (DB-PEG) kullanarak yeniden köprü.
	<ol>
<li>0.0017 g dibromomaleimide-Polietilen glikol (DB-PEG) DMF 100 µL geçiyoruz.</li>
		<li>10 mM sodyum fosfat çözüm (pH 5,00) 680 µL ekleyin.</li>
		<li>Qβ DB-PEG çözüm içine azaltılmış ekleyin ve karıştırma işlemi bir 365 nm UV lambası altında gözlemlemek. Parlak sarı floresan lambalı karıştırma üzerine bir 365 nm el UV hemen görüntülenmeyecektir.</li>
		<li>Gecede bir et lokantası (RT) Oda sıcaklığında devam tepki ver.</li>
	</ol>
</li>
	<li>Santrifüj filtre tarafından tepki karışımı arındırmak (COMW = 10 kDa) üç kez 1 x PBS de 3,283 x g 4 ° C'de 20 dk için kullanma</li>
	<li>Konjugasyon olmayan-SDS-sayfa ve yerel özel jel elektroforez azaltarak monitör. Not: Yerel özel jel sağlam parçacıklar olarak VIP çalıştırın ve onların ücret, boyutu ve şekli bağlı ayrılır.</li>
</ol>

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Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Daha küçük protein arıtma için karşılaştırıldığında, bakteriyofaj Qβ arındırıcı bir benzersiz sükroz degrade Santrifüjü adımdır. Kloroform/n-butanol ayıklama adım sonra Qβ daha fazla % 5-40 sükroz degradeler kullanarak saf. Santrifüjü sırasında parçacıklar kendi boyutları göre ayrılır. Daha küçük partiküller daha düşük yoğunluklu bölgede kalırken daha büyük partiküller yüksek yoğunluk bölgesine seyahat. Qβ için geçişin üçüncü alt seyahat ve daha küçük protei...

Nano ölçekli viral capsids kullanarak uygulamaları Biyomedikal araştırma1,2,3' te kapsamını genişletmek hedefliyor heyecan verici bir alan olarak ortaya çıkmıştır. Recombinantly ifade virüs benzeri parçacıklar (VIP) yapısal olarak virüslere karşı elde edilen ama orijinal viral genetik materyal bulaşıcı olmayan proteinaceous nano tanecikleri geliştirmelerde eksik. Yüzey özellikleri genetik programlı ve her kapsid aynı şekilde önce ve sonrasında olanlar için ifade gibi reaktif yan zincirli amino asitlerin atomistic hassasiyetle sayısını ve konumunu öğrenmek mümkündür. Birçok durumda, iç ve dış yüzeyleri pek çok feasibly bioconjugation tepkiler - Kovalent bağlar arasında bir biomolecule ve sentetik reaksiyonlar yoluyla functionalized solvent maruz amino asit kalıntıları sahip. molekül4,5.
Bioconjugation reaksiyonlar ilgi daha farklı işlevleri nispeten basit bir moda var biomolecules yardım. Molekülleri tedavi uyuşturucu6, floresan Etiketler7 ve polimerler8,9 gibi ilgi önceden sentezlenmiş ve onlar VIP'ler yüzeyinde eklenmeden önce ile karakterizedir. Özellikle ortak bir VIP içinde biyomedikal ve biyomalzeme araştırma bakteriyofaj Qβ, recombinantly olarak ifade, 28 nm icosahedral viral kapsid10temel VIP oldu. Son zamanlarda Qβ gözenekleri Haddleton-Baker tepki ile satır azaltılmış disulfides için dibromomaleimide bileşiklerin başarılı konjugasyon11 tebliğ var olsa Qβ en yaygın tepki sitelerinde geniş bir farkla lysines vardır. Reaksiyon gelirleri ile iyi verim ve eşit olarak da önemlisi, olmadan parçacıkların termal kararlılık kaybetme. Aynı zamanda, bu parçacıklar alımını hücrelere izlemek için kullanılan fiil kaynaklı floresans bu reaksiyon oluşturur. Bu çalışmada, Qβ yüzeyine bir parlak sarı fluorophore sonuç Haddleton-Baker tepki ile Polietilen glikol (PEG) konjugasyon göstermektedir. Hepsi hücreleri tarafından alınır gibi bu parçacıklar daha sonra izlenebilir. İletişim kuralı burada araştırmacılar kendi ilkeleri bir çözelti maruz disulfides içeren diğer birçok VIP'ler için uygulanabilir olmasına rağmen proteinaceous nano tanecikleri Qβ dayalı yeni floresan pegile oluşturmak yardımcı olur.

<table><tbody><tr><td>LB Broth (Miller)&amp;nbsp;</td><td>EMD Millipore</td><td>1.10285.0500</td><td></td></tr><tr><td>Tryptone, Poweder</td><td>Research Products International</td><td>T60060-1000.0</td><td></td></tr><tr><td>Yeast Extract, Poweder</td><td>Research Products International</td><td>Y20020-1000.0</td><td></td></tr><tr><td>Anhydrous magnesium sulfate</td><td>P212121</td><td>CI-06808-1KG</td><td></td></tr><tr><td>Sodium Chloride (Crystalline/Certified ACS)</td><td>Fisher Scientific</td><td>S271-10</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Elga PURELAB Flex 3 Water Purification System</td><td>Fisher Scientific</td><td>4474524</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Fisher Scientific</td><td>BP362-1</td><td></td></tr><tr><td>Potassium Phosphate Dibasic Anhydrous</td><td>Fisher Scientific</td><td>P288-500</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S25590B</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma Aldrich</td><td>I6758-1G</td><td></td></tr><tr><td>Fiberlite F10-4x1000 LEX rotor&amp;nbsp;</td><td>Fisher Scientific</td><td>096-041053</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>P212121</td><td>KW-0066-5KG</td><td></td></tr><tr><td>Chloroform</td><td>Alfa Aesar</td><td>32614-M6</td><td></td></tr><tr><td>1-Butanol</td><td>Fisher Scientific</td><td>A399-4</td><td></td></tr><tr><td>SW 28 Ti Rotor, Swinging Bucket, Aluminum</td><td>Beckman Coulter</td><td>342204: SW 28 Ti Rotor/ 342217: Bucket Set</td><td></td></tr><tr><td>Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL,</td><td>Beckman Coulter</td><td>337922</td><td></td></tr><tr><td>Coomassie (Bradford) Protein Assay</td><td>Fisher Scientific</td><td>PI23200</td><td></td></tr><tr><td>TRIS Hydrochloride</td><td>Research Products International</td><td>T60050-1000.0</td><td></td></tr><tr><td>Tetramethylethylenediamine</td><td>Alfa Aesar</td><td>J63734-AC</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma Aldrich</td><td>C4706-2G</td><td></td></tr><tr><td>2 3-Dibromomaleimide 97%</td><td>Sigma Aldrich</td><td>553603-5G</td><td></td></tr><tr><td>Polythylene Glycol</td><td>Alfa Aesar</td><td>41561-22</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>AC424375000</td><td></td></tr><tr><td>Acrylamide/bis-Acrylamide</td><td>P212121</td><td>RP-A11310-500.0</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771-100G</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>BP179-100</td><td></td></tr><tr><td>FV3000 confocal laser scanning microscope</td><td>Olympus&amp;nbsp;</td><td>FV3000&amp;nbsp;</td><td></td></tr><tr><td>Labnet Revolver Adjustable Rotator&amp;nbsp;</td><td>Thomas Scientific&amp;nbsp;</td><td>1190P25&amp;nbsp;</td><td></td></tr><tr><td>1000 mL Sorvall High Performance Bottle, PC, with Aluminum Cap&amp;nbsp;</td><td>Thermo Scientific</td><td>010-1459</td><td></td></tr><tr><td>Nalgene Centrifuge Bottles with Caps, Polypropylene Copolymer</td><td>Thermo Scientific</td><td>3141-0250</td><td></td></tr><tr><td>Nunc Round-bottom tubes; 38 mL; PC</td><td>Thermo Scientific</td><td>3117-0380</td><td></td></tr><tr><td>2 L Narrow Mouth Erlenmeyer Flasks with Heavy Duty Rim</td><td>Pyrex</td><td>4980-2L</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Units</td><td>Millipore Sigma</td><td>UFC801024</td><td></td></tr><tr><td>M-110P Microfluidizer Materials Processor</td><td>Microfluidics</td><td>M-110P</td><td></td></tr><tr><td>Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes</td><td>Thermo Scientific</td><td>3117-0380PK</td><td></td></tr><tr><td>Bottle, with Cap Assembly, Polycarbonate</td><td>Beckman Coulter</td><td>41121703</td><td></td></tr><tr><td>Cylinder, Graduated - Polypropylene 250 mL</td><td>PolyLab</td><td>80005</td><td></td></tr><tr><td>533LS-E Series Steam Sterilizers</td><td>Getinge</td><td>533LS-E</td><td></td></tr><tr><td>TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with Lid</td><td>LabSource</td><td>D36-313-CS</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tube</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>Microcentifuge Tube: 1.5mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>VWR Os-500 Orbital Shaker</td><td>VWR Scientifc Products</td><td>14005-830</td><td></td></tr><tr><td>Tetra Handcast systems</td><td>Bio-Rad</td><td>1658000FC</td><td></td></tr><tr><td>Polypropylene, 250 mL</td><td>Beckman Coulter</td><td>41121703</td><td></td></tr><tr><td>Spectrofluorometer NanoDrop</td><td>Thermo Fisher Scientific</td><td>3300</td><td></td></tr><tr><td>Long Needle&amp;nbsp;</td><td>Hamilton&amp;nbsp;</td><td>7693</td><td></td></tr><tr><td>Exel International 5 to 6 cc Syringes Luer Lock</td><td>Fisher Scientific</td><td>14-841-46</td><td></td></tr><tr><td>P1000 Pipetman</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>P200 Pipetman</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>P100 Pipetman</td><td>Gilson</td><td>F123615</td><td></td></tr><tr><td>P20 Pipetman</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>P10 Pipetman</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>Intel Weighing PM-100 Laboratory Classic High Precision Laboratory Balance</td><td>Intelligent Weighting Technology</td><td>IWT_PM100</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tube</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>4&amp;ndash;15% Mini-PROTEAN TGX Gel, 10 well, 50 &amp;micro;l</td><td>Bio-Rad</td><td>456-1084</td><td></td></tr></tbody></table>

making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

Virüs gibi parçacıklar (VIP) Biyomedikal son artış ve malzeme araştırma onların kolaylığı biyosentezi, ayrık boyut, genetik programlama ve biodegradability atfedilen. Onlar onların yüzeyine sentetik ligandlar eklemek için bioconjugation reaksiyonlar için son derece uysal, bioconjugation yöntemleri bu sulu d. capsids tarih aralığında göreceli olarak sınırlı iken. Fonksiyonel Biyomalzeme araştırma yönünü kolaylaştırmak için geleneksel olmayan bioconjugation tepkileri dikkate alınmalıdır. Bu protokol için açıklanan tepki dibromomaleimides solvent bazlı bir VIP maruz disülfür bağları bakteriyofaj Qβ yeni işlevsellik tanıtmak için kullanır. Ayrıca, nihai ürün piyasada bulunan filtre seti kullanarak izlenebilir vitro sonda üreten yararı olan flüoresan,.

Dibromomaleimide türevleri ile yoğunlaşma tepki dibromomaleimide anhidrit ve Birincil aminler15arasında sentez. Alternatif olarak, N-methoxycarbonyl aktif 3,4-dibromomaleimide kullanarak bir hafif sentetik yöntemi16 burada methoxypolyethylene glikol (PEG) için verim DB-PEG (Şekil 1) ile tepki tarafından istismar edildi. NMR Bileşik yapı (Şekil 2) tanımlamak için kullan?...

Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Konjugasyon kaynaklı floresan pegile virüs benzeri moleküller tarafından Dibromomaleimide-disülfür Kimya yapma

Burada, fluorescently Qβ VIP üzerinde disulfides dibromomaleimide ile functionalize için bir yordam mevcut. Biz Qβ ifade ve arıtma, molekülleri dibromomaleimide functionalized sentezi ve dibromomaleimide ve Qβ arasında konjugasyon tepki açıklanmaktadır. Elde edilen Sarı floresan konjuge parçacık bir floresan sonda hücrelere olarak kullanılabilir.

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

making-conjugation-induced-fluorescent-pegylated-virus-like-particles

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.8K Görüntüleme.  University of Texas at Dallas. Burada, fluorescently Qβ VIP üzerinde disulfides dibromomaleimide ile functionalize için bir yordam mevcut. Biz Qβ ifade ve arıtma, molekülleri dibromomaleimide functionalized sentezi ve dibromomaleimide ve Qβ arasında konjugasyon tepki açıklanmaktadır. Elde edilen Sarı floresan konjuge parçacık bir floresan sonda hücrelere olarak kullanılabilir.

Video: Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Alexa Fluor Etiketleme Dang Virüs görselleştirme

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

İmmünoloji ve Enfeksiyon

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Viral Nanopartiküller<em&gt; In vivo</em&gt; Tümör Görüntüleme

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Biyomühendislik

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Görüntülemede HIV-1 Zarf bağlı Virolojik Synapse ve Sentetik Lipid Bilayers üzerine Sinyal

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Bioorthogonal tıklayın Kimya ile Viral Yüzeylerin Kemoselektif Modifikasyonu

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Floresan Etiketli Vaksinia Virüs Proteinlerinin Verimli Üretimi için Metodoloji

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Tek Parçacık Düzeyinde Viral Fusion Kinetik Ölçüm yöntemi

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biyoloji

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Hepatit E virüs nano tanecikleri kimyasal konjugasyon yöntemlerle yüzey Functionalization

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Üretimi, güçlendirme ve titrasyon rekombinant solunum sinsisyal virüs

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Tek Virion Atomik Kuvvet Mikroskopisi ve Süper Çözünürlüklü Floresan Görüntüleme için Numune Hazırlama

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Konjugasyon kaynaklı floresan pegile virüs benzeri moleküller tarafından Dibromomaleimide-disülfür Kimya yapma

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