J.J.G.는 그들의 지원에 대 한 국립 과학 재단 (DMR-1654405) 및 암 예방 연구 연구소의 텍사스 (CPRIT) (RP170752s)을 인정 한다.

1입니다. 준비
<ol><li>Lysogeny 국물 (파운드) 한 천 만들고 접시12부.</li>
	<li>
변환 BL21(DE3) wtQβ 외 투 단백질 시퀀스를 포함 하는 pET28 플라스 미드와.
	<ol>
<li>대장균 BL21(DE3) 유능한 세포를 얼음 목욕에 녹여 Microcentrifuge 튜브에서 셀의 장소 50 μ.</li>
		<li>1 개의 관으로 플라스 미드의 2 μ를 추가 하 고 부드럽게 튜브를 끄 적. 다음 30 분 동안 얼음에 품 어.</li>
		<li>열-충격 45 셀 정확히 42 ° c.에 있는 물 욕조에 s 열 충격 직후 얼음 욕조에 다시 튜브를 배치 하 고 5 분 동안 품 어.</li>
		<li>어떤 항생제를 포함 하지 않는 파운드 미디어의 950 μ를 추가 합니다.</li>
		<li>37 ° c.에 60 분 200 rpm에서 문화를 흔들어</li>
		<li>(대)와 파운드 한 천 배지에서 문화의 100 μ 접시 하 고 37 ° c.에 하룻밤 접시를 품 어 필요할 때 백색 식민지를 선택 합니다.</li>
	</ol>
</li>
	<li>
슈퍼 최적의 국물 (흐 느낌) 미디어를 확인 합니다.
	<ol>
<li>압력솥 L 2 개의 삼각 플라스 크를 당황 하 게 제시카 주기.</li>
		<li>무 균 환경에서 밖으로 무게 추가 tryptone의 20.0 g, 5.0 g의 효 모 추출 물, 무수 황산 마그네슘의 2.469 g 0.584 g 염화 나트륨의 및 각 플라스 크에 염화 칼륨 0.186 g.</li>
		<li>볼륨 1 L를 함께 초순 각 플라스 크에 고압 액체 주기.</li>
		<li>흐 느낌 미디어는 압력가 마로 소독 후 실내 온도 도달 하면, 미디어의 각 리터를 대 (100 mg/mL)의 1 mL을 추가 하 고 4 ° c.에 저장</li>
	</ol>
</li>
	<li>
0.1 m M 칼륨 인산 염 버퍼 (pH 7.00)을 확인 합니다.
	<ol>
<li>초순의 500 ml에서 이수소 칼륨 68.045 g을 용 해 하 여 1 M 칼륨 이수소 솔루션을 확인 합니다.</li>
		<li>칼륨 초순 물 500 mL에 염기의 87.09 g을 용 해 하 여 1 M 칼륨 염기 솔루션을 확인 합니다.</li>
		<li>1 리터 병에 칼륨 이수소 솔루션 및 칼륨 염기의 61.5 mL 38.5 mL를 추가 합니다.</li>
		<li>7.00, 필요한 경우에 pH를 조정 하 고 1 나의 볼륨을</li>
	</ol>
</li>
	<li>
0.1 m M 칼륨 인산 염 버퍼 (pH 7.00)에 5-40% 자당 그라디언트를 확인 합니다.
	<ol>
<li>50 mL 원심 분리기 튜브에서 0.1 M 칼륨 인산 염 버퍼 (pH 7.00)에 용 해 하는 5-40% (5% 단위로 증가) 자당으로 솔루션을 준비 합니다.</li>
		<li>긴 바늘 주사기를 사용 하 여 38 mL 라운드-하단 폴 리 카보 네이트 튜브의 하단에 5% 자당 해결책의 3.3 mL를 입금 하 고 5 개의 다른 튜브에 대 한이 반복 합니다.</li>
		<li>조심 스럽게 3.3 mL 튜브의 하단에 10% 자당 해결책의 보증금과 그라디언트를 방해 하지로 바늘을 조심 스럽게 제거. 다른 5 개의 튜브를 반복 합니다. 
		 </li>
	</ol>
</li>
	<li>3.3 mL 레이어 자당 솔루션, 그라디언트를 방해 하지 않는 신중 하면서 각 관에서 40%로 15%에서 증가의 입금을 계속 합니다.</li>
	<li>완료 되 면, 호 기온 변화도의 정상을 커버 하 고-80 ° c.에 저장</li>
</ol>2입니다. Qβ의 표현
<ol><li>1:1 표 백제/에탄올 벤치 영역을 닦으십시오.</li>
	<li>무 균 환경에서 흐 느껴 울 다 미디어의 3 mL에 대장균 BL21(DE3)의 단일 식민지를 추가 하 여 무 균 환경에서 2 개의 3 mL 스타터 문화를 확인 합니다.</li>
	<li>37 ° C, 0% 상대 습도 (rH) 룸에서 250 rpm에서 통에 하룻밤 성장.</li>
	<li>
개 자식 미디어에서 선발 문화 예방:
	<ol>
<li>두 3 mL 스타터 문화 셰이 커 고, 무 균 환경에서 2 중으로 각 시 동기 문화를 부 어 2 L 1 l 각 신선한 흐 미디어의 삼각 플라스 크를 당황 하 게.</li>
		<li>37 ° C, 0 %rH 룸에서 250 rpm에서 통에 접종된 미디어를 배치 합니다.</li>
	</ol>
</li>
	<li>OD600 0.9-1.0에 도달할 때까지 37 ° C, 0 %rH 룸에서 250 rpm에서 통에 박테리아를 성장.</li>
	<li>1 M 이소프로필 β-D-1 thiogalactopyranoside (IPTG) P1000 피 펫을 사용 하 여 단백질 발현을 유도 하는 것의 1 개 mL를 추가 합니다.</li>
	<li>37 ° C, 0 %rH 룸에서 250 rpm에서 셰이 커에는 미디어를 하룻밤 둡니다.</li>
	<li>미디어에서에서 제거 흔드는 다음 아침 및 원심 분리기를 사용 하 여 1000 mL 병 20,621 × g에서 4 ° C에서 1 시간에 대 한 셀을 수확 합니다.</li>
	<li>
삭제는 상쾌한 고 셀 펠 릿을 수집 합니다.
	<ol>
<li>박테리아를 죽 일 백제의 약 5 mL로 플라스 크에는 상쾌한 하 거 라. 이것은 폐기물 이다.</li>
		<li>주걱을 사용 하 여 원심 분리기 병의 하단에서 셀 펠 릿을 긁 고 50ml 원심 분리기 튜브에 펠 릿을 넣어.</li>
	</ol>
</li>
</ol>3입니다. Qβ의 정화
<ol><li>Resuspend 셀 펠 릿 ~ 20-30 mL의 0.1 M 칼륨 인산 염 버퍼 (pH 7.00).</li>
	<li>물의 resuspension 없는 덩어리는 다는 것을 확인 하 고 제조업체의 프로토콜에 따라 microfluidizer 프로세서를 사용 하 여 세포를 lyse ( 재료의 표참조). 입자의 수확량을 증가 시키기에 두 번 이상 세포를 lyse.</li>
	<li>4 ° c.에 1 시간에 250 mL 20,621 x g에서 원심 분리기 병에는 lysate 원심</li>
	<li>펠 릿을 삭제 하 고 상쾌한 ml에서의 볼륨을 측정 한다. 0.265로 그 값을 곱하면 고는 상쾌한 황산 암모늄의 g의 금액 추가.</li>
	<li>단백질에 밖으로 침전 시키기 위하여 200 rpm에서 저 어 접시에 적어도 1 시간에 4 ° C에서 저 어.</li>
	<li>20,621 x g 4 ° c.에 1 시간에 250 mL 병에서 원심</li>
	<li>상쾌한 무시 하 고 약 10 mL의 0.1 M 칼륨 인산 염 버퍼 (pH 7.00)와 펠 릿을 resuspend.</li>
	<li>몇 초 동안 vortexing에 의해 원유 샘플 및 혼합 1:1 클로 프롬/n-butanol 동등한 양의 추가 합니다.</li>
	<li>4 ° c.에 30 분 동안 20,621 × g에서 38 mL 튜브에 원심</li>
	<li>파스퇴르 피 펫을 사용 하 여 최고 수성 층을 복구 합니다. 수성 및 유기 층 사이 형성 했다 레이어 같은 젤을 사용할 않기로 주의 해야 합니다.</li>
	<li>6 5-40 %pre-made 자당 기온 변화도 녹여 고 약 2 mL에 각 추출의 로드 합니다.</li>
	<li>무료 감속으로 4 ° C에서 16 h 99,582 x g에서 ultracentrifuge.</li>
	<li>발광 다이오드 (LED) 조명 각 튜브 아래 빛나는 고 블루 밴드 표시 한다. 긴 바늘 주사기이 입자를 복구 합니다.</li>
	<li>Ultrapellet 370,541 x g 2.5 h 4 ° c.에 대 한에서 입자</li>
	<li>상쾌한 무시 하 고 0.1 M 칼륨 인산 염 버퍼 (pH 7.00) 순화 된 입자의 투명 한 펠 릿 resuspend.</li>
</ol>4. 정량화 및 제품의 확인
<ol><li>Bradford 분석 실험을 사용 하 여 계량 제품13.</li>
	<li>실행 줄이고 비 감소 나트륨 라우릴 황산 polyacrylamide 젤 전기 영동 (SDS-PAGE) 제품14확인. 참고:; 외 투 단백질의 분자량을 확인 하는 데 사용은 SDS 페이지 감소 비 줄이는 SDS 페이지 더 높은 순서 구조를 확인 하는 것은.</li>
</ol>5. 어근 Qβ에 DB 화합물
<ol><li>
Qβ에 disulfides 줄일 수 있습니다.
	<ol>
<li>해산의 0.0020 g tris(2-carboxyethyl) 초순 100 x 재고 솔루션의 1 mL에 phosphine (TCEP). 참고: 신선한 TCEP 감소 하기 전에 준비.</li>
		<li>Microcentrifuge 튜브에 Qβ (5 mg/mL)의 200 µ L를 추가 합니다.</li>
		<li>100 x TCEP 재고 솔루션의 20 µ L을 추가 하 여 따라.</li>
		<li>1 시간에 대 한 실 온에서 품 어.</li>
	</ol>
</li>
	<li>
다시 dibromomaleimide 폴 리 에틸렌 글리콜 (PEG DB)을 사용 하 여 감소 이황화 다리.
	<ol>
<li>Dibromomaleimide-폴 리 에틸렌 글리콜 (DB-PEG) DMF의 100 µ L의 0.0017 g을 분해.</li>
		<li>10 m m 나트륨 인산 염 해결책 (pH 5.00)의 680 µ L를 추가 합니다.</li>
		<li>추가 DB-말뚝의 솔루션으로 Qβ를 감소 하 고 365 nm UV 램프에서 혼합 과정을 관찰. 밝은 노란색 형광 혼합 시 365 nm 휴대용 UV 램프로 즉시 구상 될 수 있다.</li>
		<li>하룻밤 실 온 (RT)는 불고기 집에 진행 하는 반응 하 게.</li>
	</ol>
</li>
	<li>원심 필터 반응 혼합물을 정화 (COMW = 10 kDa) 세 번 사용 하 여 1 x PBS 3,283 x g에서 4 ° c.에서 20 분</li>
	<li>줄임으로써 비-SDS 페이지 및 네이티브 agarose 젤 전기 이동 법은 활용을 모니터링 합니다. 참고: VLPs 네이티브 agarose 젤에 그대로 입자로 실행 하 고 그들은 그들의 충전, 크기와 모양에 따라 구분 됩니다.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+Virus-like+Particles+with+Poly(2-oxazoline)s.">Stabilization of Virus-like Particles with Poly(2-oxazoline)s.</a> Angew. Chem. Int. Ed. 50 (11), 2601-2605 (2011).</li><li>Chen, Z., Li, N., Chen, L., Lee, J., Gassensmith, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Functionalized+Bacteriophage+Q&#946;+as+a+Photocaged+Drug+Carrier.">Dual Functionalized Bacteriophage Q&#946; as a Photocaged Drug Carrier.</a> Small. 12 (33), 4563-4571 (2016).</li></ol>

저자 들은 아무 경쟁 금융 관심사 선언 합니다.

작은 단백질 정화에 비해, 살 균 소 Qβ 정화는 독특한 단계 자당 기온 변화도 원심 분리입니다. 클로 프롬/n-butanol 추출 단계 후 Qβ 5-40% 자당 기온 변화도 사용 하 여 더 순화 이다. 원심 분리, 중 입자는 그들의 크기에 따라 구분 됩니다. 더 큰 입자 작은 입자는 낮은 밀도 영역에 체류 하는 동안 더 높은 밀도 영역에 여행. Qβ는 그라데이션의 더 낮은 세 번째에 여행 하 고 더 작은 단백질 불순물은 원...

바이러스 성 capsids 나노 크기를 사용 하 여 생물 의학 연구1,2,3응용 프로그램의 범위를 확대 하는 것을 목표로 하는 흥미 진 진한 필드로 떠오르고 있다. Recombinantly 표현된 바이러스 같은 입자 (VLPs)는 구조적으로 바이러스에서 파생 된 하지만 그들은 그들에 게 비 전염 성 배치할 나노 입자를 만드는 원래 바이러스 유전 물질 부족 합니다. 표면 기능 프로그램 유전자는 고 각 capsid 앞뒤 사람에 게 동일 하 게 표현 된다, 위치 및 원자 정밀도로 아미노산의 반응 측면 체인의 수를 알아야 가능 하다. 대부분의 경우에서 외부와 실내 표면 bioconjugation 반응-는 biomolecule와 합성 사이 공유 결합을 형성 하는 반응을 통해 functionalized feasibly 수 있습니다 용 매 노출된 아미노산 잔류물의 많은 종류를가지고 분자4,5.
Bioconjugation 반응 생체의 비교적 간단한 패션에서 더 다양 한 기능을가지고 도움이 됩니다. 분자의, 치료 약6, 형광 태그7 등 폴리머8,9 미리 합성 하 고 특징 VLPs의 표면에 연결 하기 전에 수 있습니다. 생물 의학에서 특히 일반적인 VLP 바이오 소재 연구, recombinantly 표현으로 28 nm icosahedral 바이러스 성 capsid10살 균 소 Qβ에 따라 VLP 되었습니다. 비록 우리가 최근에 Haddleton 베이커 반응을 통해 Qβ의 숨 구멍 줄 감소 disulfides dibromomaleimide 화합물의 성공적인 활용11 전달 Qβ에 가장 일반적인 반응 사이트 넓은 여백에 의해 lysines 있습니다. 반응 좋은 진행 항복 하 고, 동등 하 게 중요 한 것은, 입자의 열 안정성을 잃고 없이. 동시에이 반응을 활용 유도 형광, 셀으로이 입자의 통풍 관을 추적 하는 데 사용할 수 있습니다 생성 합니다. 이 작품에서는, 우리 Haddleton 베이커 반응, 밝은 노란색 fluorophore를 통해 Qβ의 표면에 폴 리 에틸렌 글리콜 (PEG)의 활용을 보여 줍니다. 이러한 입자 세포에 의해 그들은 촬영으로 추적할 수 있습니다. 본 프로토콜 연구원 원리 용 노출된 disulfides 포함 된 많은 다른 VLPs 중 하나에 적용 되는 새로운 형광 PEGylated 배치할 나노 입자를 기반으로 Qβ, 생성 도움이 됩니다.

<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

바이러스 같은 입자 (VLPs) 생물 의학에 있는 최근 증가 및 재료 연구 생 합성, 개별 크기, 유전자 프로그래밍 및 biodegradability의 그들의 용이 함에 표시 될 수 있습니다. 그들은 그들의 표면에 합성 ligands를 추가 하기 위한 bioconjugation 반응에 매우 의무가 있어, 이러한 수성 태어난된 capsids에 bioconjugation 방법론에서 범위 상대적으로 제한 됩니다. 촉진 하기 위해 기능성 생체 재료 연구의 방향, 비 전통적인 bioconjugation 반응은 고려 되어야 한다. 이 프로토콜에서 설명 하는 반응 dibromomaleimides를 사용 하 여 살 균 소 Qβ를 기반으로 한 VLP의 노출된 이황화 결합 용 매에서 새로운 기능을 소개. 또한, 최종 제품은 형광, 추적이 가능한 생체 외에서 프로브를 상업적으로 사용할 수 있는 필터 세트를 사용 하 여 생성의 이점이 있다 이다.

Dibromomaleimide 파생 상품 dibromomaleimide 무수 물 및 1 차 아민15사이 응축 반응을 통해 합성 될 수 있다. 또는 N-methoxycarbonyl 활성화 3, 4-dibromomaleimide를 사용 하 여 가벼운 합성 방법16 methoxypolyethylene 글리콜 (PEG) 수익률 DB-페그 (그림 1)에 반응 하 여 여기 악용 했다. NMR 화합물 구조 (그림 2)를 식별 하?...

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

Dibromomaleimide-아 황산 화학에 의해 형광 PEGylated 바이러스 같은 입자 활용 유도 만들기

여기, 선물이 붙일 functionalize dibromomaleimide와 Qβ VLP에 disulfides에 절차. 우리는 Qβ 식 및 정화, dibromomaleimide 기능성된 분자의 합성 및 dibromomaleimide와 Qβ 사이 활용 반응 설명합니다. 결과 노란색 형광 활용된 입자 셀 안에 형광 프로브로 사용할 수 있습니다.

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

Research

JoVE Journal

Bioengineering

6.8K 조회수.  University of Texas at Dallas. 여기, 선물이 붙일 functionalize dibromomaleimide와 Qβ VLP에 disulfides에 절차. 우리는 Qβ 식 및 정화, dibromomaleimide 기능성된 분자의 합성 및 dibromomaleimide와 Qβ 사이 활용 반응 설명합니다. 결과 노란색 형광 활용된 입자 셀 안에 형광 프로브로 사용할 수 있습니다.

비디오: 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.

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리

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

연구

면역학 및 감염병학

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.

에 대한 바이러스 나노 입자<em&gt; 생체에</em&gt; 종양 이미징

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

생체공학

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.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

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 클릭 화학을 통해 바이러스성 표면 Chemoselective 변경

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.

형광 태그 바시니아 바이러스 단백질의 효율적인 생성을위한 방법론

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.

단일 입자 수준에서 바이러스성 퓨전 속도론의 측정 방법

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

생물학

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.

간염 E 바이러스 나노 화학 활용 방법을 사용 하 여의 표면 기능화

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.

생성, 증폭 및 재조합 호흡 Syncytial 바이러스의 적정

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.

단일 비리온 원자력 현미경 검사및 초분해성 형광 이미징을 위한 샘플 준비

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.

Dibromomaleimide-아 황산 화학에 의해 형광 PEGylated 바이러스 같은 입자 활용 유도 만들기

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