1. Nicotiana benthamiana 식물에서 바이러스 접종과 VLPs 정화 <ol><li> 감자 바이러스 X (PVX) 기반 들고 벡터 plasmids의 상한선 T7-RNA 기록을 생산 MRFV 야생 타입 (중량) 및 Cys-변이 코트 단백질 (CP) 유전자 12 Ambion의 T7-mMessage mMachine 키트를 사용합니다. </li><li> 각 T7의 성적 반응의 경우, N.의 두 완전히 확장 잎의 예방 10 μl 반응과 25에서 빛 (25,000-30,000 룩스) 16 시간 60 %의 습도에서 온실에서 10 일 동안 식물을 길러 benthamiana 20 ° C과 어두운 8 시간 ° C. </li><li> Harves t N. benthamiana 바이러스에 감염된 잎 10 일 후 접종 (DPI)는 얼음에 식물 조직 (50g)와 장소를 무게. </li><li> 0.5 M 오세영 - 구연산 버퍼, pH를 5.5 (사용이 식물 조직 g 당 버퍼의 ML)의 차가운 솔루션의 100 ML의 존재에 믹서기에서 식물 자료를 균질화. </li><li> cheesecl 3 층을 통해 homogenate를 필터링oth 30 분에 27,000 XG에서 원심 분리하여 명확히. 차가운 졸업 실린더로 표면에 뜨는을 전송, 볼륨을 측정하고 차가운 멸균 비이커에 전송할 수 있습니다. 펠렛을 폐기하십시오. </li><li> 4 ° C에서 15 분 동안 저어, 10 % 폴리에틸렌 글리콜 8 천 (PEG 8,000)를 추가하여 표면에 뜨는을 처리, 4에서 추가 45 분에 품다 ° C 바이러스 같은 입자 (VLPs) PEG 행렬을 침전 할 수 있습니다. </li><li> 4 ° C에서 20 분에 27,000 XG에서 샘플을 원심 분리기하고 표면에 뜨는을 제거합니다. </li><li> 0.05 M 오세영 - 구연산 버퍼, 5.5 2 % 포함 튼 X-100 산도 8 ML을 추가하여 펠렛을 처리합니다. 소용돌이 원심 분리기 튜브는 잘 펠릿을 해제합니다. 차가운 멸균 비커에 펠렛과 버퍼를 전송합니다. 같은 버퍼 2 ML 즉시 튜브를 씻어 비커에 추가 할 수 있습니다. 펠릿은 (일반적으로 1 시간) 용해 아르 ° C까지 4에 정지 저어. </li><li> 27,000 XG에서 5 분 동안 원심 분리하여 명확히, 펠렛을 버리고, 및 P4의 2 시간에 140,000 XG에서 원심 분리하여 rocess는 표면에 뜨는 ° C. </li><li> 한 ML 0.05 M 오세영 - 구연산 버퍼, 산도 5.5에서 펠렛을 Resuspend하고 0.05 M 오세영 - 구연산 버퍼, 산도 5.5에서 만든 10-40%의 자당 기울기 위의 정지를 놓습니다. </li><li> 4에 140,000 XG에 3 시간의 기울기를 원심 분리기 ° C. </li><li> 원심 분리기 튜브의 상단을 통해 빛을 밝히고 튜브의 상단에서 4cm의 광 산란 상단 밴드를 수집, 4에서 140,000 XG에서 3 시간에 0.05 M 오세영 - 구연산 버퍼, 산도 5.5 및 원심 분리기로 희석 ° C. </li><li> 최대 6 개월까지에 대해 4 ° C에서 멸균 물과 매장의 0.5 ML에서 펠렛을 Resuspend. </li><li> 브래드 포드 단백질 분석을 수행하여 VLPs 농도를 Quantitate. VLPs의 수율은 식물 조직의 1 ~ 3 μg / g입니다. </li></ol> 2. VLPs의 Fluorescein-5-maleimide-라벨 반응 <ol><li> 50 MM 나트륨 phosphat에 fluorescein-5-maleimide (427.37 g / 사마귀)의 1 ㎜ 솔루션을 준비전자 버퍼, 산도 7.0, 1 MM EDTA (에틸렌 다이아 민 테트라 초산). 소용돌이를 사용하여 혼합하고 완전한 해산을 확인합니다. 알루미늄 호일을 사용하여 빛으로부터 관을 보호합니다. </li><li> 단계 1과 믹스에서 생산 VLPs에 fluorescein-5-maleimide을 추가합니다. sulfhydryls의 몰 금액에 대한 fluorescein-5-maleimide의 25 배를 초과 몰을 사용합니다. 일반적으로, 1 μg / μl과 fluorescein-5-maleimide 솔루션 60 μl의 농도에 VLPs의 30 μl를 사용하여 허용 결과를 생산하고 있습니다. </li><li> 4 ° C.에서 상온이나 야간 2 시간의 반응을 품다 </li><li> 50 mm의 최종 농도에 dithiothreitol (DTT)를 추가하여 반응을 종료 할 수 있습니다. </li><li> 제거되지 않은 반응 상온에서 1,500 XG에서 2 분을위한 탈염 스핀 컬럼 (열 과학 Inc의, 록 포드 IL)과 원심 분리를 사용하여 fluorescein. </li><li> SDS-PAGE 분석은 각 반응의 10 μl로 2 X SDS-PAGE Laemmli 샘플 버퍼 10 μl를 추가합니다. 포기 개월에 대해 4 ° C에서 빛으로부터 보호 표시된 단백질을 저장최대 3 개월까지 -20 ° C에서 단일 사용 aliquots 인치 </li></ol> 3. 비오틴의 설립의 반응과 결정에 라벨 지정 <ol><li> 물에서 인산염 버퍼 생리 (PBS) 1 단계에서 생산 VLPs의 산도 7.0에 버퍼 교환을 수행하는 탈염 스핀 열을 (열 과학 주식회사, 록 포드 IL)을 사용합니다. </li><li> 아래로 pipetting과에 의해 maleimide-PEG2 - 비오틴과 혼합 없음 - 체중에 PBS의 190 μl를 추가하여 maleimide-PEG2 - 비오틴의 20 MM 주식 솔루션을 준비합니다. </li><li> VLPs 및 혼합에 maleimide-PEG2 - 비오틴을 추가합니다. VLPs 솔루션을 시약의 10 배 어금니 초과를 사용 ≤ 2 밀리그램 / ML. </li><li> 4 시간 동안 실온에서 반응을 품다. </li><li> 제거되지 않은 반응 상온에서 1,500 XG에서 2 분을위한 탈염 스핀 컬럼 (열 과학 Inc의, 록 포드 IL)과 원심 분리를 사용하여 maleimide-PEG2 - 비오틴합니다. </li><li> 피어스 비오틴 Quantitation 키트 (열 과학)과 구경을 사용하여 VLPs의 첩자 당 비오틴의 첩자를 결정제조업체의 지시 사항을 llowing. </li></ol> 4. Cys 2 VLPs와 F 펩타이드의 상호 연결 <ol><li> 2 단계 (Cys 2 VLPs)에 교차 연결에 사용할 수 시스테인 잔류 물이 발생 VLPs는 다음 단계에 사용됩니다. 디메틸 sulfoxide의 680 μl에 NHS-PEG4-maleimide의 가교제의 10 밀리그램을 용해하여 신선한 간 연결 재고 솔루션 (28.63 MM)를 준비합니다. </li><li> 17 (F)를 분해 - 아미노 활용 버퍼 50 μl (1 개 PBS, pH를 7.2, 1 MM EDTA (에틸렌 다이아 민 테트라 초산))에서 산 긴 펩타이드 (LLPNMOKDKEACAKAPL) 0.1 mm의 농도에. </li><li> 1 ㎜ 최종 농도 (이는 펩타이드 솔루션의 0.1 MM의 10 배를 초과 몰입니다)에 용해 된 펩타이드 (단백질 NH2)의 50 μl에 가교제 4 μl를 추가합니다. </li><li> 4 ° C.에서 2 시간의 반응을 품다 </li><li> 1500 XG에서 원심 분리하여, 활용 버퍼 (1 MM EDTA (에틸렌 다이아 민 테트라 초산) 1 개 PBS, pH를 7.2)와 equilibrated 탈염 열 (열 과학 주식회사, 록 포드 IL)를 사용하여 교차 연결된 펩타이드를 정화상온에서 1 분에. </li><li> VLPs (단백질 SH) 및 반응과 VLPs과 펩타이드 사이의 분자량의 차이에 참여 thiols와 활성 아민의 수에 의해 결정 몰 비율로 교차 연결된 펩티드 (단백질 NH2)를 결합 반응 믹스를 설정합니다. </li><li> 4 ° C.에서 2 시간 동안 실온에서 반응 혼합물을 품다 </li><li> 서양 얼룩 분석을 위해, Laemmli 버퍼, 10 분에 종기가있는 결과로 복합 제품 1:1 혼합 10~20% 트리스 - 글리신 젤 (Invitrogen) 미리 캐스트에 SDS-PAGE를 수행에 electroblotting 다음 니트로 셀룰로스 막 (Invitrogen). 1 개 PBS, pH를 7.2이 0.05 % 십대 초반 - 2​​0 (PBS-십대 초반)와 적절한 인산 가수 분해 효소 - 라벨 차 항체에 이어 펩타이드 특정 항체와 5% 탈지 분유와 프로브를 포함하는으로 세포막을 차단합니다. </li></ol>

<ol>
	<li>Barnhill, H., Reuther, R., Ferguson, P. L., Dreher, T. W., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnip+yellow+mosaic+virus+as+a+chemoaddressable+bionanoparticle.">Turnip yellow mosaic virus as a chemoaddressable bionanoparticle.</a> Bioconj. Chem. 18, 852-859 (2007).</li><li>Chapman, S., Kavanagh, T., Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+X+as+a+vector+for+gene+expression+in+plants.">Potato virus X as a vector for gene expression in plants.</a> Plant J. 2, 549-557 (1992).</li><li>Chen, C., Kwak, E. S., Stein, B., Kao, C. C., Dragnea, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+gold+particles+in+viral+capsids.">Packaging of gold particles in viral capsids.</a> J. Nanosci. Nanotechnol. 5, 2029-2033 (2005).</li><li>Fowler, C. E., Shenton, W., Stubbs, G., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+liquid+crystals+as+templates+for+the+interior+design+of+silica+mesophases+and+nanoparticles.">Tobacco mosaic virus liquid crystals as templates for the interior design of silica mesophases and nanoparticles.</a> Advanced Materials. 13, 1266-1269 (2001).</li><li>Gazit, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biomolecular+templates+for+the+fabrication+of+metal+nanowires.">Use of biomolecular templates for the fabrication of metal nanowires.</a> FEBS. J. 274, 317-322 (2007).</li><li>Gillitzer, E., Wilts, D., Young, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+a+viral+cage+for+multivalent+presentation.">Chemical modification of a viral cage for multivalent presentation.</a> Chem. Commun. , 2390-2391 (2002).</li><li>Hammond, R. W., Hammond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+capsid+proteins+assemble+into+virus-like+particles+in+Escherichia+coli.">Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli.</a> Virus Res. 147, 208-215 (2010).</li><li>Hermamson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate techniques. , (1991).</li><li>Kaiser, C. R., Flenniken, M. L., Gillitzer, E., Harmsen, A. L., Harmsen, A. G., Jutila, M. A., Douglas, T., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+studies+of+protein+cage+nanoparticles+demonstrate+broad+tissue+distribution+and+rapid+clearance+in+vivo.">Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.</a> Int. J. Nanomed. 2, 715-733 (2007).</li><li>Knez, M., Bittner, A. M., Boes, F., Wege, C., Jeske, H., Maisse, E., Kern, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotemplate+synthesis+of+3-nm+nickel+and+cobalt+nanowires.">Biotemplate synthesis of 3-nm nickel and cobalt nanowires.</a> Nano Lett. 3, 1079-1082 (2003).</li><li>Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+CuCl2+concentration+on+the+aggregation+and+mineralization+of+Tobacco+mosaic+virus+biotemplate.">Effect of CuCl2 concentration on the aggregation and mineralization of Tobacco mosaic virus biotemplate.</a> J. Colloid. Interface. Sci. 297, 554-560 (2006).</li><li>Lvov, Y., Haas, H., Decher, G., Mohwald, H., Mikhailov, A., Mtchedlishvily, B., Morgunova, E., Vainshtein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successive+deposition+of+alternate+layers+of+polyelectrolytes+and+a+charged+virus.">Successive deposition of alternate layers of polyelectrolytes and a charged virus.</a> Langmuir. 10, 4232-4236 (1994).</li><li>Natilla, A., Hammond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+virus-like+particles+expressed+in+tobacco+plants:+a+new+platform+for+cysteine+selective+bioconjugation+peptide+display.">Maize rayado fino virus virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display.</a> J. Virol. Methods. 178, 209-215 (2011).</li><li>Rae, C. S., Khor, I. W., Wang, Q., Destito, G., Gonzalez, M. J., Singh, P., Thomas, D. M., Estrada, M. N., Powell, E., Finn, M. G., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+trafficking+of+plant+virus+nanoparticles+in+mice+via+the+oral+route.">Systemic trafficking of plant virus nanoparticles in mice via the oral route.</a> Virology. 343, 2224-2235 (2005).</li><li>Raja, K. S., Wang, Q., Gonzalez, M. J., Manchester, M., Johnson, J. E., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+virus-polymer+materials.+Synthesis+and+properties+of+PEG-decorated+Cowpea+mosaic+virus.">Hybrid virus-polymer materials. Synthesis and properties of PEG-decorated Cowpea mosaic virus.</a> Biomacromolecules. 4, 472-476 (2003).</li><li>Royston, E., Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silica-coated+Tobacco+mosaic+virus.">Characterization of silica-coated Tobacco mosaic virus.</a> J. Colloid Interface Sci. 298, 706-712 (2006).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+in+the+Tobacco+mosaic+virus.">Dual-surface modification in the Tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Young, M., Willits, D., Uchida, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+as+biotemplates+for+materials+and+their+use+in+nanotechnology.">Plant viruses as biotemplates for materials and their use in nanotechnology.</a> Annu. Rev. Phytopathol. 46, 361-384 (2008).</li></ol>

간행물의 상용 제품의 상표 이름의 언급은 특정 정보를 제공 할 목적으로 만 있으며, 농업의 미국 부에 의해 추천이나 승인을 의미하지 않습니다.

여기에 제시된 방법은 공장 제작 VLPs의 표면에뿐만 아니라 다른 단백질 단지에 존재 반응 cysteine​​s 매우 민감하고 빠른 분석이 가능합니다. Maleimides 안정적인 thioether 채권을 형성하기 위해 무료로 설프 하이 드릴 함유 분자와 반응하여 thiol 특정 시약입니다. 이 방법은 다른 아미노산과 상호 작용에 관여하지 설프 하이 드릴 그룹과 반응 할 수있는 maleimides의 특이성에 그립니다. VLPs의 기본 구조...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Thinwall, 울트라 클리어 튜브 </td><td> Beckman </td><td> 344059 </td><td></td></tr><tr><td> mMESSAGE mMACHINE T7 키트 </td><td> 생활 Tecnologies </td><td> AM1344M </td><td></td></tr><tr><td> Fluorescein-5-Maleimide </td><td> 온도 과학 생활 기술 </td><td> 46,130 F150 </td><td> 46130은 F150와 함께 주문 대신 부족합니다 </td></tr><tr><td> 피어스 비오틴의 Quantitation 키트 </td><td> 열 과학 </td><td> 28,005 </td><td></td></tr><tr><td> EZ-링크 Maleimide - PEG2 - 비오틴, 형식 없음 - 체중 </td><td> 열 과학 </td><td> 21,901 </td><td></td></tr><tr><td> SM (PEG) N Crosslinkers </td><td> 열 과학 </td><td> 22,107 </td><td></td></tr><tr><td> 10-20% 트리스 - 글리신 젤 </td><td> Invitrogen </td><td> EC61352 </td><td></td></tr><tr><td> Laemmli 버퍼 </td><td> 바이오 RAD </td><td> 1610737 </td><td></td></tr><tr><td> 트리스 글리신 SDS는 버퍼를 실행 </td><td> Invitrogen </td><td> LC2675 </td><td></td></tr><tr><td> 트리스 글리신 전송 버퍼 </td><td> Invitrogen </td><td> LC3675 </td><td></td></tr><tr><td> 니트로 셀룰로스 막 필터 종이 샌드위치 </td><td> Invitrogen </td><td> LC2001 </td><td></td></tr><tr><td> 토끼 IgG에 항체를 정제 인산 가수 분해 효소 레이블 선호도 </td><td> Kirkegaard 및 페리 연구소 </td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP 인산 가수 분해 효소 기판 </td><td> Kirkegaard 및 페리 연구소 </td><td> 508107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

바이러스 속성과 물리 및 물리적 특성을 모방하고 활용하는 것은 세계에서 가장 급박 한 문제 중 일부에 대한 해결책을 제공 할 약속을 보유하고 있습니다. 깎아 지른듯한 범위와 자신의 흥미 특성과 결합 바이러스의 종류는 잠재적으로 바이러스 기반 기술의 응용 프로그램 끊임없는 기회를 제공합니다. 바이러스는 온도와 pH의 조건의 다양한 미만의 이산 모양과 크기, 대칭의 특이성, polyvalence, 안정적인 특성을 가진 입자로 자기 조립 할 수있는 능력을 갖추고 있습니다. 당연히, 속성의 이러한 놀라운 범위, 바이러스는 biomaterials 9, 백신 14, 15, 전자 재료, 화학 도구 및 분자 전자 컨테이너 4, 5, 10, 11, 16, 18, ​​12에 사용하기 위해 제안합니다. 나노 기술에 바이러스를 이용하기 위해, 그들은 새로운 기능을 가르친다하기 위해 자연적인 형태에서 수정해야합니다. 이 도전 홍보ocess는 유전 바이러스 게놈의 수정 및 화학적으로 바이러스 입자 반응 그룹 8 외국인 또는 원하는 분자를 부착 등 여러 가지 메커니즘을 통해 수행 할 수 있습니다. 바이러스를 수정하는 기능은 주로 바이러스의 physiochemical과 물리적 특성에 따라 달라집니다. 또한, 유전자 또는 physiochemical 수정에 나쁜 바이러스 기본 구조 및 바이러스 기능에 영향을주지 않고 수행해야합니다. 옥수수 rayado fino 바이러스 (MRFV) 코트 단백질은 단백질 단백질에 의해 안정화 아르 안정적이고 빈 VLPs을 생산 대장균에 자기 조립 상호 작용하고는 바이러스 기반 기술 응용 프로그램 8에서 사용할 수 있습니다. 담배 공장에서 생산 VLPs는 펩티드의 다양한 종류의 covalently 13 표시 할 수있는 발판으로 조사되었다. 여기, 우리는 바이러스 capsid에있는 용매의 접근이 cysteine​​s 어떤이 modifi 사용할 수 있습니다 결정) 1 단계를 설명양이온, 그리고 2) 수정 capsids에 펩티드를 bioconjugate. 네이티브 또는 mutationally - 삽입 아미노산 잔류 물 및 표준 커플 링 기술을 사용하여 재료의 다양한 종류 등 Brome 모자이크 바이러스 3, 카네이션의 반점 바이러스 12 Cowpea chlorotic 반점 바이러스 6, 담배 모자이크와 같은 식물 바이러스의 표면에 표시되어 바이러스 17, 순무 대가리 노란색 모자이크 바이러스 1, MRFV 13.

N.의 돌연변이 MRFV 코트 단백질 (CP) 유전자의 일시적 표현 PVX 기반의 벡터 생산 VLPs의 benthamiana 공장은 그림 1에 설명되어 있습니다. 수정 MRFV 코트 단백질 유전자는 PVX 기반 벡터, pP2C2S 2, (D. Baulcombe, 세 인스 베리 연구소, 노리치, 영국의 선물)에서 중복 subgenomic의 CP 프로모터의 전사 제어 PCR에 의해 증폭 한 후 배치됩니다.에서가 체외 RNA 전사는 N.을 ?...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

의 시스테인 잔류의 thiol 그룹의 용매 접근성을 분석하는 방법<em&gt; 옥수수 rayado fino 바이러스</em펩타이드 가교 반응에 이어&gt; (MRFV) 바이러스 같은 입자 (VLPs)이 설명되어 있습니다. 방법은 특정 반응에 타겟이 될 수있는 VLPs의 표면에 여러 화학 그룹의 가용성을 활용합니다.

시스테인 잔류 물을 용매 접근성에 대한 분석<em&gt; 옥수수 rayado fino 바이러스</em에서 생산&gt; 바이러스 같은 입자<em&gt; Nicotiana benthamiana</emVLPs에 펩티드의&gt; 식물과 교차 연결

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

analysis-solvent-accessibility-cysteine-residues-on-maize-rayado-fino

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

11.0K 조회수.  Agricultural Research Service, United States Department of Agriculture. 의 시스테인 잔류의 thiol 그룹의 용매 접근성을 분석하는 방법 옥수수 rayado fino 바이러스 (MRFV) 바이러스 같은 입자 (VLPs)이 설명되어 있습니다. 방법은 특정 반응에 타겟이 될 수있는 VLPs의 표면에 여러 화학 그룹의 가용성을 활용합니다.

비디오: Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

에 의해 제작 상피 손상의 분석<em&gt; Entamoeba 아메바</em&gt; 감염

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

연구

면역학 및 감염병학

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

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

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

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

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

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Tetracistronic Minigenomes를 포함하는 바이러스 입자와 바이오 안전성 수준이 조건에서 에볼라 바이러스의 수명주기를 모델링

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

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

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

새로운 바이러스 게놈을 발견 감염 된 잎에서 RNA 분석 원유 Virion 추출에 DNA의 결합 분석

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

바이러스 성 감염 및 초파리 Melanogaster 상호 작용 호스트-바이러스의 분석의 설립

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

선생님 FcγR Cross-linking에 대 식 세포에서 생산의 흐름 Cytometric 측정

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

시스테인 잔류 물을 용매 접근성에 대한 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

에 의해 제작 상피 손상의 분석