Простая и надежная В естественных условиях И В пробирке Подход к изучению вируса Ассамблеи
Summary
Простой, эффективный и надежный способ синхронизировать поставки несколько вирусных компонентов в клетках растений с помощью<em> Agrobacterium</em> Опосредованного переходные выражения описаны. Такой подход дает возможности изучения репликации encapsidation следует<em> В пробирке</em> Сборка не-вирусных компонентов в геном обедненного оптических вирусных призраки подходит для биомедицинских приложений.
Abstract
In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.
Protocol
Discussion
Agroinfiltration здесь подход может широко применяться для широкого спектра вирусов растений. Отличительная особенность этого подхода синхронизированы поставку нескольких agroconstruct в ту же камеру, что является главным недостатком, обычно связанные с обычно используется механический посев ви?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить нескольких членов лаборатории за их ценные предложения по развитию agroinfiltration и в лабораторных анализах сборки. Эта работа финансируется за счет гранта из Калифорнийского университета. Это исследование было поддержано в части за счет гранта Национального научного фонда (CBET-1144237).
Materials
| Name of the reagent | Company | Catalog number |
| MES Sodium salts | Sigma-aldrich | M2993 |
| Indocyanine green | Sigma-aldrich | I2633 |
| Beckman ultracentrifuge | Beckman Coulter | Model: L8-70M |
| Centricon-100 column | Millipore | YM-100 |
| Spectrophotometer | Cary 50, Varian Inc. | Part number 10068900 |
| Spectrofluorometer | Fluorolog 3, Jobin-Yvon. | Part number FL3-21 |
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