黃瓜花葉病毒
ATCC? Number:PVAS-549?
Organism: Cucumber mosaic virus deposited as Cucumber mosaic cucumovirus
Designation: CMV-48 [CMV Antiserum]
Depositors: CC Jacono, FW Zettler
Biosafety Level:1
Shipped: freeze-dried
Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.
Comments: Cucumber, spinach, muskmelon, pumpkin, squash, watermelon, cowpea, celery, carrot Virus culture is free of satellite RNA. Host plant from which virus was purified: Cucurbita pepo
References: 56206: . . CMI/AAB Descriptions of Plant Viruses 213: 1979..
方法：（黃瓜病葉勻漿粗病毒提取并濃縮后）
Ⅰ. CSCL自形成梯度
用平均密度近1.37的CSCL，（每毫升加0.5克），日立CP100MX離心機(jī)，P100AT2轉(zhuǎn)頭，6.5PA管，樣品量0.5～1ml均勻混在 CSCL中，加滿離心管， 78，000rpm ，離心6小時(shí)，20℃，加速9減速5。離心后CMV沉降在1.37附近，接近離心管中部，如果在DGF—U用上取法連接帶流動(dòng)池的分光光度計(jì)測(cè)OD，可得到沿管長(zhǎng)分布的OD—管長(zhǎng)度曲線，1.37附近的峰就是CMV峰，透析后得純CMV，CSCL可按下法回收。（見(jiàn)附件）如沒(méi)有DGF—U（日立自動(dòng)梯度儀），可用手工做，每管分成50～100管，分別測(cè)OD繪曲線圖，確定CMV所在的收集離心管。
Ⅱ. Percoll自形成梯度
CMV在Percoll中浮密度大約是1.06～1.065。先用二管試驗(yàn)，P70AT轉(zhuǎn)頭，用初密度為1.06的等密度Percoll，［用2.5份Percoll 、7.5份0.25M Sucrose配成Percoll等滲液，每管總量36ml，即Percoll 9ml，進(jìn)口分裝的（sigma）0.25M 蔗糖27ml］，樣品2ml混在Percoll梯度液中，100，000xg（31，500rpm），50分鐘20℃，加速9，減速5
Ⅲ. CMV在蔗糖中浮密度我不確定，估計(jì)在1.15～1.20之間。您可以先制備一個(gè)1.05～1.25的密度梯度（先做1.05，1.10，1.15，1.20，1.25，計(jì)算容量，靜置過(guò)夜即可成連續(xù)梯度）用P40ST轉(zhuǎn)頭，13mlPA管，梯度液約11ml，梯度形成后加樣至距管口3mm，200，000xg（33，500rpm），5℃，離心1小時(shí)，加速7，減速5。離心后用上取法每管收集成50管，分別測(cè)OD，繪成OD—管數(shù)曲線，可找到CMV峰并確定它在哪個(gè)收集管中。
建議：
1. 你可以看到，如果沒(méi)有自動(dòng)梯度儀，你必須每次分別測(cè)50～100個(gè)樣品的OD再繪成曲線根據(jù)峰位置確定CMV所在的收集管。如果你有日立DGF—U，再加一個(gè)帶流動(dòng)的分光光度計(jì)和分部收集器（國(guó)產(chǎn)）就可以組成完全自動(dòng)化的收集線。
2. 做試驗(yàn)前建議先看看我的文章（用戶培訓(xùn)的19篇中第2，3，3a及18篇等。搞清楚后再做實(shí)驗(yàn)。
3. 以上推薦的方法用于初試，確定了CMV在CSCL，Surcrose，Percoll中的浮密度準(zhǔn)確值后可用階梯梯度進(jìn)行容量較大的分離（過(guò)程簡(jiǎn)化）
4. 粗離：黃瓜花葉搗碎過(guò)濾后，高速離心機(jī)8x50ml轉(zhuǎn)頭，4，000rpm，5℃，20分，去沉淀，上清液濃縮后加緩沖液用于超離。
附：CsCl回收方法：
Protocal of recovery of caesium chloride after use ：
1. Starting with about a litre of CsCl gradient solution，extract it six times with an equal volume of acetone.
2. Filter off the crystals and suck dry.
3. Separate the aqueous filtrate from the acetone and heat the remaining aqueous solution in a fume hood with stirring to reduce the volume to about 150 ml.
4. Allow the solution to cool and filter off a second crop of crystals.
5. Combine the first and second crop of crystals and heat them in a silica dish to 600℃ to burn off all the organic material.
6. Dissolve the CsCl in a minimal amount (4-500ml) of distilled water and filter through Whatman No 54 filter paper to remove the carbon.
7. Concentrate the filtrate by heating in an evaporating dish and when a skin of crystals starts to form on the surface allow the solution to cool.
8. Filter off the crystals and concentrate the filtrate further by repeating Step 7.
9. Filter off a second crop of crystals，discard the filtrate，combine the two crops of crystals，and dry them at 130℃.