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本文提出了一个改进了的大麦黄花叶病毒提纯方法。病大麦叶片在高浓度(0.5M)磷酸钾缓冲液pH7.0(内含0.1%ME,0.01M EDTA)中匀浆,经1/4体积四氯化碳澄清后,病汁液用6%PEG,3%NaCl和1%TritonX-100混合物沉淀,高浓度(0.5M)同种缓冲液(含0.5M尿素,0.1%ME和0.01M EDTA)悬浮,接着进行20%蔗糖垫(含0.3%Triton X-100)超迷离心去除寄主细胞成分,10—40%蔗糖密度梯度离心进一步纯化。所获得的BaYMv提纯制剂A260/A280,A260/A240比值分别为1.20和1.04,纯病毒产量约为5.5—8.0mg/kg病叶。提纯病毒制剂在电镜下看不到有寄主杂质存在。
This article presents an improved method for the purification of barley yellow mosaic virus. Diseased barley leaves were homogenized in high concentration (0.5 M) potassium phosphate buffer pH 7.0 (containing 0.1% ME, 0.01 M EDTA). After clarification with 1/4 volume of carbon tetrachloride, the diseased juice was treated with 6% PEG , A mixture of 3% NaCl and 1% Triton X-100, suspended in a high concentration (0.5 M) of the same buffer containing 0.5 M urea, 0.1% ME and 0.01 M EDTA, followed by a 20% X-100) superficially centrifuged to remove the host cell components, 10-40% sucrose density gradient centrifugation for further purification. The obtained BaYMv purified preparation A260 / A280, A260 / A240 ratio of 1.20 and 1.04, respectively, pure virus yield of about 5.5-8.0mg / kg diseased leaves. Purified virus preparations do not see the presence of host impurities under electron microscopy.