西瓜花叶病毒-2新疆株的纯化

PURIFICATION OF WATERMELON MOSAIC VIRUS-2 XINJIANG

研究了西瓜花叶病毒 2新疆株的纯化 .感病西葫芦叶组织在含有w =0 .2 %巯基乙醇和 0 .0 1mol·L- 1乙二胺四乙酸二钠 (EDTA)的 0 .5mol·L- 1的磷酸钾缓冲液中匀浆 ,用匀浆体积 1/4的氯仿澄清 ,粗提取液中的病毒经含有 φ =1%TritonX 10 0和w =3%NaCl的w =6 %聚乙二醇沉淀 ,并以含有 0 .5mol·L- 1尿素、w =0 .1%的巯基乙醇和 0 .0 1mol·L- 1EDTA的 0 .5mol·L- 1磷酸钾缓冲液悬浮 ,后经含有w =0 .1%巯基乙醇和 0 .0 1mol·L- 1EDTA的w =10 %～ 4 0 %蔗糖密度梯度离心 ,获得了经电子显微镜、紫外检测和接种试验证实侵染活性为 94 .8%的纯化病毒 .纯化病毒的A2 6 0 /A2 80 =1.2 1,A2 6 0 /A2 4 5=1.15 ,纯化病毒产量约为每kg西葫芦感病叶组织中可提取 16mg .

A study on the purification procedure for watermelon mosaic virus 2 Xinjiang(WMV 2 XJ) is carried out. WMV 2 XJ infected pumpkin leaves are homogenized with 0.5 mol·L -1 potassium phosphate buffer pH7.5 containing w =0.2% mercaptoethanol and 0.01 mol·L -1 editic acid. After clarification with chloroform ( V /4 of homogenate), the virus in the crude extracts is concentrated by precipitation with w =6% polyethylene glycol containing φ =1% Triton X 100 and w =3% NaCl and resuspended with 0.05 mol·L -1 potassium phosphate buffer containing 0.5 mol·L -1 urea, w =0.1% mercaptoethanol and 0 01 mol·L -1 editic acid and then followed by w =10%～40% sucrose density gradient centrifugation containing w =0.1% mercaptoethanol and 0.01 mol·L -1 editic acid. Highly purified WMV 2 XJ preparation with high infectious activity(94.8%), confirmed by electron microscopy, ultraviolet absorption spectrum and inoculation test, is achieved by this purification. The A 260 / A 280 ratio and A 260,max / A 245,min for the purified WMV 2 XJ preparation are 1.21 and 1.15 respectively. The yield of purified WMV 2 XJ is about 16 mg·kg -1 infected pumpkin tissue.