【摘要】 本研究工作中,建立了一个有效的甜菜坏死黄脉病毒的分离提纯程序,解决了该病毒粒体易于聚集难以提纯的问题,其操作要点是,（1）通过Sepharose 2B柱层析代替超离心,有效地除去一些小分子量核酸杂质;（2）经PEG再次沉淀浓缩后,调整pH至酸牲（pH3.0）,使病毒充分悬浮以减少凝聚;（3）在病毒等电点（pH4.8～4.9）条件下,进一步沉淀以纯化病毒。根据病毒提取物的OD260/OD280比值,算出核酸含量约4.5％。核酸电泳出现4条带,分子量分别为:2.25×10^?,1.8×10^?,1.05×10^?,0.75×10^?道尔顿。病毒提取物经超速离心出现4个界面,沉淀系数分别为,200.8S,165S,125.8S,100S。说明甜菜坏死黄脉病毒可能是4组分病毒粒体。病毒粒体含一蛋白亚基,分子量约为2.05±0.05×10⁴道尔顿,由16种共199个氨基酸组成。更多还原

【Abstract】 An improved purification procedure for Beet Necrotic Yellow Vein Virus ( BNYVV ) was described, including the following four important steps. ( 1 ) Fresh Tetragonia expeusa infected leaves harvested right after the symptom appeared were homogenized with borate buffer. ( 2 ) The rough extract was purified by using Sepharose 2 B chromatography instead of ultracentrifu-gation to remove some small molecular weight nucleic acids. ( 3 ) Concentrated with 6% PEG again, and adjusted the preparation to acidity to avoid virus aggregate. ( 4 ) The preparation was further purified by means of isoe-lectric point precipitation, the isoeletric point of BNYVV was at pH 4.8-4.9. After agarose gel electraphoresis, four nucleic acid bands were detected. Their molecular weight were 2.25× 106, 1.8×108, 1.05 × 106 and 0. 75 × 108 Dalton respectively. During ultracentrifugation, four boundaries were developed in the interface, with sedimentation coefficient of 200.8S, 165S, 125.8S a nd 100S respectivly. We suggest that BNYVV is a four component virion contai- ning a single species of coat protein subunit with MW about 2.05±0.05×104 Dalton. The amino acied composition of the coat protein was analysed.更多还原