小麦梭条斑花叶病毒(WSSMV)提纯、粒子性质和血清学

PURIFICATION, SOME PROPERTIES AND SEROLOGY OF WHEAT SPINDLE STREAK MOSAIC VIRUS(WSSMV)

采用冰冻患病小麦叶片,经0.5M磷酸钾缓冲液pH7.0抽提,1/4体积四氯化碳澄清,6%聚乙二醇,3%NaCl和1%Triton X—100沉淀,二次差速离心(含20%蔗糖垫,0.3%Triton X—100).一次10—50%蔗糖密度梯度离心,最后经一次超速离心浓缩,成功地获得部分提纯制剂。经电镜观察,可见大量线状病毒粒子,很少有杂质。病毒粒子在中性条件下稳定,在酸性条件下变形。病毒粒子直径13—14nm.长度250—1800nm,其中在300—400nm,550—700nm之间具有二个主要分布峰。经紫外读数,病毒制剂表现为典型的核蛋白吸收特性.其中OD260/OD244为1.19,OD260/OD280为1.29。病毒粒子外壳蛋白有二个组分,分子量分别约为30kd和27kd。在免疫电镜实验中,小麦梭条斑花叶病毒(WSSMV)和小麦黄花叶病毒(WYMV)抗血清不仅均能显著而有效地捕获汁液中的WSSMV粒子,而且还可在粒子外面包上一层厚厚的抗血清外套。

Wheat spindle streak mosaic virus (WSSMV) was purified from frozen infected wheat leaves by extracting in 0.5M potassium phosphate buffer pH 7.0, clarifying in 1/4 v carbon tetrachloride, precipitating with 6% PEG(wt. 6000),3% NaCl added 1% Triton X-100, followed by two cycles of differential centrifugation with 20% sucrose cushion containing 0.3% Triton X-100 and centrifuged in 10-50% sucrose density gradients and finally concentrated by a ultracentrifugation. The purified preparation contained filamentous particles with a bimodal length distribution of 300-400 nm and 550-700 nm, and showed a typical UV spectrum of a nucleoprotein with max. and min. absorption at 260 nm and 244 nm respectively. The ratios of OD_(260)/OD_(244), OD_(260)/ OD_(280) was 1.19 and 1.29 each, The virus has two components of coat proteins with wt, of c. 30kd and c. 27kd. In ISEM tests, the virus particals were not only trapped effectively from saps but also decorated strongly by both WSSMV-antisera and WYMV-antisera.