中国禽呼肠孤病毒番鸭分离株的纯化及其核酸与结构蛋白分析

将番鸭呼肠孤病毒国内分离株在番鸭成纤维细胞 (DEF)上克隆纯化 3次后 ,在鸡成纤维细胞 (CEF)上增殖 ,收获的细胞培养物经 3次冻融 ,采取差速离心、超速离心、非线性蔗糖密度梯度离心和脱糖处理相结合的方法进行提纯 ;对不同梯度区带的蛋白提取物经电镜观察、毒价(TCID50 )和含毒量测定得到病毒纯化物 ;最后对病毒纯化物进行琼脂糖核酸电泳和SDS PAGE蛋白电泳分析。结果 ,核酸电泳得到 10条RNA带 ,分列呈“334”形式的 3组 ,其迁移率除M 3基因外无明显差异 ;蛋白电泳得到分子质量分别为 14 9.0、12 9.0、111.0、79.0、75 .0、5 0 .0、4 2 .0、39.0、37.0、35 .9、32 .0、2 9.0ku的 12条蛋白带 ,其中 5个蛋白的分子质量与参考株S1133相近。该分离株具有禽类呼肠孤病毒核酸和蛋白电泳图谱的共同特征。

Purification and analysis of genomes and structural proteins of Muscovy duck reovirus isolated in China

The strain of Muscovy duck reovirus isolated in China was multiplicated in semiconfluent monolayers of chicken embryo fibroblast(CEF)by plague technique after purification three times in Muscovy duck embryo fibroblast(DEF). The infected cells was disrupted by three freeze-thaw cycles,and then differential centrifugation, ultracentrifugation and sucrose discontinious gradient ultracentrifugation were adopted to concentrate and purify the virus. Electron microscopy(EM),TCID_(50)test and spectrometer were used to detect the purified virus.The extracted RNA of the virus was analysised with agarose electrophorisis. The result confirms that the genomes of the virus consist of 10 RNA fragments and the rates of RNA migration in agarose gels have no differeneces except forM3 gene. Analysis of the viral polypeptides by SDS-PAGE shows that5 of12 polypeptides molecularweightsoccurring in gels are almost the same as that of avian reovirus S1133 referred as control, and the above results demonstrates that DRV-YHhas a common electrophoretic mobilities of avian reovirus.