应用差速离心法提纯禽脑脊髓炎病毒及电镜观察

DIFFERENTIAL CENTRIFUGATION PURIFICATION AND ELECTRON MICROSCOPE EXAMINATION OF AVIAN ENCEPHALOMYCLITIS VIRUS

为了建立一种有效的方法分离禽脑脊髓炎病毒(A v ian Encepha lom ye litisV irus,AEV),本实验通过SPF鸡胚首先来增殖AEV.将1:5倍稀释的AEV-NH 937株和阴性对照磷酸缓冲盐溶液分别经卵黄囊接种于6日龄SPF鸡胚,继续孵化10d后,收集尿囊液.比较接种组和健康对照组鸡胚的大小,结果显示,健康对照组鸡胚明显大于接种组.取接种组的胚脑、消化道和胰腺,经匀浆后与同组尿囊液配成1:5的组织悬液.应用差速离心法从该组织悬液中分离纯化AEV,经纯化的AEV被磷钨酸染色后,电镜下可观察到大量AEV粒子.结果表明,差速离心法提纯禽脑脊髓炎病毒效果良好.

In order to establish an effective method for isolation of avian encephalomyelitis virus, AEV NH-937 strain was multiplicated in chicken embryos. A 1 ： 5 dilution of isolate-NH937 of AEV was noculated to susceptible 6-day-old chicken embryos of the test group while phosphate-buffered saline （PBS） was inoculated to those of the control group. After 10-days＇ incubation,the embryos were killed and allantoic fluid was collected. A comparison of chicken embryo size was made between the test group and control group. Results showed that the size of the control group＇s embryos was obviously bigger than that of the test group＇s. Embryo brains,digestive tract and pancreas of the test group were collected. The tissue homogenate of the test group was diluted 1 ： 5 with allantoic fluid of the same group. By means of differential centrifugation, virions were isolated from the tissue homogenate and purified. Prepared virions were stained with phosphotungstic acid. Then,an electron microscope was used for viral morphologic characterization. Numerous virions were seen under the electron microscope. Results show that this method is convenient and effective.