摘要
目的:建立新分离呼肠病毒(reovirus)的空斑技术,进行病毒纯化,为呼肠病毒生物学和分子生物学的研究奠定基础.方法: 4株新分离呼肠病毒的早代毒种经L929细胞连续传代,当产生细胞病变后进行空斑试验,加营养琼脂培养6 d后加中性红,24 h内挑取空斑,扩增一次后做PCR鉴定.取呼肠病毒PCR阳性病毒进行第2次克隆纯化.结果:4株新分离呼肠病毒经L929细胞连续传2～4代,均能观察到明显的细胞病变.用10-3和10-4接种L929单层细胞,第6～7天均能产生空斑,空斑呈圆形,直径为0.2～1.0 mm,并分别测定其空斑滴度(PFU/ml).两次纯化后进行PCR检测,呼肠病毒基因扩增阳性、脊髓灰质炎病毒基因扩增阴性.结论:4株呼肠病毒的空斑出斑规律,通过两次克隆,获得纯化的病毒.
Objective：To develop a plaque assay for the new isolated reovirus strains, purify them, and lay foundation for study of their biological and molecular biological characters. Methods： The early generations of four new isolated reovirus strains serially passaged through L929 cells, and the plaque test was carried out after cytopathic effect（CPE） appeared. Neutral red was added to the cell cultures on the sixth day after nutritious agar had been added. Then plaques were selected within 24 hours, multiplied once and identified by PCR reaction. The PCR positive generations were selected to clone and purify once again. Results：The obvious CPE of the four reovirus strains were observed after 2 to 4 serial passage through L929 cells. The plaques of these reovirus were revealed after 6 to 7 days on L929 cell monolayer, which was infected with 10^-3 to 10^-4 diluted reovirus. The plaques were round in shape, 0. 2 - 1.0 mm in diameter, and their plaque titers （PFU/ ml） were measured separately. Purified viruses, with reovirus gene positive and poliovirus gene negative, were all obtained through purification for two times. Conclusion ：The plaque appears regularly for all four new isolated reovirus strains, and purified reovirus strains are obtained by cloning for two times.
出处
《军事医学科学院院刊》
CSCD
北大核心
2005年第5期418-420,共3页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金课题(30471555)
军事医学科学院院创新启动基金(200408183)
