【作者】 张桂红；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 采用传代细胞(非洲绿候肾细胞)从猪腹泻粪便中分离出1株猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV) ,命名为PEDV LJB/O3株。该病毒接种Vero细胞时,在其维持液中加入3μg/ml胰酶,通过稳定的细胞病变、病毒的TCID50测定、RT-PCR扩增试验、间接免疫荧光试验、间接ELISA试验等方法对该分离毒进行了鉴定。实验结果表明:病毒盲传20代,未出现明显的病变,第21代48h出现可见CPE, 72h收获病毒液。随着传代代数的增加,出现CPE的时间缩短,细胞病变程度亦随之加剧。该毒株传到40代时,接种病毒的细胞产生规律性病变,病毒培养24h,细胞肿胀变圆,折光性增强,颗粒物质增多;培养36h,细胞大量脱落崩解,细胞间隙变大;培养48h,细胞脱落达到80%,细胞拉成网状,而对照细胞直到培养48h还保持致密的单层,呈长梭形,轮廓清晰,细胞脱落极少。为进一步证明这种病变是由PEDV所引起以及病毒在细胞中得以繁殖,本实验以每隔10个代次采用RT-PCR技术扩增PEDV M基因,对第1、10、20、30和40不同代次的细胞培养物进行的RT-PCR检测,结果均为阳性。间接免疫荧光实验结果表明,经PEDV感染40h后的Vero细胞,在细胞中检测到病毒抗原的存在,可见绿色荧光,而未被PEDV感染的Vero细胞中,看不见任何绿色荧光。间接ELISA检测,P/N平均比值为7.6,判断此细胞培养毒为阳性。对第40代病毒测定TCID50结果为10-5.2/0.1 mL。由以上的试验可以证明从粪便里分离出的这株病毒为猪流行性腹泻病毒,且此病毒能在含有胰酶维持液的Vero细胞培养物上生长繁殖。更多还原

【Abstract】 A porcine epidemic diarrhea virus (PEDV) designated LJB/03 was isolated from the feces of diarrheaed piglets and cultured in Vero cell propagated in maintenance medium(MM) containing 3μg/ml trypsin.The isolated virus was identified by stable cytopathic effects (CPE), TCID50, RT-PCR, immunofluorescence, ELISA ect. The experimental results indicated that the bland passages of 20 genrations were undertaken untill the CPE could be seen on 21 genrations in 48 h and were harvested in 72h. CPE was shorten and intensified with increased passages.On passage 40 in Vero cell culture, CPE characterized by cellular swelling , refracted reinforcement and particulate matter increaser was detected microscopically after 24h; A great quantity of cells defluxioed and the airspace amang cells was enlarged after 36h; the cells having defluxioed reached 80% and Vero cells reticulated after 48h.But the cell control still retained a pyknomonolayer and there was a small quantity of cells having defluxioed . At passage levels 1, 10, 20, 30,and 40, PEDVs were identified through RT-PCR for every 10 passage on the M gene in order to certificated that the CPE caused by PEDV and the virus propagated in Vero cells. The results were masculine.Indirect immunofluorescence test indicated that viral antigen could be detected and could be seen green fluorescence in Vero cells. No green fluorescence could be seen with the negative control of mock-infected Vero cell.Indirect ELISA detected that P/N was 7.6.It was justified that the virus in Vero cell culture was masculine. Titer of the virus was10-5.2TCID50/0.1 ml at 40 passage.Above experiments certified that it was PEDV from the feces and it propagated in Vero cell culture in medium containing trypsin.更多还原