【作者】 郑全；

【导师】 王树志；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本研究用吉林省某鹿场母鹿流产胎儿的肝脏病料，经MDBK细胞适应培养，获得一株病毒（命名为MDVCC-deer6）。该分离毒能在MDBK细胞上形成有规律的病变，其病变形态与病变时间同标准株牛病毒性腹泻-粘膜病病毒一致。电镜负染观察，病毒粒子呈典型粘膜病病毒形态。血凝试验表明，该病毒对鸡、兔、猪和绵羊的红细胞均无血凝性；毒力测定，该病毒的TCID₅₀为10^-5.5。理化学研究表明，该病毒对氯仿、乙醚敏感；胰酶试验中，经37℃，1小时处理的病毒，仍然能够在MDBK细胞上生长，但毒力下降3.2个滴度。耐酸性试验中，病毒在pH3.0经37℃作用1小时，毒力下降4.4个滴度；耐碱性试验中，病毒在pH9.0经37℃作用1小时，毒力下降0.4个滴度。耐热性试验中，该病毒在恒定温度50℃，设定不同时间，从30分钟到70分钟，毒力均有不同程度下降。其中，50℃作用30分钟，病毒平均下降0.6个滴度；50℃，70分钟，病毒平均下降3.7个滴度；恒定温度56℃，设定不同时间，从30分钟到70分钟，毒力也均有不同程度下降。其中，56℃作用30分钟，病毒平均下降2个滴度；56℃，70分钟，病毒完全灭活。中和试验表明，粘膜病病毒特异阳性血清能完全中和该分离毒。综上实验结果，可初步确定该分离毒属于粘膜病病毒。 分子生物学鉴定方面，选用MDV标准株的高度保守的P125区序列设计引物。合成该引物，选择梅花鹿母鹿的流产胎儿肝组织原代病料和该分离毒的各代细胞培养物，均成功地扩增出了约400bp目的片段，与国际标准毒株一致，而未接毒MDBK对照细胞PCR的扩增结果为阴性。 本研究首次从梅花鹿流产胎儿中分离出MDV，揭示梅花鹿亦可感染MDV。MDV不仅能引起梅花鹿的腹泻，同时还能造成梅花鹿母鹿的流产。本研究为梅花鹿的粘膜病防制工作提供了重要的信息资料和理论依据，同时也丰富了粘膜病病毒的研究内容。更多还原

【Abstract】 In this study, an unknown virus designated MDVCC-deer6 was isolated from livers of a aborted fetus using MDBK cell line. The virus could multifly on the MDBK cells,and induce regular cytopathic effect (CPE).Observed by negative staining electron microscope, the virus could be seen with typical MDV virions. In biological assay, the hemagglutination reaction test proved that this virus had no any reaction to erythrocyte of chook,rabbit, pig and sheep. In the test of virulence determination of isolated MDVCC-deer6, the result of TCID50 is 10-5.5. Physicochemical assays showed that this viurs was sensitive to choroform and ether. In trpsin tolerance assay,this virus could resist to 1% trpsis at 37C in an hour and multifly ,but the average infection titre of the virus decreased 3.2 titre. In acid tolerance assay,this virus was sensitive to pH3.0 at 37C in 1 hour, and the average infection titre of the virus decreased 4.4 titre. In alkali tolerance assay , this virus was resistant to pH9.0 at 37C in 1 hour, and the average infection titre of the virus decreased little.In heat assay, at 50C,the virus was processed from 30 minutes to 70 minutes and at each condition the viral virulence reduced to some certain degree.Among these conditions,when at 50C in 30 minutes,the average infection titre of this virus decreased 0.6 titre,and when at 50C in 70 minutes, the average infection titre of this virus decreased 3.7 titre.at 56C,the virus was processed from 30 minutes to 70 minutes and at each condition the viral virulence reduced to some certain degree too. Among these conditions, when at 56C in 30 minutes,the average infection titre of this virus decreased 2 titre, and when at 56C in 70 minutes ,CPE of this virus disappeared and the virus lost the multiplication capacity completely. By neutrolizaion assay,MDVCC-deer6 could be identified as a kind of MDVBy RT-PCR method,we selected a pair of primers named p1 and p2 which located in highly conserved P125 nucleotide region . we could get 402 bp nucleotide fragement in C24V and 672 bp nucleotide fragement in NADL after amplifying by the pair of primers, we amplified the target fragement of about 400bp from the passages of culture of this isolated virus and the the passages of culture of this C24V. However, the control cell showed negative result.From the results described above, it can be concluded that the strain MDVCC-deer6 is a new strain of deer-origined MDV.更多还原