犬贾第虫dsRNA病毒的鉴定及特性

Identification and Characterization of Giardia canis dsRNA Virus

对分离到的 6株犬贾第虫 ( Giardia canis)包囊进行核酸分析 ,在其中 1株犬贾第虫核酸的琼脂糖凝胶电泳图谱上观察到 1条长度约 7.0 kb的片段。经鉴定 ,该核酸不能被 DNA酶 ( 1 0 0 mg/ L)降解 ,对质量浓度低的 RNA酶 ( 0 .1 mg/ L)不敏感 ,但可被质量浓度高的 RNA酶 ( 1 0 mg/ L)降解。纯化包囊经液氮冻融后 ,磷钨酸负染 ,电镜观察发现有球形、直径约为 33nm的病毒样粒子存在 ,包囊超薄切片在胞质中也观察到该病毒样粒子存在。RNA依赖 RNA聚合酶活性测定结果表明 ,该病毒具有 RNA依赖 RNA聚合酶的活性。犬贾第虫 ds RNA病毒核酸经 RT-PCR扩增后得到 1条预计扩增大小的片段 ,将其回收后连接到 p MD1 8-T载体上进行克隆并测序 。

Total nucleic acids from six strains of Giardia caniscysts were extracted with phenol/chloroform and Trizol reagent kit respectively. A particular extrachromosomal about 7.0 kb band was observed after agarose gel elec- trophoresis with both extraction methods. The nucleic acid was notsensitive to DNase ,however,the stability of the nucleic acid to RNase A digestion depended on the concentration of RNase A.When the nucleic acid was treated with dif- ferentconcentrations of RNase A,ithad been found to be more sensitive to high concentration of RNase A( 1 0 mg/L) than to low concentration of RNase A ( 0 .1 mg/L) . The supernatantwas negatively stained with 2 %Uranyl acetate and observed by TEM after the Giardia canis cysts were disrupted by freeze/thaw cycle ( 5 min at -1 96℃ and 1 0 min at 2 3℃ ) ,then centrifuged at low speed.The spherical virus-like particles of33-37nm in diameter were observed.The cross-section of Giardia canis cysts examined by TEM revealed that the virus-like particles were present in the cyto- plasm.The activity of RNA dependent RNA polymerase was also detected in crude lysates of Giardia caniscysts.The polymerase activity was abolished in the presence of EDTA suggesting a divalent cation requirement and it was not in- hibited by proteinase K.By using specific synthetic primers corresponding to the published sequences of the Giar- diavirus,a fragment of6 70 bp was amplified from the purified7.0 kb ds RNA band by RT-PCR.The products of RT- PCR were linked into p MD1 8-T vector,then cloned and sequenced.