隐孢子虫(Cryptosporidium)病毒的鉴定及其特性

Identification and Characterization of Cryptosporidium dsRNA

根据已发表的隐孢子虫病毒的序列 ,设计合成 2对引物 ,对小球隐孢子虫 ( Cryptosporidium parvum)、鼠隐孢子虫 ( C.muris)、贝氏隐孢子虫 ( C.baileyi)、火鸡隐孢子虫 ( C.meleagridis)进行 RT-PCR扩增 ,结果发现仅小球隐孢子虫 ( C.parvum)含有大小约为 1 .7× 1 0 3nt和 1 .3× 1 0 3nt2个片段的 ds RNA病毒。将其PCR产物连接到 p MD1 8-T载体上进行克隆测序 ,经与 Gen Bank比较 ,含这 2个片段的 ds RNA病毒与已发表的该虫病毒的同源性分别为 96%和 98%。电泳鉴定发现 ,该病毒对低浓度 RNA酶不敏感 ,且不能被 DNA酶降解。依赖 RNA的 RNA聚合酶活性测定结果表明 ,该病毒具有该聚合酶的活性。电镜观察未发现存在病毒样粒子。

The two pairs of primer were designed and synthesized based on the sequences of C.parvum dsRNA virus published by Khramtsov(1997).The different species of Cryptosporidia were amplified by RT PCR.The results showed that only C.parvum contained about 1.7×10 3 nt and 1.3×10 3 nt dsRNA virus.The products of PCR were linked into pMD18 T vector,then cloned and sequenced. The homology of the two C.parvum dsRNA viruses to the sequences published by Khramtsov(1997) in GenBank was 96% and 98% respectively. The two dsRNA viruses were sensitive to RNaseA but not to RQ1 while the nucleic acid were treated with the enzymes. The activity of RNA polymerase was showed while 32 P UTP was added into reaction solution.There were no virus like particles to be observed by TEM which indicating that the virus is not to be encapsulated.