微小隐孢子虫18SrRNA部分基因克隆和序列分析

CLONING AND SEQUENCE ANALYSIS OF PARTIAL 18S rRNA GENE OF THREE CRYPTOSPORIDIUM PARVUM ISOLATES

分别从长春小白鼠、徐州人和南京黄牛的粪便中分离纯化了 3株微小隐孢子虫 (C .parvum)卵囊 ,根据C .parvum 18SrRNA基因序列设计合成引物 ,用PCR扩增卵囊基因组DNA ,其大小为 5 86bp的片段 ,PCR产物用试剂盒回收纯化后直接测序。将测得的序列用DNASTAR软件分析并与国外已发表的相应序列进行同源性比较 ,并绘制系统发育进化树。结果表明 ,徐州人源株与长春鼠源株及徐州人源株与南京黄牛源株同源性均为 97 6 % ;长春鼠源株与南京黄牛源株相应序列同源性为 99 6 %。此 3株C .parvum与国外株相应序列同源性为 81 4 %～ 10 0 0 %。限制性内切酶酶切位点分析仅发现徐州株有 1个特殊的DdeⅠ位点。

To assess the genetic diversity among isolates,three domestic isolates of C.parvum from mouse in Changchun,cattle in Nanjing and child in Xuzhou had been obtained respectively.A pair of primers was synthesized based on C.parvum 18S rRNA sequence published.The C.parvum genes of three domestic isolates were amplified by PCR. The 586bp fragment was obtained and sequenced respectively.The sequences had been compared to the corresponding genes published by DNASTAR software.The results demonstrated that the similarity of nucleotide sequence between isolate from child in Xuzhou and isolate from mouse in Changchun or isolate from cattle in Nanjing was 97 6% respectively and the similarity of isolate from mouse in Changchun and isolate from cattle in Nanjing was 99 6%.While the similarity of three domestic isolates and nine foreigh isolates was from 81 4% to 100 0%.Additionally,a special DdeⅠ site has been found by restriction endoenzyme analysis.So the study is good for the diagnosis of cryptosporidiosis parvum,molecular epidemiology and survey of infe ction resource for human body.