五株隐孢子虫18S rRNA部分基因克隆和序列分析

Cloning and Sequence Analysis of Partial 18S rRNA Gene of Five Cryptosporidium Isolates in China

设计Nested PCR引物扩增牛源微小隐孢子虫Cryptospordium parvum、羊源微小隐孢子虫C. parvum、牛源安氏隐孢子虫C. andersoni、鸡源贝氏隐孢子虫C. baileyi及猪源隐孢子虫C. suis 18S rRNA基因突变区,PCR产物经克隆测序,其片段大小分别为212、213、213、213和210 bp.将测得的序列用DNAStar软件分析并与NCBI数据库中相同与相近种株序列进行相似性比较, 进行相似性分析并用TREECON软件绘制系统发育进化树.结果表明测得的5株隐孢子虫与各自相同种相似性为98.1%～100%,与其他种相似性为90.1%～98.6%.分析显示在此突变区设置特异酶切位点能区分开C. parvum, C. andersoni, C. baileyi与C. suis,位点分别是TaqI、BstUI、MseI.本研究为我国隐孢子虫分类、分子流行病学研究提供了新的方法.

Polymorphous regions of C. parvum, C. suis, C. andersoni and C. baileyi were amplified using two pairs of nested PCR primers based on Cryptosporidium 18S rRNA gene. The amplified fragments of 212, 213, 213, 213 and 210 bp were respectively cloned and sequenced. The obtained sequences were compared with corresponding genes published in GenBank using DNASTAR software. The results of multiple alignment demonstrated that the homology among the same species was 98. 1% -100% , among the different species was 90. 1% -98.6%. Furthermore, the species-specific restriction enzyme sites of TaqI, BstUI and Msel were found by restriction endo-enzyme analysis. The present study provided a novel assay for the diagnosis and molecular epidemiology survey of cryptosporidiosis.