基于凝集素基因对部分隐孢子虫种类的分子鉴定

目的 为了解隐孢子虫凝集素基因在不同分离株的序列差异及其遗传进化关系,以SSU rRNA基因作参照,评价Lectin基因位点作为隐孢子虫基因分型和进化关系分析的可行性。方法 采用巢式PCR,分别在Lectin和SSU rRNA位点处对本实验室分离保存的多个隐孢子虫分离株进行PCR的扩增。用Clustalx对扩增序列与参考序列进行比对,用MEGA5中的邻近法(Neighbor joining method NJ),进行进化树的构建。结果 基于Lectin基因位点,在C. parvum、C. hominis、C. cuniculus及其在SSU rRNA 处与人源C. hominis极为相近的horse genotype、驴源C. hominis均成功扩增出了大小在450 bp左右的目的 条带,并进行了进化树的分析,不同种类和同种不同动物来源的隐孢子虫分布在不同的分支上。结论 Lectin基因位点可很好的区分SSU rRNA序列极为相似的几种隐孢子虫,有望为人兽共患隐孢子虫分类和遗传研究提供新的基因靶标。

Molecular identification of some species of Cryptosporidium based on Lectin gene

In order to understand the sequence differences and their genetic evolution of Lectin gene in different Cryptosporidium isolates, the Lectin gene locus was evaluated as the feasibility of Cryptosporidium gene typing and evolutionary relationship with SSU rRNA gene as reference. The nested PCR was used to amplify the Cryptosporidium isolates isolated from the laboratory at Lectin and SSU rRNA loci. ClustalX was used to compare the amplified sequence with the reference sequence, and the phylogenetic tree was constructed by the Neighbor joining method NJ in MEGA5. Lectin gene locus was used as a typing tool to amplify C. parvum, C. hominis, C. cuniculus and horse genotype, C. hominis from donkey which were very similar to C. hominis from Human. These isolates at Lectin gene was amplified successfully(about 450 base pair)and then was carried out a phylogenetic tree analysis. Results indicated that, different Cryptosporidium species and the same species derived from different animals were in different branches. Lectin gene locus is very likely to be a new Cryptosporidium classification tool which provides a reference basis for the further development of zoonotic Cryptosporidium classification and genetic research. Therefore, it is worthy to do more study on the gene. The possibility of using Lectin gene as a genotyping tool was explored by amplifying the SSU rRNA and Lectin loci of Cryptosporidium species, respectively. The study is to provide new molecular tools for the epidemiological investigation of Cryptosporidium, analysis of genetic structure and the origin of epidemic disease. The results of sequence alignment and phylogenetic tree show that Lectin gene can be used to identify the species of Cryptosporidium which similar to the SSU rRNA sequences. It is expected to add a new molecular tool for molecular epidemiology and population genetic analysis of Cryptosporidium, and it is a useful supplement to the classification tool of Cryptosporidium.