微小隐孢子虫CRIP基因克隆与分析

Cloning and analysis of cyclophilin-RNA interacting protein gene of Cryptosporidium parvum

目的比对分析微小隐孢子虫南京(NJ)株亲环素-RNA相互作用蛋白(CRIP)与其他隐孢子虫株CRIP基因序列的差异。方法根据GenBank微小隐孢子虫IowaⅡ株CRIP基因序列设计并合成2对引物,应用巢式聚合酶链反应(PCR)技术从微小隐孢子虫NJ株基因组DNA中扩增CRIP基因,并将其克隆到pMD18-T载体上,将重组质粒pMD18-T-CpCRIP进行PCR和双酶切鉴定后测序,应用生物信息学方法分析微小隐孢子虫NJ株CRIP基因与其他种属的CRIP基因序列同源性。结果巢式PCR扩增获得的特异性CRIP基因序列,经PCR及双酶切鉴定确为pMD18-T-CpCRIP重组质粒;测序结果显示,该序列有119 bp,微小隐孢子虫NJ株CRIP基因全长为909bp,编码302个氨基酸;测序结果和同源性分析显示,中国微小隐孢子虫NJ株CRIP基因序列与国外的微小隐孢子虫Iowa II株同源性为98%;该隐孢子虫NJ株CRIP基因序列获GenBank登录号JQ396883。结论微小隐孢子虫NJ株CRIP基因与其他微小隐孢子虫株存在基因变异。

Objective To obtain nucleotide sequences of cyclophilin-RNA interacting protein（CRIP） of Crygptosporidium parvum（ C. parvum） Nanjing（NJ） strain by cloning and to analyze the difference in CRIP gene sequence between NJ strain and other strains. Methods According to known CRIP C. parvum gene sequences in GenBank, we designed and synthesized two pairs of primers to amplify the CRIP genes from the C. parvum NJ strain by nested PCR technique and cloned it into the pMD18-T vectors. Then the recombinant plasmids were sequenced by PCR and double enzyme digest method. We used bioinformatics methods to find out the homologies of the CRIP gene between C. parvum NJ strain and other strains. Results CRIP specific gene sequences were amplified by nested PCR and the correct recombinant plasmids were identified by PCR and double enzyme digest method. The results of nucleotide sequencing showed that the amplified sequence was I 119 bp and the CRIP gene of the C. parvum NJ strain was 909 bp encoding 302 amino acids. The sequence analysis showed that there was a 98% homology in amino acids between NJ strain and the Iowa II strain abroad. Conclusion The CRIP gene of C. parvum NJ strain was cloned successfully.