微小隐孢子虫20K亲环蛋白基因克隆及分析

Cloning and analysis of 20K cyclophilin gene of Cryptosporidium parvum

目的克隆微小隐孢子虫(Cp)南京株(NJ)的20K亲环蛋白(CyP)基因,并对其核苷酸和氨基酸序列进行分析。方法采用昆明种小鼠建立微小隐孢子虫NJ株感染模型,根据微小隐孢子虫Iowa II株20K CyP基因序列设计合成2对引物,应用巢式PCR技术从微小隐孢子虫NJ株基因组DNA中扩增20K CyP基因,并将其克隆到pMD18-T载体上,将阳性克隆重组质粒进行菌落PCR及双酶切鉴定;应用生物信息学方法分析微小隐孢子虫NJ株20K CyP基因与其他虫株核苷酸和氨基酸序列差异,并分析该基因蛋白结构域。结果巢式PCR扩增得到特异的20K CyP基因,经PCR及双酶切鉴定获得了正确的pMD18-T-20K CyP重组质粒;测序结果显示,微小隐孢子虫NJ株20K CyP基因全长519 bp,编码173个氨基酸,该基因已登录GenBank,登录号为JQ284431;氨基酸序列分析表明,微小隐孢子虫NJ株与Iowa II株20K CyP基因编码的氨基酸序列具有100%同源性;对20K Cyp基因进行蛋白结构域分析,显示该基因序列所编码的蛋白具有特异性类亲环蛋白A、B、H的肽脯氨酰顺反异构酶(PPIase)区域,该区域与人的亲环蛋白A、B、H具有相似性。结论成功克隆微小隐孢子虫NJ株20K CyP基因,该基因具有高度保守性。

Objective To acquire 20K cyclophihin（CyP） gene of Cryptosporidium parvum（C.parvum） and to analyze the nucleotide and amino acid sequences of the gene.Methods We constructed animal models of NJ strain of C.parvum infection with Kunming mice.We designed two pairs of primers based on the known gene sequences of 20K CyP of C.parvum Iowa Ⅱ strain and amplified it from the C.parvum NJ strain by nested PCR method,then cloned it into the pMD18-T vectors.The positive recombinant plasmid pMD18-T-20K CyP was acquired after the identification by PCR and double enzyme digestion methods.Results We achieved right recombinant plasmid pMD18-T-20K CyP by the identification of PCR and double enzyme digestion method.The sequencing results of the nucleotides showed that the 20K CyP gene of the C.parvum NJ strain was 519 bp in full length,encoding 173 amino acids.The gene had been logined to GenBank database with the accession number of JQ284431.And the sequence analyses indicated that it had 100% homology in amino acids with the Iowa II strain.After analyzing its protein structure domain,there were specific cyclophilin A,B,Hlike PPIase domain which were similar to human cyclophilins A,B,and H.Conclusion The 20K CyP gene of C.pravum NJ strain was successfully cloned,and the structure of 20K CyP is highly conserved in C.parvum.