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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 clone the 20K Cyclophilin (CyP) gene of Cryptosporidium parvum (NJ) and analyze its nucleotide and amino acid sequence. Methods Kunming mice were used to establish the infection model of Cryptosporidium parvum NJ. Two pairs of primers were designed and synthesized based on the sequence of 20K CyP of Cryptosporidium parvum Iowa II. Nested PCR was used to amplify genomic DNA of Cryptosporidium parvum NJ The 20K CyP gene was amplified and cloned into pMD18-T vector. The positive clones were identified by colony PCR and double enzyme digestion. The 20K CyP gene of Cryptosporidium parvum NJ isolates was analyzed by bioinformatics method. Nucleotide and amino acid sequence differences, and analyze the gene protein domain. Results The specific 20K CyP gene was amplified by nested PCR. The correct pMD18-T-20K CyP recombinant plasmid was obtained by PCR and double enzyme digestion. The sequencing results showed that the full length of 20K CyP gene of Cryptosporidium parvum NJ strain was 519 bp , Encoding 173 amino acids. The gene was registered in GenBank with the accession number of JQ284431. Amino acid sequence analysis showed that the homology of the amino acid sequence encoded by Cryptosporidium parvum NJ strain with the 20K CyP gene of Iowa II strain was 20% Protein domain analysis revealed that the protein encoded by this gene sequence has a specific PPIase region of the like cyclophilin A, B, H, which interacts with human cyclophilin A , B, H have similarities. Conclusion The 20K CyP gene of Cryptosporidium parvum NJ was successfully cloned and the gene was highly conserved.