论文部分内容阅读
目的克隆微小隐孢子虫(C.parvum)南京株(NJ)亲环素型肽脯氨酰顺反异构酶基因,测序并分析C.parvumNJ株与其他隐孢子虫分离株CyP-type PPIase基因序列的差异以及亲缘关系。方法根据GenBank上C.parvumIowa II株CyP-type PPIase基因序列,设计2对引物,应用巢式PCR扩增目的基因,并将其克隆入pMD18-T载体,对重组子进行双酶切和菌落PCR鉴定并测序,应用生物信息学方法分析C.parvum NJ株CyP-type PPIase基因与其它隐孢子虫株的核苷酸和氨基酸序列差异。结果巢式PCR扩增获得特异的目的基因,双酶切和菌落PCR鉴定获得正确的重组子,测序表明,扩增基因组DNA序列为746bp,含有目的基因ORF全长570bp,编码190个氨基酸。序列分析表明,我国C.parvum NJ株与国外分离的Iowa II株CyP-type PPIase基因的氨基酸序列具有99%的同源性。进化树分析表明,C.parvumNJ株与Iowa II株CyP-type PPIase基因之间的亲缘关系最近,与脑膜炎双球菌的亲缘关系最远。结论成功克隆C.parvumCyP-type PPIase基因,C.parvumCyP-type PPIase基因在氨基酸水平高度保守。
Objective To clone the gene encoding progesterone prolyl cis - trans isomerase from Nanjing parvum (NJ) strain of C.parvum and to sequence and analyze the CyP - type PPIase gene of C.parvumNJ and other Cryptosporidium isolates Sequence differences and kinship. Methods Two pairs of primers were designed according to the CyP-type PPIase gene sequence of C.parvum Iowa II strain in GenBank. The target gene was amplified by nested PCR and cloned into pMD18-T vector. The double digestion and colony PCR Identified and sequenced. The nucleotide and amino acid sequence differences between the CyP-type PPIase gene of C. parvum NJ strain and other Cryptosporidium strains were analyzed by bioinformatics method. Results The specific target gene was amplified by nested PCR. The correct recombinant was obtained by double digestion and colony PCR. The sequencing showed that the amplified genomic DNA sequence was 746 bp, and the ORF of the target gene was 570 bp in length and encoded 190 amino acids. Sequence analysis showed that the C.parvum NJ strain in China has 99% homology with the amino acid sequence of the CyP-type PPIase gene in Iowa II strain isolated abroad. Phylogenetic tree analysis showed that C.parvumNJ strain had the closest genetic relationship with Iowa II strain CyP-type PPIase gene and had the closest relationship with N. meningitidis. Conclusion The C.parvum Cytoplasmic PPIase gene was cloned successfully, and the gene of C.parvum Cyt c-type PPIase was highly conserved at the amino acid level.