微小隐孢子虫亲环素型肽脯氨酰顺反异构酶基因克隆与分析  

Cloning and analysis of cyclophilin type peptidyl-prolyl cis-trans isomerase of Cryptosporidium parvum

作　　者：付芹芹[1] 杨光[2] 王穗湘[2] 孙小会[1] 郭志云[2] 张丽菊[1] 陈川[2] 刘国宁[3] 荆春霞[1] 

机构地区：[1]暨南大学医学院流行病学教研究,广州510632 [2]暨南大学医学院寄生虫系,广州510632 [3]暨南大学医学院卫生学教研室,广州510632

出　　处：《中国人兽共患病学报》2012年第9期885-889,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30901249,81101267);广东省自然科学基金(10151063201000036,S2011010002526);广东省医学科研基金(B2007097)联合资助~~

摘　　要：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。The aim is to clone cyclophilin type peptidyl-prolyl cis-trans isomerase(CyP-type PPIase) gene of C.parvum NJ strain and analyze the sequence differences between NJ strain and other strains.Based on the given CyP-type PPIase gene sequences of C.parvum Iowa II in GenBank,we designed two pairs of primers to amplify the CyP-type PPIase genes from the C.parvum NJ strain by Nested PCR method,and clone it into the pMD18-T vectors.The positive recombinant plasmid pMD18-T-CyP-type PPIase gene was sequenced after the identification of PCR and double-enzyme digests methods.We used bioinformatics methods to find out the nucleotide and amino acids sequence differences of the CyP-type PPIase gene between the C.parvum NJ strain and other strains.The CyP-type PPIase gene could be specifically amplified by Nested PCR,and the correct recombinant plasmid pMD18-T-CyP-type PPIase gene was constructed after the identification by PCR and double-enzyme digests methods.The amplification sequence was 746 bp,including the whole CyP-type PPIase gene ORF 570 bp of the C.parvum NJ strain and encoding 190 amino acids.The sequence of amino acids and homology analysis showed that the homology of CyP-type PPIase gene of C.parvum NJ strain and C.parvum Iowa II strain was 99%.Evolutionary tree analysis showed that CyP-type PPIase gene of C.parvum NJ strain had the closest relationship with C.parvum Iowa II strain,and has the furthest relationship with diplococcus meningitidis.The CyP-type PPIase gene of C.parvum NJ strain was successfully cloned,and the CyP-type PPIase gene has higher homology with other strains.

关 键 词：微小隐孢子虫 亲环素型肽脯氨酰顺反异构酶基因 克隆 

分 类 号：R382.3[医药卫生—医学寄生虫学]