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付芹芹, 荆春霞, 杨光, 郭志云, 孙小会, 王穗湘, 李月琴, 周天鸿. 微小隐孢子虫腺苷酸激酶基因克隆及分析[J]. 中国公共卫生, 2011, 27(7): 845-848. DOI: 10.11847/zgggws2011-27-07-14
引用本文: 付芹芹, 荆春霞, 杨光, 郭志云, 孙小会, 王穗湘, 李月琴, 周天鸿. 微小隐孢子虫腺苷酸激酶基因克隆及分析[J]. 中国公共卫生, 2011, 27(7): 845-848. DOI: 10.11847/zgggws2011-27-07-14
FU Qin-qin, JING Chun-xia, YANG Guang, . Cloning and bioinformatics analysis of adenylate kinase of Cryptosporidium parvum[J]. Chinese Journal of Public Health, 2011, 27(7): 845-848. DOI: 10.11847/zgggws2011-27-07-14
Citation: FU Qin-qin, JING Chun-xia, YANG Guang, . Cloning and bioinformatics analysis of adenylate kinase of Cryptosporidium parvum[J]. Chinese Journal of Public Health, 2011, 27(7): 845-848. DOI: 10.11847/zgggws2011-27-07-14

微小隐孢子虫腺苷酸激酶基因克隆及分析

Cloning and bioinformatics analysis of adenylate kinase of Cryptosporidium parvum

  • 摘要: 目的 获得微小隐孢子虫(Cryptosporidium parvum,Cp)南京(NJ)株的腺苷酸激酶(adenylate kinase,AK)基因的核苷酸和氨基酸序列,比对分析与其他隐孢子虫株AK基因序列的差异。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GenBank微小隐孢子虫Iowa II株AK基因已知序列设计合成2对引物,应用巢式PCR方法从微小隐孢子虫NJ株基因组DNA中扩增AK基因,并将其克隆入pMD18-T载体;对重组质粒pMD18-T-CpAK经PCR及酶切鉴定后测序,应用生物信息学方法分析微小隐孢子虫NJ株AK基因与其他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AK基因序列,酶切及PCR鉴定为正确的pMD18-T-CpAK重组质粒,扩增序列为903bp,含隐孢子虫AK全长基因663 bp;核苷酸序列测定及同源性分析表明,微小隐孢子虫NJ株AK基因与人隐孢子虫TU502 type 2株AK的同源性为99%,与微小隐孢子虫Iowa II株AK序列同源性为98%;进化树分析表明,微小隐孢子虫NJ株的AK基因与人隐孢子虫TU502 type 2株AK基因亲缘关系最近。结论 成功克隆到微小隐孢子虫NJ株AK基因并获得GenBank基因登录号(HM067440),该AK基因在不同物种间高度保守。

     

    Abstract: Objective To analyze nucleotide and amino acids sequences of adenylate kinase(AK) of Cryptosporidium parvum and to compare the differences of the sequences between NJ strain and other strains.Methods Kunming genus mice were used to build Cryptosporidium parvum infection model.Based on the gene sequences of Cryptosporidium parvum Iowa Ⅱ AK gene in GenBank,we designed two pairs of primer to amplify the AK genes from the Cryptosporidium parvum NJ strain by nested PCR method,and cloned it into the pMD18-T vectors; the positive recombinant plasmid pMD18-T-CpAK was sequenced after the identification of PCR and double enzyme digest method.We used bioinformatics methods to find out the difference in nucleotide and amino acids sequences of the AK gene between the Cryptosporidium parvum NJ strain and other strains.Results The CpAK gene was specifically amplified with nested PCR and the correct recombinant plasmid pMD18-T-CpAK was constructed after the identification with PCR and double enzyme digest method.The augmentation sequence is 903bp,including the AK gene of the Cryptosporidium parvum NJ strain of 663 bp.The sequence of nucleotides and homology analysis show ed that the homology of AK of Cryptosporidium parvum NJ strain and the Cryptosporidium hominis TU502 type 2 AK was 99% and the homology of AK of Cryptosporidium parvum NJ strain and the Cryptosporidium parvum Iowa Ⅱ strain was 98%.Evolutionary tree analyses show ed that the AK of Cryptosporidium parvum NJ strain had the closest relation with Cryptosporidium hominis TU502 type 2 AK.Conclusion The AK gene of Cryptosporidium parvum NJ strain was successfully cloned and registered with a registration GenBank accession number(HM067440).The AK gene has higher gene homology with other strains.

     

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