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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。
Objective To obtain the nucleotide and amino acid sequence of adenylate kinase (AK) gene of Nanjing (NJ) strain of Cryptosporidium parvum (Cp), and to compare the nucleotide and amino acid sequences of AK gene with other Cryptosporidium strains difference. Methods Kunming mice were used to establish the infection model of Cryptosporidium parvum NJ. According to the known sequence of AK gene of GenBank Iowa II strain, two pairs of primers were designed and synthesized. The genomic DNA of Cryptosporidium parvum NJ strain was amplified by nested PCR The AK gene was amplified and cloned into pMD18-T vector. The recombinant plasmid pMD18-T-CpAK was identified by PCR and restriction enzyme digestion. The bioinformatics method was used to analyze the relationship between the AK gene of Cryptosporidium parvum NJ strain and other insects Strain nucleotide and amino acid sequence differences. Results The specific AK gene sequence was amplified by nested PCR. The correct pMD18-T-CpAK recombinant plasmid was identified by restriction enzyme digestion and PCR. The amplified sequence was 903 bp and the full length AKP of Cryptosporidium was 663 bp. The nucleotide sequence Determination and homology analysis showed that the homology between the AK gene of Cryptosporidium parvum NJ strain and AK of Tc502 type 2 strain was 99%, and that of AK gene of Cryptosporidium parvum NJ strain was 98% Phylogenetic tree analysis showed that the AK gene of Cryptosporidium parvum NJ strain was closest to AK gene of human Cryptosporidium parvum TU502 type 2 strain. Conclusion The AK gene of Cryptosporidium parvum NJ strain was successfully cloned and the GenBank accession number (HM067440) was obtained. The AK gene was highly conserved among different species.