Cloning and bioinformatics analysis of adenylate kinase of Cryptosporidium parvum

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.