Molecular Characterization and Prokaryotic Expression of Toxocara canis Superoxide Dismutase
To study the molecular characteristics of Toxocara canis superoxide dismutase(Tc-sod),the full-length cDNA of Tc-sod gene was amplified as template according to the genomic data of Tc-sod(Gen-Bank:AAB00227.1)and sequence analysis and multiple sequence alignment were performed.The pro-karyotic expression vector Tc-sod/pET-32a was constructed and induced by IPTG.The recombinant pro-tein was purified and polyclonal antibody was prepared.The results showed that the full-length sequence of the Tc-sod gene was 573 bp,which encode 190-amino acids.Multiple alignment found that the amino acid sequence of Tc-SOD shared a conserved domain of SOD-Cu with that of Schistosoma mansoni,Ani-sakis simplex,Necator americanus,Brugia malayi and Ancylostoma caninum.The phylogenetic tree analysis suggested that Tc-SOD was closely related to the sequence of B.malayi.SDS-PAGE showed that the recombinant protein Tc-SOD was about 37 ku in size and expressed in soluble form.The protein was purified by Ni-NTA affinity chromatography.The high purity protein was obtained by elution with 70 mmol/L imidazole,and used to immunize the New Zealand white rabbits to prepare polyclonal antibody.Indirect ELISA showed that the antibody titer was>1∶320 000,indicating that the recombinant protein had good immunogenicity.The Western Blot result showed that the antibody could specifically bind to Tc-SOD,indicating that the antibody had good specificity.
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