| Ancylostoma ceylanicum is a zoonotic animal-derived hookworm.Tissue inhibitors of metalloproteinases(TIMPs)are specific inhibitors of matrix metalloproteinases(MMPs),which are widely distributed in vertebrates and invertebrates.They have important physiological functions,such as enhancing erythrocyte activity and promoting cell growth.Based on the importance of TIMP for organisms,the cloning,expression and characterization of TIMP gene of dog-derived Ancylostoma ceylanicum was studied in this paper.Firstly,the hookworm larvae were cultured by the "T" filter paper method and the double glass culture method,and the worms were collected after 7 days of culture.The results showed that the "T" filter paper method enriched the larvae more than the double glass culture method,indicating "T" filter paper method was superior to the double glass culture method.The primers were designed according to the TIMP(Ace-TIMP)gene sequence annotated in NCBI database.The Ace-TIMP gene was amplified by RT-PCR using c DNA as template,and its sequence was analyzed by online software.The results showed that the ORF sequence of Ace-TIMP gene was 648 bp,encoding 215 amino acids.Among them the signal peptide sequence was 48 bp,encoding 16 amino acids.The protein is a hydrophilic protein with a signal peptide and no transmembrane domain.It consists of one alpha helix and seven beta chains folded.The pET32 a expression plasmid was used as the prokaryotic expression vector,and the correct mature peptide sequence and expression vector were digested with Eco R I and Hind III,then the recombinant expression plasmid p ET32a-TIMP-MP was constructed,and transformed into E.coli Transetta(DE3),induced to express by IPTG,and the expressionproduct was identified by SDS-PAGE and Western blot.The target protein was purified by ion affinity chromatography column and the protein concentration was determined.The results showed that the protein was a soluble protein,which was mainly expressed in the supernatant,and its molecular mass was about 42 k Da.Western blot analysis showed that the recombinant protein specifically bind to the His-tag murine monoclonal antibody.The purer recombinant proteins were obtained by nickel ion affinity chromatography from the20 m M imidazole eluate.The concentration of the fusion protein was 0.9 mg/m L as determined by the BCA method.Kunming mice were immunized with the purified recombinant protein to prepare polyclonal antibodies,and the antibody titer was determined by indirect ELISA.The specificity of polyclonal antibody was analyzed by Western blot.The inhibitory activity of Ace-TIMP protein to MMP-2 was analyzed by substrate zymography.The results showed that the antiserum titer was as high as 1:51200,and the prepared polyclonal antibody could specifically bind to the His-tagged antibody,and the substrate zymography showed a blue band containing the Ace-TIMP in the lane,it indicated that the TIMP from A.ceylanicum could inhibit the degradation activity of MMP-2 on gelatin.In this study,the TIMP gene was first cloned from dog-derived A.ceylanicum and its prokaryotic expression vector was constructed and expressed in vitro.The sequence was subjected to multiple alignment and genetic evolution analysis,and its antigenicity and the inhibitor activity of MMP were analyzed,which laid a foundation for further study on biological function and immune potential of A.ceylanicum TIMP. |
