Cloning,Expression And Biological Characteristics Of Aspartyl Protease Inhibitor From Ancylostoma Ceylanicum

Ancylostoma ceylanicum can infect dogs,cats and humans,which is an animal-derived zoonotic hookworm.Aspartyl proteinases inhibitors(API)are commonly found in intestinal parasitic nematodes,which have strong immunogenicity and are potential vaccine candidate antigens.Currently,the cloning,expression and immunogenicity of Ac-API-1 from Ancylostoma caninum have been reported,while the molecular properties and biological functions of Ancylostoma ceylanicum API(Ace-API)are poorly understood.Therefore,this paper studies the cloning and expression,tissue localization,immunogenicity of Ace-API and its effects on pepsin activity,which lays a foundation for exploring the biological functions of the protein as a candidate molecule for anti-hookworm vaccine.1.Amplification,expression and sequence analysis of Ace-API gene.The specific primers were designed based on the Ace-API-1 gene(No.AY705929)sequence in Gen Bank,and the Ace-API gene sequence was amplified by RT-PCR using the cDNA of adult Ancylostoma ceylanicum as the template.Bioinformatics and phylogenetic relationship of Ace-API amino acid sequence were analyzed using online software.The expression of its mRNA in adult and infectious stage(L3)was detected by Quantitative real-time PCR.The results showed that Ace-API cDNA sequence was 684 bp long and encoded 228 amino acids.Its amino acid sequences was 96.93% and 96.49% similar to Ac-API-1 and Adu-API-1,and they clustered into one branch in the phylogenetic tree.The gene was expressed in both adults and L3,but the expression level in adults was significantly higher than that in L3.2.Prokaryotic expression and tissue localization of Ace-API.The Ace-API cDNA sequence was cloned into the prokaryotic expression vector pET32 a,transformed into E.coli BL21(DE3),and expressed under the induction of isopropyl-β-Dthiogalactopyranoside(IPTG),and purified by Ni column affinity chromatography.The reactivity of the recombinant protein was detected by Western-blot method using Ace-API antiserum(1: 2000),infected dog serum(1: 2000)and healthy dog serum(1: 2000)as primary antibodies,respectively.Anti-Ace-API polyclonal antibody was prepared from Kunming mice,and its titer was detected by ELISA.Adults of Ancylostoma ceylanicum were fixed,embedded in paraffin and sliced.The adult slices were reacted with the polyclonal antibody as the primary antibody,and goat anti-mouse FITC-IgG as the secondary antibody,and observed under a fluorescent microscope.The results showed that the recombinant protein was mainly expressed in a soluble form,and its expression was the highest when induced with 1.0 mM IPTG for 14 h at 37℃.Purified protein was obtained after purification.The protein can be recognized by anti-Ace-API serum and serum of infected dog.The titer of the prepared polyclonal antibody was 1: 51200.Ace-API was mainly localized in the epidermis,secretory glands,and pseudocoelomic fluid of the adult.3.Immunogenicity and effects of Ace-API on pepsin activity.Lymphocytes were isolated from the peripheral blood of healthy dogs and co-incubated with Ace-API recombinant protein of different concentrations,and the proliferation was detected with CCK-8.Meanwhile,the total RNA of the lymphocytes was extracted and transcribed into the first-strand cDNA,and the expression of cytokines was detected by fluorescent quantitative PCR.The recombinant protein was incubated with pepsin,and then reacted with its substrate hemoglobin using trypsin as control.All reaction products were analyzed by SDS-PAGE.The results showed that the recombinant protein had a strong stimulating effect on lymphocyte proliferation and cytokine expression at 20 μg / mL,and the expression levels of cytokines IL-4,IL-10,IL-12,IL-13 and IFN-γ significantly increased compared with the control group.The addition of Ace-API reduced the decomposition of pepsin to hemoglobin,indicating that the protein has an inhibitory effect on pepsin activity.In this study,the Ace-API gene was first cloned from dog-derived A.ceylanicum and expressed in vitro.Ace-API has a high expression level in adults,and is localized in the epidermis,secretory glands and pseudocoelomic fluid.It has the function of inhibiting pepsin,and can stimulate the proliferation of peripheral blood lymphocytes and high expression of cytokines IL-4、IL-10、IL-12、IL-13 and IFN-γ.The above-mentioned results indicated that Ace-API has the potential value in developing anti-Ancylostoma ceylanicum vaccine,which provides a new idea for the control of hookworm infection.