Eukaryotic expression and immunogenicity of Ancylostoma ceylanicum calreticulin |
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| Institution: | 1. Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China;2. Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt;1. Malaria Functional Genomics Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA;2. Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand;1. Institute of Pharmacy, Department of Pharmaceutical Biology, University of Greifswald, 17491 Greifswald, Germany;2. School of Pharmacy, College of Health Sciences, Addis Ababa University, Churchill Street, 1176 Addis Ababa, Ethiopia;3. Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, 160062 Mohali, India;1. Department of Biology, National Changhua University of Education, Changhua 500, Taiwan;2. Endemic Species Research Institute, Council of Agriculture, Nantou, 552, Taiwan;3. Department of Biological Resources, National Chiayi University, Chiayi 600, Taiwan;1. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry and University Hospital, Palacky University Olomouc, Czech Republic;2. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden;3. A.V. Bogatsky Physico-Chemical Institute of National Academy of Sciences of Ukraine, Odessa, Ukraine;4. I.I. Mechnikova Odessa National University, Odessa, Ukraine |
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| Abstract: | Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes human and animal intestines, causing malnutrition and iron-deficiency anemia. Calreticulin is a multifunctional protein involved in all stages of parasitic infection. Studies have found that parasites can secret calreticulin to regulate the host's immune response. To explore the immunogenicity of the eukaryotic expression plasmid of Ancylostoma ceylanicum calreticulin (Ace-CRT), we constructed a recombinant Ace-CRT eukaryotic expression plasmid (pEGFP-N3-Ace-CRT). Successful expression of the target protein in Human Embryonic Kidney (HEK) 293 T cells was confirmed by indirect immunofluorescence and Western blot analysis. BALB/c mice were immunized with pEGFP-N3-Ace-CRT plasmid. Measuring IgG antibody levels in immunized mice sera by ELISA showed that the recombinant plasmid stimulated IgG antibody production in mice. Spleen lymphocytes were collected from vaccinated mice to determine the proportion of T cell subsets and the expression levels of cytokines. Flow cytometry revealed that the percentage of CD3 + CD4+ and CD3 + CD8+ T cells in mice spleen in the immunization group was significantly higher than that in the control group. Recombinant plasmid immunization increased IL-4, IL-10, IL-12, and IL-13 expression while decreasing IL-5, IL-6, and INF-γ in mice spleens. These results indicate that the eukaryotic plasmid constructed in this study had good immunogenicity and mainly induced a T helper 2 response in the host, laying a foundation for screening candidate molecules for anti-hookworm vaccines. |
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