{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T13:16:18Z","timestamp":1774271778260,"version":"3.50.1"},"reference-count":47,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T00:00:00Z","timestamp":1747612800000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,5,1]],"date-time":"2025-05-01T00:00:00Z","timestamp":1746057600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100013221","name":"Razi Vaccine and Serum Research Institute","doi-asserted-by":"publisher","award":["12\u201018\u201018\u2010106\u201096045\u2010961025"],"award-info":[{"award-number":["12\u201018\u201018\u2010106\u201096045\u2010961025"]}],"id":[{"id":"10.13039\/501100013221","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Veterinary Medicine &amp;amp; Sci"],"published-print":{"date-parts":[[2025,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Pathogenic strains of spirochetes of\n                      <jats:italic>Leptospira<\/jats:italic>\n                      spp. cause a globally distributed zoonotic disease called leptospirosis. The disease has several clinical manifestations, ranging from asymptomatic and subclinical infection to fatal and severe forms.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Hypothesis\/objectives<\/jats:title>\n                    <jats:p>\n                      The aim of this study was to produce a recombinant Leptospiral immunoglobulin\u2010like surface protein\u2010A (r\u2010LigA) antigen of\n                      <jats:italic>Leptospira interrogans<\/jats:italic>\n                      in a prokaryotic expression system and to assess its efficacy in a mouse model.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Materials and methods<\/jats:title>\n                    <jats:p>\n                      The optimal epitopes of the LigA protein were identified via bioinformatics studies. The pET32a\n                      <jats:sup>+<\/jats:sup>\n                      \u2010LigA plasmid construct was cloned into\n                      <jats:italic>E. coli<\/jats:italic>\n                      Top10\u2010DH5\u03b1, expressed in\n                      <jats:italic>E. coli<\/jats:italic>\n                      pLysS strains, and subjected to different IPTG concentrations at different times and temperatures. The expressed r\u2010LigA was purified using nickel\u2010affinity (Ni\u2010NTA) chromatography from the insoluble fraction and reassessed by SDS\u2010PAGE, western blotting, dot blotting, and Bradford assay. Female Balb\/C mice were immunised subcutaneously with r\u2010LigA alone or emulsified in Freund's adjuvant and subsequently boosted at 2 and 4 weeks. Specific antibody levels were evaluated by indirect ELISA.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Bioinformatics analysis identified the key antigenic region of LigA spanning amino acids 852 to 1210. Colony PCR and digestion confirmed the successful transformation. Induction using 0.5\u00a0mM IPTG at 30\u00b0C for 5\u00a0h was found to be optimal. Overexpression of r\u2010LigA under optimised conditions accumulated proteins as inclusion bodies. Purification of r\u2010LigA under native conditions using optimised Ni\u2010NTA yielded 1050 \u00b5g\/mL protein and high immunogenicity by effectively stimulating the immune system in female Balb\/C mice.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>These findings support r\u2010LigA as a strong candidate for future leptospirosis diagnostic tools and subunit vaccine development.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/vms3.70360","type":"journal-article","created":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T11:12:27Z","timestamp":1747653147000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Expression and Immunogenicity Analysis of Recombinant\n                    <i>Leptospira Interrogans<\/i>\n                    Surface Protein LigA in Mouse 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