title: Characterization of transmissible gastroenteritis coronavirus S protein expression products in avirulent S. typhimurium delta cya delta crp: persistence, stability and immune response in swine.
abstract: The spike protein from transmissible gastroenteritis virus (TGEV) was expressed in attenuated S. typhimurium delta cya delta crp delta asd chi 3987. Three partially overlapping fragments of TGEV S gene, encoding the amino-terminal, intermediate, and carboxy-terminal end of the protein, as well as the full length gene were inserted into the asd+ plasmid pYA292 to generate recombinant plasmids pYATS-1, pYATS-2, pYATS-3, and pYATS-4, respectively, which were transformed into S. typhimurium chi 3987. Recombinant S. typhimurium chi 3987 (pYATS-1) and chi 3987 (pYATS-4) expressing constitutively a 53 kDa amino-terminal fragment of the S protein and the full length protein (144 kDa), respectively, showed high stability. After 50 generations in vitro 60% and 20% of the bacteria transformed with pYATS-1 and pYATS-4, respectively, expressed the S-protein antigen. Since S. typhimurium chi 3987 (pYATS-1) showed a better level of expression and stability in vitro, this recombinant strain was selected as a potential bivalent vector to induce both immunity to Salmonella and TGEV in swine. In order to study colonization of swine tissues by S. typhimurium delta cya delta crp, a gene conferring resistance to rifampicin was cloned into the chromosome of S. typhimurium chi 3987, generating chi 4509 strain. Both S. typhimurium chi 4509 (pYA292) and chi 4509 (pYATS-1) colonized the ileum of orally inoculated swine with clearance of bacteria between days 10-20 post-infection. The expression of the amino-terminal fragment of the S protein diminished the ability of S. typhimurium chi 4509 (pYATS-1) to colonize deep tissues. The recombinant strain S. typhimurium chi 3987 (pYATS-1) induced TGEV specific antibodies in both serum and saliva of orally inoculated swine.
authors.affiliation: Department of Molecular and Cell Biology, Campus Universidad Autónoma, Madrid, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Smerdou
authors.forename: A
authors.initials: A
authors.lastname: Urniza
authors.forename: R
authors.initials: R
authors.lastname: Curtis
authors.forename: L
authors.initials: L
authors.lastname: Enjuanes
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000917:Antibody Formation; D000942:Antigens, Bacterial / Q000008:administration & dosage; D003001:Cloning, Molecular; D005761:Gastroenteritis, Transmissible, of Swine / Q000276:immunology / Q000821:virology*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012486:Salmonella typhimurium / Q000235:genetics / Q000276:immunology* / Q000821:virology; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D014764:Viral Proteins