ABSTRACT
The HSD-I gene codes a human sperm membrane protein (hSMP-1) and has been assigned the accession number U12978. The gene is located on human chromosome 9, region p12-p13. When the 1.7-kb cDNA of HSD-I was digested sequentially with EcoRI, BamHI, and HindIII, a 550-bp cDNA fragment was formed, which codes for the extracellular domain. This fragment was cloned into the asd+ vector pYA3149 to construct pYA3149R. The recombinant plasmid was used to transform an avirulent deltacva, deltacrp, deltaasd vaccine strain of Salmonella typhimurium chi4550. The hSMP-1 component was localized on the surface of the head of mature rat spermatozoa by an immunofluorescence technique using polyclonal anti-hSMP-1 antibodies. Since rat sperm contain hSMP-1, this rodent can be used to assay the immunogenicity of pYA3149R. Female Wistar rats were immunized by oral administration of the recombinant Salmonella. Anti-hSMP-1 antibodies in blood and vaginal washes of immunized animals were determined. Both body fluids contained significant amounts of the antibodies, showing that the recombinant Salmonella is an effective oral immunogen in rats.
Subject(s)
Membrane Proteins/immunology , Salmonella typhimurium/immunology , Spermatozoa/immunology , Vaccines, Attenuated , Vaccines, Synthetic , Administration, Oral , Animals , Antibody Formation , Antigens/immunology , Antigens, Surface , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin A/analysis , Male , Membrane Proteins/analysis , Membrane Proteins/genetics , Molecular Sequence Data , Rats , Rats, Wistar , Restriction Mapping , Salmonella typhimurium/genetics , Spermatozoa/cytology , Vagina/immunologyABSTRACT
A synthetic oligonucleotide, HSD-2a, encoding a peptide segment of the extracellular domain of a human sperm membrane protein, YWK-II, was inserted by blunt-end ligation at the EcoRV site in the hypervariable, antigenically determinant region IV of Salmonella flagellin gene fliC (d). The recombinant plasmid (pLS408-H1) was transferred into the nonpathogenic aroA live vaccine Salmonella dublin strain SL5928, which is flagellin-negative. Location of the HSD-2a protein on the surface of the flagella was demonstrated by ELISA and by immunogold-labeling electron microscopy using mouse anti-YWK-II antiserum. A 60 kD protein, corresponding to the chimeric flagellin, was isolated from the recombinant S. dublin and found to contain the expressed HSD-2a peptide determined by Western blot analysis using anti-YWK-II antiserum. The Salmonella strain carrying the recombinant plasmid-pLS408-H1 may thus be a potential source of antifertility vaccine.