Recombinant peptides as immunogens: a comparison of protocols for antisera production using the pGEX system.
Using an inducible vector system that directs high-level production and rapid purification of recombinant protein, we have immunized mice with peptides prepared by several methods: 1) samples fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) subsequently transferred to PVDF membrane and subcutaneously implanted in mice; 2) samples cut directly from SDS-PAGE gels and injected intraperitoneally; 3) injection of recombinant protein bound to agarose beads; and 4) injection of log-phase E. coli transformed with recombinant vector. All four strategies yielded specific antisera reacting with both the parental fusion protein and the recombinant fragment as determined by enzyme-linked immunosorbent assay and immunoblot analysis. Specific recognition of the recombinant fragment was demonstrated by a competitive inhibition assay in which the parental fusion protein abrogated reactivity of serum with the isolated recombinant fragment.
Oettinger
H F
HF
Harvard Medical School, Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA 02115.
Pasqualini
R
R
Bernfield
M
M
D000180:Acrylic Resins; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology; D005822:Genetic Vectors; D007106:Immune Sera / Q000096:biosynthesis*; D007114:Immunization / Q000379:methods*; D008562:Membrane Glycoproteins / Q000276:immunology; D008567:Membranes, Artificial; D051379:Mice; D011145:Polyvinyls; D011509:Proteoglycans / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D053667:Syndecans; D014614:Vaccines, Synthetic / Q000276:immunology* / Q000302:isolation & purification; D000099166:Polyacrylamides; D005465:Fluorocarbon Polymers
D000180:Acrylic Resins; D007106:Immune Sera; D008562:Membrane Glycoproteins; D008567:Membranes, Artificial; D011145:Polyvinyls; D011509:Proteoglycans; D011994:Recombinant Proteins; D053667:Syndecans; D014614:Vaccines, Synthetic; D000099166:Polyacrylamides; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers