Recombinant Opc meningococcal protein, folded in vitro, elicits bactericidal antibodies after immunization.
The meningococcal Opc protein has been expressed as inclusion bodies in Escherichia coli. After cell disruption and successive washing of the insoluble fraction, insoluble proteins were solubilized in presence of the chaotropic agent guanidium hydrochloride. The extract was applied to a Reverse Phase High Performance Liquid Chromatography (RP-HPLC)-C4 column, for further purification. The obtained recombinant Opc protein was refolded in vitro, by the addition of several compounds to the resuspended solution. Over time, the progress of renaturation was tested by immunoblot with the human monoclonal antibody LuNm03 against the meningococcal Opc protein. LuNm03 recognizes a conformational epitope on the native meningococcal Opc protein. Having established the optimal conditions of renaturation. Balb/c mice were immunized to study the humoral immune response. The human at immune response elicited in mice was measured by ELISA and immunoblot, while the functional activity of these antibodies was assayed in a bactericidal test. According to our results, it was possible to obtain a recombinant Opc protein folded in vitro, with a conformation suitable enough to generate functional antibodies in mice, capable of killing meningococci in the presence of human complement.
Musacchio
A
A
Division of Vaccines, Center for Genetic Engineering and Biotechnology, Habana, Cuba.
Carmenate
T
T
Delgado
M
M
González
S
S
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli; D005285:Fermentation; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D011489:Protein Denaturation; D017510:Protein Folding; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D014614:Vaccines, Synthetic; C074517:opc protein, bacteria