Construction of recombinant polypeptides based on beta antigen C (Bac) protein & their usage for protection against group B streptococcal infection.
OBJECTIVE
immunocompromized adults. Approximately 50 per cent of the GBS strains carry and express the gene of BAC antigen which is capable to bind IgA. Gene encoding for the BAC antigen has been cloned and sequenced but actual IgA binding region on the protein has not been detected. The aim of the present work was to localize the region of IgA binding on Bac protein, to evaluate the role of one of the Bac protein regions MLKKIE in IgA binding, and to investigate the ability of Bac based recombinant proteins to generate protective antibodies against GBS infection.


METHODS
Recombinant proteins based on beta antigen C were generated after PCR amplification of the fractions of bac gene with the following cloning of the PCR products into expression plasmids. Recombinant peptides were tested for IgA binding by immunoprecipitation and Western blot. One of the recombinant proteins expressing IgA binding was used as an antigen for immunization of mice and for GBS protection studies.


RESULTS
Several bac gene constructs were generated. Their ability to bind IgA varied dramatically depending on the size of the construct and location of the fragment on the bac gene map. The smallest peptide expressing IgA binding was 14 kD in size. Amino acid substitutions in MLKKIE region facilitated IgA binding ability. Immunization of mice with recombinant Bac based peptide induced the appearance of anti-GBS antibody with high affinity level providing protection against GBS infection.


CONCLUSIONS
Size dependence of Bac based recombinant peptides proved that the effective IgA binding required specific folding of the protein binding IgA. Region MLKKIE could not be considered as region, responsible for IgA binding. Generation of antibodies against Bac based recombinant peptides with high titre and affinity makes these proteins a potent candidates for generating a vaccine against GBS infection.
Suvorov
A
A
Institute of Experimental Medicine, Academy of Medical Sciences, Pavlova 12,St. Petersburg 197376, Russia. lab@surorov.spb.su
Ustinovitch
I
I
Meringova
L
L
Grabovskaya
K
K
Leontieva
G
G
Vorobieva
E
E
Totolian
A
A
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D001428:Bacterial Vaccines / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D007231:Infant, Newborn; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D013290:Streptococcal Infections / Q000276:immunology / Q000517:prevention & control*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D017931:DNA Primers; D011994:Recombinant Proteins