A total of 1-2 x [10.sup.3] cells/well in 100 [micro]L RPMI 1640 supplemented with 10% FBS were stimulated with 20 [micro]g/mL specific antigen (TcpA, TcpB, and FlaA) or 1 [micro]g/mL PHA (phytohemagglutinin) concentration (as a mitogen) used for T-cell activation and proliferation.
The amplified DNA fragments (TcpA, TcpB, and FlaA) were cloned in pET32a and pGEX4T1 vectors.
TcpA, TcpB, and FlaA proteins were produced in Escherichia coli.
Serum collected after the last vaccination was evaluated against the purified recombinant proteins TcpB, TcpA, and FlaA with regard to IgG2a and IgG1 antibody titers using ELISA method.
However, TcpB antigen drives immune response away from the humoral immune response.
TcpA and TcpB are the main subunits of pilus in Vibrio cholerae which can be seen on its outer surface .
TcpB is another component of pilus which helps with attachment of Vibrio cholerae to intestinal cells.
The vaccinated groups (six groups) included mice vaccinated with TcpA, TcpB, FlaA, TcpA+TcpB, and TcpA+TcpB+FlaA.
Evaluation of cytokine responses after stimulation of spleen cells with recombinant proteins TcpA, TcpB, and FlaA exhibited the ability of these proteins to produce IFN-gamma and IL5.
Nevertheless, the level of IL5 in the cell culture of the TcpB group was lower than the other groups.
Thus, according to the obtained results regarding cytokine (in the lymphocyte culture), it is indicated that the immune system in TcpA and FlaA groups tends toward humoral immune responses, while TcpB drives the immune responses away from humoral immunity.
Increased level of IgG1 titer was also noted in the TcpB group.