Evaluation and comparison of antibody titers against single recombinant proteins, mixtures and chimer CTXB, TCPA and CTXB-TCPA

Cholera as diarrheal illness is one of the most important causes of death and people's disability in different societies. Colonization factor pili (tcpA) and cholera toxin are the most important factors in the pathogenesis of Vibrio Cholera. B subunit of cholera toxin (ctxB) and tcpA have the ability to induce immune responses. The aims of this study was production of CTXB, TCPA, CTXB-TCPA recombinant protein and evaluation of antibody titers against separately, cocktail and chimeric protein in mice.
In this research study, ctxB ¡tcpA and ctxB-tcpA genes were cloned in pET28a and pET32a vectors. Recombinant plasmids was transformed to Escherichia coli (E.coli) BL21 DE3 and expression was induced with IPTG. The protein expression were evaluated by SDS-PAGE and Western Blotting analysis. The recombinant proteins were purified using Ni–NTA affinity chromatography. Mice immunization were done subcutaneously or intraperitoneally. Antibody titer was determined by ELISA in immunized mice sera.
SDS PAGE and western blotting confirmed expression and purification of recombinant proteins. The yield of purified CTXB, TCPA, CTXB-TCPA proteins was 15/570, 11/533 and 33/100 mg/L, respectively. ELISA results showed satisfactory immunization of mice. There was no significant difference in antibody titers against CTXB-TCPA protein and CTXB, TCPA cocktail. Also, no significant difference was observed in titers between subcutaneously or intraperitoneal injection.
The low differences in the antibody titer may be related to the longevity of memory cells and also their injection method. Due to the advantages of chimeric proteins, CTXB-TCPA protein could be a good alternative instead of protein cocktail to stimulate the immune system.