Expression and purification of recombinant chimeric protein contains CtxB and TcpA from Vibrio cholera and investigation of antibody titer in mouse

Authors
M. Amerian
S. Nazarian
Department of Biology, Faculty of Science, Imam Hossain University, Tehran, Iran
Abstract
Objective: The TcpA colonization factor of pili A and the cholera toxin are the most important pathogenesis factors of Vibrio cholera that have the ability to stimulate the immune system. The aim of this study is a bioinformatics analysis of the expression of CtxB-TcpA recombinant chimeric protein in E. coli, and production of antibody against it in mice.
Methods: We designed a gene cassette that contained the CtxB and TcpA genes, and a spacer linker by using bioinformatics. Characteristics that include the structure of the chimer protein and epitopes were studied. In order to build a gene cassette, TcpA and CtxB genes were proliferated and cloned in pET28a(+). CtxB-TcpA gene expression was induced by IPTG. The produced CtxB-TcpA recombinant protein was confirmed by SDS-PAGE and Western blot analyses. Antibody produced from mice serum was isolated and confirmed by ELISA.
Results: The codon adaptation index of the optimized gene was 0.9. The prevalence ratio codons increased to 74% through codon optimization. Enzyme analysis verified the chimeric gene CtxB-TcpA cloning in the pET28a (+) expression vector. A protein with a molecular weight of 35 kDa was seen on SDS-PAGE. Its reaction with anti-histidine antibodies was confirmed by Western blot. The purified protein was 33.100 mg/l. Immunization of mice induced a serum antibody response.
Conclusion: The chimeric protein can be considered a good candidate for effective immunity against cholera.

Keywords

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Pathobiology Reserach
Volume 20, Issue 3
October 2017
Pages 33-48