Expression and purification of recombinant chimeric protein contains CtxB and TcpA from Vibrio cholera and investigation of antibody titer in mouse | ||
| Pathobiology Reserach | ||
| Article 3, Volume 20, Issue 3, 2017, Pages 33-48 PDF (1.39 M) | ||
| Authors | ||
| Milad Amerian; Shahram 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 | ||
| Colonization factor; Vibrio cholera; Bioinformatics; Recombinant chimeric protein | ||
| References | ||
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