Volume 20, Issue 3 (2017)
mjms 2017, 20(3): 33-48 |
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Amerian M, Nazarian S. Expression and purification of recombinant chimeric protein contains CtxB and TcpA from Vibrio cholera and investigation of antibody titer in mouse. mjms 2017; 20 (3) :33-48
URL: http://mjms.modares.ac.ir/article-30-6779-en.html
URL: http://mjms.modares.ac.ir/article-30-6779-en.html
1- Department of Biology, Faculty of Science, Imam Hossain University, Tehran, Iran
Abstract: (10244 Views)
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.
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.
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