بررسی کلونینگ ؛ تعیین توالی و بیان فرگمنت ‭c ‬توکسین کلستریدیوم تتانی سویه واکسینال موسسه رازی

Background: Fragment C is C-terminal domain of tetanus toxin heavy chain that can be promote immune response against lethal dose of tetanus toxin, thus it can be considered as a vaccine candidate against tetanus infection, occures by Clostridium tetani . In this study DNA of tetc was prepared, cloned into pTZ57R ,subcloned into pET expression vector and the recombinant protein (fragment C) was over expressed in E. coli. Methods: Bacteria were grown in Modified Mueller's Media, the total DNA (cells and plasmids) was prepared for PCR and by using specific primers, tetc DNA was amplified and ligated into pTZ57R ,subcloned in two pET vectors (22b and 28 a). The recombinant vectors were transferred into competent E.coli strain BL21 (DE3) pLysS and after selection of proper colony, which carried the target DNA within the vector; cells were cultured and induced with IPTG, in order to express protein (TetC). The cultures were tested for presence of TetC by SDS-PAGE and western blot. Results and Conclusion : Molecular techniques such as PCR and sequencing which showed exact defined size of the tetc (1356 bp) and SDS-PAGE and western blot of over expressed protein, confirmed the correct cloning and expression of TetC. In this research, full length of tetc was obtained from Harvard CN49205 strain of C.tetani ,vaccinal strain of Razi institute. sequencing this fragment showed that 100% identity with Harvard and Massachusetts strains in Genbank. over expression in E. coli which, can be used for further studies, including purification and neutralizing antibody production against TetC.