همسانه سازی و بیان ناحیه کاتالیتیک نوروتوکسین بوتولینوم تیپ E در باکتری E.coli

Background and purpose: Clostridium botulinum bacteria produces seven types of botulinum neurotoxins among which types A, B, E and F are responsible for human botulism. One of the treatments for botulism is the inhibition of botulinum neurotoxins catalytic domain activity by inhibitors. In this study, botulinum neurotoxin type E catalytic domain has been cloned in pET28a vector and expressed in E. coli BL21 (DE3). Materials and methods: In order to cloning of the catalytic domain, the genomic DNA was extracted. The sequence was amplified by Polymerase chain reaction (PCR) and was inserted into pGEM-T Easy vector. Then, the recombinant vector was transferred to E. coli DH5α cells. Afterwards, the cloning product was removed from pGEM-T Easy vector and inserted into pET28a vector using ligation reaction. Finally, the recombinant pET28a was transferred into E. coli BL21 (DE3) cells. Expression of catalytic domain was studied in standard conditions. Results: The results of enzymatic digestion and PCR reaction confirmed that cloning and subcloning occurred in pGEM-T Easy and pET28a vectors, respectively. The process was verified by sequencing. Finally, expression of this sequence was confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Conclusion: The cloning of the sequence was accurately conducted in pGEM-T Easy and pET28a vectors. Also, the results showed that the expression of this sequence has been performed properly.