designing and producing a proper fusion construction is the most important problem of producing large quantities of functional protein. this construction should have all necessary components of a real gene. a good designed fusion gene construction could be cloned into a good and suitable host. designed epsilon alpha fusion gene construction have 2358bp in length which nucleotides 1 to 984 is complete epsilon phototoxin, 985 to 1020 (36 bp) is linker sequence which is optimized for e. coli and residue sequence is alpha mature toxin. development and submission of the synthetic construct sequence was reported previously.
the aim of the present study was to clone and sequence clostridium perfringens type d and clostridium septicum epsilon alpha fusion gene for expression and production fusion protein.
Methods
Epsilon alpha fusion gene was duplicated and ligated into pgem_b1 cloning vector. e. coli/top10 competent cells was transformed using the ligation product and the recombinant clones were chosen on lb ampicillin medium. at the end, pgem_b1 recombinant plasmid was extracted from bacterium host and sequenced using specific primers.
Results
The result of sequencing and blas analysis showed that fusion gene ligated in recombinant vector was matched to accession number ku726861 previously deposited and clearly indicated that the designed epsilon-alpha fusion gene could be clone and express in a suitable host cell.
Conclusion
Based on the latest information, this is the first cloning of epsilon-alpha fusion gene, which could be used for producing of a recombinant epsilon-alpha fusion protein vaccine.
