Cloning Influenza m2e- Vibrio cholera ctxB fusion fragment in pET28a plasmid

Aim and background. The extra domain of the M2 protein of influenza is a weak immunogen, Hence this is not a suitable candidate for the vaccine development. The B subunit of the Cholera enterotoxin is nontoxic causing attachment of the holotoxin to the GM1 at the surface of the epithelial intestine cells, while, CTB is highly immunogenic. So fusion of these two genes would be very applicable in influenza vaccine development. using PCR with specific primers including restriction sites ctxB was amplified. Standard strain of influenza type A (A/ PUERTO RICO /8/34) was prepared and M2e was amplified using RT-PCR with specific primers. The second PCR reaction was performed for the fusion of the M2e and ctxB using the F primer of M2e and R primer of ctxB. PCR product was digested with BamH1 and EcoRI and cloned into the pET28a. Verification of the cloning was performed using sequence analyzing. After analysis, the recombinant pET28a/M2e-ctxB was transferred into E.coli Top10. The final confirmation was performed using colony PCR, double digesting and sequence analysis. Sequence analysis showed that M2e has been fused to ctxB in an exact frame. Moreover the fused gene was cloned into the restriction site of the BamH1 and EcoR1 in pET28a. Despite stability of the sequence of M2e it is not immunogen but fusion to strong immunogens such as CTB would be a new suggestion for Influenza recombinant vaccine production.