[Prokaryotic expression of chimeric trimer protein [3M[2]e-HA2-NP] derived from conserved domains of influenza virus A/H1N1 as a promising universal subunit vaccine]

Background and Aim: Influenza A virus is an important respiratory pathogen which can cause high rates of morbidity and mortality during seasonal epidemics and pandemics. Current vaccines are not capable of producing effective immunity against different influenza virus subtypes. Designing universal vaccines using conversed domains of influenza virus antigens can overcome this limitation. The ectodomain of influenza M[2] protein [M[2]e], the hemagglutinin stalk domain [HA2], and nucleoprotein [NP] are the most conserved sequences among subtypes of influenza A viruses. The aim of this study was to attach part of the NP gene into the binary structure of 3M[2]e-HA2 and assessment of expression of a chimer trimer protein in prokaryotic system. This recombinant protein is considered as a promising antigenic candidate for a universal vaccine production

Materials and Methods: First, part of the NP gene segment of human influenza A/H1N1[PR/8/34]was amplified by PCR using designed specific primers. This amplified gene was cloned into pGEM-TEasy cloning vector. Then, the confirmed segment of NP gene was subcloned into PET28a/3M[2]e-HA2 recombinant expression vector, downstream of the HA2 segment. After confirmation of cloning, the chimer protein was expressed in E.coli BL21[DE3]

Results: The results of colony PCR, restriction enzyme digestion and sequencing indicated that the NP gene segment was correctly cloned into PET28a/3M[2]e-HA2. Chimer protein expression was analyzed by SDS-PAGE and confirmed by Western blotting

Conclusion: Design and production of recombinant protein [3M[2]e-HA2-NP] could be an important step towards the development of a universal influenza vaccine

Effects of protoplast fusion on S-endutoxin production in bacillus thuringiensis Spp. [H14]

In this study, mutant forms of Bacillus thuringiensis spp. israelensis [H14] were produced. These mutants were identified when the cells were cultured on chloramphenicol plates and stained with crystal violet. Protoplasts of the mutants were isolated by enzymatic digestion [lysozyme] of the cell walls at the presence of an osmotic stabilizer. The protoplasts were induced to fuse to each other in the presence of PEG 6000. The frequency of regeneration and recombination was 80% and 2 10-4, respectively. In order to survey the effect of protoplast fusion on production of toxin, anti-serum against pure toxin was raised in rabbit and was used in single radial immunodiffusion. The comparison of -endotoxin concentration between B. thuringiensis fusion and the wild type strains showed that B. thuringiensis fusion has 1.48 time more toxin than wild type