Influenza A virus nucleoprotein production in Escherichia coli

CARGNELUTTI D.E.; SANCHEZ M.V.; SCODELLER E.A.

Mendoza

Congreso; Reunión Científica Conjunta de la Sociedad de Biología de Cuyo,; 2010

Sociedad de Biología de Cuyo

Actual influenza vaccines are made by a production process that uses fertilized chicken eggs. Although the egg method may yet prove useful for production of a pandemic vaccine, we need new, more flexible manufacturing approaches. Vaccine based on highly conserved antigens can provide protection against different influenza A strains and subtypes. The use of influenza A virus recombinant nucleoprotein (rNP) as a vaccine antigen, stems from the fact that NP show less antigenic variation than the influenza virus surface glycoproteins. The object of this work is to describe the production of rNP in a prokaryotic system. Plasmid pET30a-NP was constructed by cloning the PCR products of the gene from A/PR/8/34 (H1N1) influenza virus strain into the plasmid expression vector pET30a. The plasmid was then used to transform E coli BL21 (DE3) for expression. Bacteria were grown to log phase, and protein expression was induced by adding IPTG to a final concentration of 0,1 mM. After 12 hs of incubation at 37°C, the cells were pelleted and resuspended in lisis buffer and sonicated. Lysated were clarified by centrifugation and the soluble rNP was purified with a nickel-charged sepharose affinity column with a purity greater than 95%. By this system we produce the rNP as a antigen candidate for an influenza vaccine, in a faster and cheaper way than the technology that use chicken eggs.