A Study On The Improvement Of Immunogenicity Of Streptococcus Pneumoniae PsaA DNA Vaccine By Prime And Boost Strategy

Objective Streptococcus pneumoniae,a principal agent for bacterial pneumonia, bacteremia,and meningitis,has caused great morbidity and mortality throughout the world,especially in young children,the elderly and population with underlying disease.It is also a major agent for highly prevalent but less serious infection,such as acute otitis media(AOM) and sinusitis.Antibiotics are often prescribed for the treatment of pneumococcal infection.However,the recent emergence of penicillin-and multi-drug resistant S.pneumoniae strains complicates the treatment of disease and increases the burden on public health systems.The effective means to control pneumococcal disease will rely on the preventive strategies,such as vaccination.Current pneumococcal vaccines(7 or 11-valent polysaccharide and protein conjugate vaccines)provide limited coverage against more than 90 different capsular serotypes.Another shortcoming of these vaccines is cost,which limits usage in developing countries.PsaA is one of the virulence factors of Streptococcus pneumoniae,it's produced by all types of Streptococcus pneumoniaea and is a conserved protein.It is a 37-kDa protein composed of 309 amino acid residues,and belongs to the family of metal binding lipoprotein.Plasmid DNA vaccination is emerging as a safe and cost-effective method in vaccinology.DNA vaccines have shown to induce strong immune response in small animals,but in some large animals,induced limited immune response.This study is mainly on the improvement of immunogenicity of Streptococcus pneumoniae psaA DNA vaccine by the prime-boost stategy.Methods This research was bank on the sequence of psaA in the Genebank,a pair of primers was designed and psaA gene was amplified by PCR from the genomic DNA of Streptococcus pneumoniae.By gene recombination technology in vitro, sequence psaA was cloned into prokaryotic expression vector pET28a to construct pET28a-psaA recombinant plasmid and eurokaryotic expression vector pVAX1 to construct pVAX1-psaA recombinant plasmid.After being confirmed by DNA sequencing,then transform E.coli BL21(DE3),expressed antigen protein was purified by affinity chromatography and the antigenicity of the antigenicity of the expressed protein was confirmed by Western blotting assay.In our study,BALB/c mices were immunized by intramuscularly(i.m.) injection of pVAX1-psaA DNA vaccine.Recombinant PsaA protein boost without adding adjuvant was applied two weeks after third DNA vaccination.Results The psaA gene was amplified and subcloned successfully.The constructed psaA DNA vaccine was confirmed by DNA sequcencing,and the recombinant PsaA protein was purified by the one-step Ni²⁺ affinity chromatography.The animal experiment results showed that the anti-PsaA level of the DNA prime-protein boosting mice was higher significantly than the other groups.Conclusion The immunogenicity of Streptococcus pneumoniae psaA DNA vaccine could be improved significantaly by the DNA prime and protein boost strategy.