RESUMO
Objective To prepare a conjugate vaccine by linking Haemophilus influenzae type b (Hib)polysaccharide to PsaA protein carrier and evaluate the immunogenicity and efficacy of the conjugate vaccine. Methods A recombinant protein rPsaA,expressed by using the genetic engineering technology, was used as a protein carrier to prepare conjugate vaccine together with Hib polysaccharide. Ten mice at age of 3 weeks were immunized with the conjugate vaccine,while another 10 age-matched mice were immunized with Hib-tetanus toxoid(Hib-TT)vaccine which was produced formerly as a control. The mice treated with equal volume of PBS were set up as the negative control. The IgG antibodies in serum samples against PsaA and Hib polysaccharide were detected in two weeks after the final immunization. A suspension of Pneumococ-cus was injected into the middle ears of mice from experiment and control group. Histopathological analysis was performed to measure the clearance of bacteria in the middle ears and the severity of infection on days 3 and 7 after bacterial challenge. Results The rPsaA protein was prepared by the genetic engineering tech-nology and purified successfully with anion-exchange column. The Hib polysaccharide-PsaA protein conju-gate vaccine was prepared through a series of amide condensation reactions. The detection of IgG antibodies against PsaA protein and Hib polysaccharide in the immunized mice demonstrated that there was no signifi-cant difference with the titer of IgG against Hib polysaccharide between the mice immunized with the Hib-PsaA conjugate vaccine and those immunized with the Hib-TT vaccine. Less Pneumococcus strains were de-tected in the middle ears of mice immunized with the conjugate vaccine than those mice immunized with the Hib-TT vaccine three days after challenge. The mice from control group showed severe inflammation in the middle ears than those from experiment group. The Hib polysaccharide-PsaA protein conjugate vaccine im-proved protection against Pneumococcus infections as compared with the Hib-TT vaccine. Conclusion The rPsaA protein could be produced by genetic engineering technology and purified by anion-exchange column. The Hib polysaccharide was successfully conjugated with the rPsaA protein through amide condensation reac-tion. Both anti-PsaA and anti-Hib immune responses were induced in young mice by the injection of Hib pol-ysaccharide-PsaA protein conjugate vaccine. Apart from providing protection against Hib infection,the con-jugate vaccine might also be used for the prevention of acute otitis media caused by Pneumococcus infection.
RESUMO
Objective To express and purify the pneumococcal surface adhesin A(PsaA) protein,discuss its application as a protein carrier in conjugates vaccine. Methods The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. The PCR product was then cloned into the prokaryotic expression vector pET-28a and the recombinant was transformed into host cell E. coli BL21 (DE3). The expression of the recombinant protein(rPsaA) was induced by IPTG and purifled by using DEAE anion-exchange chromatography. The rPsaA was successfully conjugated with group A meningococcal polysaccharide(GAMP). The mice were immunized subcutaneously with the conjugate and the immune responses against GAMP and PsaA were detected by ELISA. Results The recombinant PsaA was expressed as a 37 × 103 soluble protein without His-Tag. The rPsaA was successfully conjugated with GAMP. In addition to the immune response against PsaA, The antibody response against GAMP was significant improved in the mice immunized with conjugate vaccine in comparison with those immunized with GAMP alone. Conclusion The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and CAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.