Prokaryotic Expression And Preliminary Study On Immunizing Protection Of Pneumococcal Surface Adhesin A Of Streptococcus Pneumoniae

Objective: Streptococcus pneumoniae is a kind of conditioned pathogens to establish pharynx nasalis of 40% healthy crowd, and lead to pneumonia, otitis media, bacteremia, meningitis diseases and so on. It is a leading cause of morbidity and mortality in developed and developing countries. Each year S.P causes approximmately1.2 million deaths worldwide from pneumonia especially for old people and children. In developing countries, 5 million less 5 years old children died of pneumonia of Streptococcus pneumoniae every year. Streptococcus pneumoniae exten- sively distributes in nature, have 90 kinds of serotypes and patho- genicity for men mostly 30 serotypes in them, is one of main pathogens of bacterial pneumonia. At present capsular bolysaccharides vaccines are mostly used, but these difficultly contain all serotypes and fail to protect to infants and the elderly. Therefore, development of effective protein-based vaccines is pretty urgent.PsaA is a kind of lipoprotein located in surface of Streptococcus pneu- moniae, and bring into main play during Streptococcus pneumoniae adhesiveing mucous membrane of respiratory tract, therefore PsaA concerns with invasion and virulence of Streptococcus pneumoniae. PsaA is a kind of surface protein antigen, which is possesed by all kinds of strains, genetic conservative and species characteristic. Coding open reading frame of psaA gene is 930 bp, which codes PsaA protein of 37KD. Spatial framework of mature PsaA is one a helix linking two (β/α)4 structural domains. We will construct custom-crafted gene, and obtain PsaA protein by means of expres-sion, and then identify its nature with Western blot for studying PsaA protein function in pathopoiesis of Streptococcus pneumoniae. Eventually we will immune animals with expressed protein, and investigate antibody.Methods: 1 PsaA of Streptococcus pneumoniae gene and prokaryotic expression carrier pET32a were linked after cutting of two kinds of enzyme by gene recombination method in vitro, and were identificated by sequenc- ing. Eventually, the plasmids were transformed into Escherichia coli BL21 (DE3).The proteins were expressed with IPTG induction. Recombinant protein was detected with SDS-PAGE and purified with saturated ammon- ium sulfate. The antigenicity of proteins was confirmed with anti- Streptoc- occus pneumoniae mouse blood serum by Western blot.2 Active immunization protection assay 40 kunming mice were divided 2 groups randomly: control group and PsaA proteins experimental group. PsaA proteins experimental group each mouse was received 10 ug of every time on day 0, 14, 21 by fold inguen hypodermic injection. Control group mice injected isotonic Na chloride. Each mouse was infected fatal dose S.P by abdominal cavity after immunity completion 7th day. These mice were then monitored for death over 21 days, and the median survival time of each mouse was recorded.Results:1.The results of tacho-PCR, restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene psaA had been cloned into the plasmid pET32a (+). The length of the psaA gene was 870bp. Compared to the sequence of GeneBank, no mutation was found. A recombinant protein about 57kD was expressed in BL21 (DE3) after induction of IPTG. SDS- PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 60% of total baterial protein and the purity of target protein was up to 80%. Western blot suggested that expressed protein was confirmed PsaA and displayed satisfactory antigenicity.2.Active immunization protection assays In this experiment, the median survival times of PsaA proteins experimental group was signifi- cantly longer than control group(p<0.001). Conclusion:1.The psaA genes of S.P were cloned successfully into the plasmid pET-32a. Sequencing and sequencing analysis were done and no mutation was found. The expressed and purified recombinant proteins were proved with immunogenicity. The recombinant protein can be tentative antigen of rapid diagnosis for pneumonia of Streptococcus pneumoniae.2.PsaA protein could elicit protection against fatal dose S.P aggression for Kunming mice. The recombinant protein can be candidate protein antigen of vaccines against S.P. It established foundation for investigation of protein vaccines of Streptococcus pneumoniae.