ABSTRACT
Objective To evaluate the protective effectiveness of intranasal immunizations with recombinated-pneumococcal autolysin(Re-LytA), which protects mice against local and systemic Streptococ- cus pneumoniae(Sp) infection. Methods Testing group (group A): CpG as an adjuvant, the mice were intranasally immunized with purified Re-LytA, obtained by affinity chromatograph. The negative control group(group B) were intranasally immunized with sterile saline. And the positive control group (group C) were received 23-valent polysaccharide commercial vaccine through intramuscular injection. All the samples were collected 2 weeks post the last immunization. The levels of antibody was determined by ELISA. Then the mice were challenged intraperitoneally and intranasally with Sp, respectively. The infection and coloniza- tion was followed by monitoring colony-forming units of Sp in the blood, homogenized lung, and nasopharyn- geal lavage fluid 4 days post intranasal immunization. The mice were observed daily to note the livability of each group. Results The level of the LytA antibody (IgG, IgA, slgA) in group A were higher than that in group B and C (P < 0.05). Neither the LytA nor polysaccharide antibody could be detected in group B. Polysaccharide antibody could be detected in group C. After challenged intraperitoneally there was no signifi- cant difference in survival rates between group A and group C (P > 0.05), which was significant higher than that in group B (P <0.05). After challenged intranasally, compared with the group A, the geometric mean colony-forming units washed from the nasopharyngeal lavage fluid of the group B and group C were signifi- cantly higher (P <0.05). Conclusion lntranasal immunizations with Re-LytA can protect mice against lo- cal and systemic pneumococcal infection, and the protective immunity may be related to sIgA.
ABSTRACT
Objective To clone the gene of autolysin(LytA) which are from different clinical strains of Streptococcus pneumoniae and express them in Escherichia coli. Methods The LytA gene was amplified by PCR from the total DNA of S.pneumoniae. Primers were designed according to the LytA gene sequence of R6. Recombinant plasmids were constructed and the sequences of different clinical strains were analyzed through method of bioinformatics. The cloned genes were expressed in E.coli and detected by SDS-PAGE. Results Complete LytA gene were amplified from all of the different clinical strains of S.pneumoniae and recombinant plasmids pGEX-4T-1-LytA were constructed successfully. After comparing the sequence of DNA and supposed protein, we find some differences. Induced by IPTG, LytA gene was expressed effectively in E.coli Jm109. Result of SDS-PAGE showed that the molecular weight of expressed protein was 62 kD, the same as calculated. Conclusions The sequences encoding LytA from different clinical strains of S.pneumoniae were cloned, the recombinant plasmids pGEX-4T-1-LytA was constructed successfully. Sequence analysis showed that there have difference among the gene and amino acid sequences of LytA from different clinical strains. Further studies should be focused on whether the difference contributes to activity of autolysin and the drug-resistance of S.pneumoniae.