Study Of The Protective Effect Of Mucosal Immunity ?A146ply-SP0148 Fusion Protein In Mice Infected With Streptococcus Pneumoniae

Objective:To purify the expression of three recombinant proteins of Streptococcus pneumoniae(S.pn)?A146Ply,SP0148 and ?A146Ply-SP0148,immunize the C57BL/6 mice with the purified recombinant proteins through nasal mucous,and further discuss the protective effect and mechanism of recombinant protein on the mice fatally infected with D39 streptococcus pneumoniae(S.pn),and its effect on the nasal airway colonization of mice with 19 F streptococcus pneumoniae(S.pn),so as to provide the experimental basis for the study and development of protein vaccine.Methods:?A146Ply and SP0148 genes were amplified with PCR technology,to obtain the?A146Ply-SP0148 gene through gene synthesis.The target gene was linded with the pET28 a plasmid vector and transformed into the BL21 competent cell.After expression was induced by IPTG and purification was made by Ni-NTA column,three recombinant proteins ?A146Ply,SP0148 and ?A146Ply-SP0148 with purification above 80% were finally obtained.Such three proteins were used as the antigen to immunize C57BL/6 mice through nasal mucous,then the titer of specific antibody IgG in serum and IgA in saliva was evaluated.D39 streptococcus pneumoniae(S.pn)was used to attack the immunized mice and their survival rate was monitored for 21 continuous days,to evaluate the protective effect of recombinant proteins on the C57BL/6 mice fatally infected with streptococcus pneumoniae(S.pn).19 F streptococcus pneumoniae(S.pn)was given to the mice through nasal cavity,and streptococcus in nasal lavage fluid and lung homogenate was counted,to evaluate the effect of ?A146Ply-SP0148 fusion protein on colonization of streptococcus pneumoniae(S.pn)in nasal cavity and lung of mice.Results:This study successfully constructed the pET-28a(+)recombinant plasmid of ?A146Ply,sp0148,and ?A146Ply-SP0148 fusion gene,and three highly purified and high-content recombinant proteins ?A146Ply,SP0148 and ?A146Ply-SP0148 were finally obtained.Compared with the CT adjuvant group,the high-titer specific antibodies lgG and lgA were generated in the serum of C57BL/6 mice of ?A146Ply group and SP0148 group which were treated through nasal administration(P<0.01respectively).Especially for the mice in group ?A146Ply-SP0148,high-titer specific antibodies lgG and lgA could be stimulated(P<0.001).The evaluation on protective effect on the fatally infected mice indicated that?A146Ply-SP0148 recombinant protein has better immune protection response on the mice fatally infected with D39 streptococcus pneumoniae(S.pn)through mucosa(compared with positive control PPV23 group,P>0.05).Compared with PPV23 positive control group,streptococcus in nasal lavage fluid and lung homogenate in?A146Ply-SP0148 group was relatively high,but there was no apparent difference between groups(P>0.05).The antibody complement bactericidal experiment confirmed that ?A146Ply,SP0148,?A146Ply-SP0148 stimulated the specific antiserum produced by the mice.And the killing ability of the activated complement to S.pn was significantly higher than that of the Anti-CT group(P<0.01 respectively).Conclusion:Conclusion: ?A146Ply-SP0148 fusion protein can induce the mucosal immune response of mice infected with streptococcus pneumoniae(S.pn),and can apparently reduce the colonization of 19 F streptococcus pneumoniae(S.pn)in nasopharynx and lung of mice.?A146Ply and SP0148 can both induce the immune response of Th1 and Th17 cells,indicating that ?A146Ply-SP0148 fusion protein will be a new optional vaccine for streptococcus pneumoniae(S.pn).