Study On Pneumococcal Serine-Threonine Kinase Protein(StkP) As Vaccine Candidate

Streptococcus pneumoniae(Pneumococcus) is responsible for life-threteaninginvasive diseases(meningitis,bacteriemia,pneumoniae， otitis),especially at theextremities of age.Because of antibiotic resistance and numerous serotype issues,theresearch of Pneumococcus has became a main method. World Health Organisitionrecommend the pneumococcal polysaccharide vaccine and polysaccharide proteinbinding vaccine to prevent pneumococcal infection effectively. However, thesevaccines habour the problems such as limited serotype covered and limitedpopulation，which lead to the research of protein-based vaccine.This thesis selectedpenumocuccal protein StkPC314as pnemococcus vaccine candidate and evaluate itspreclinical study.This thesis selected C-terminal tail for314aa of pneumococcal proteinStkP,Serine-Threonine kinase protein，as protein vaccine.StkPC314gene sequence isdesigned and synthesized according to Escherichia coli codon optimization,then isconstructed into pET20b-StkPC314prokaryotic expression plasmid by molecularclone methods.After high expression of recombined vector,the StkPC314protein ispurified by three chromatographic columns.Through mice immune assay of StkPC314protein,the Polyclonal Antibody is detected by indirect ELISA assay,the high titer ofantiserum titers illustrate the high humoral immune responses to antigen protein.Themice is challenged the Streptococcus pneumoniae ATCC6301after immune ofproteinStkPC314protein,the survival rate is obviously higher than control group,whichillustrate the protein vaccine can prevent the infection caused bypneumococcus.Besides,by animal model two conclusions can be made:the optimalimmune adjuvant is freund,the optimal immune dose is20μg/mice.The mice animalmodel shows the protein vaccine obtain the high Immunogenicity and Protective Efficacy，which shows the potential as pneumococcus protein vaccine candidate.In order to establish the acurate quantitive method to detect StkPC314. Wescreened5hybridoma cells which can secrete StkPC314-specific monoclone antibodyby Hybridoma Technique. Through cultivation,collection,purification,the McAb isprepared,and is anaylized for appetency calculation,binding sites,Ig subtypes.Usingthe mixture of several McAbs, by establishing indirect ELSIA methods,we canquantity the polyclonal antibody concentration of mice antiserum.In order to quantifythe antigen protein,we establish the double-antibody sandwich ELISA methods bybiotin labeling for McAb.the accurate protein concentration for quantification is12.2-97.6ng/ml,the lowest detection limit (LOD)12.2ng/ml.