Construction Of Self-biomineralization Live Attenuated Streptococcus Pneumoniae Vaccine

Streptococcus pneumoniae can cause a variety of diseases,including otitis media,pneumonia,sepsis,and meningitis,and is especially harmful to the elderly and children.Due to an increasing number of bacterial antibiotic resistance,the Streptococcus pneumoniae vaccine has become an effective strategy to prevent and control Streptococcus pneumoniae infection.Currently available vaccines for Streptococcus pneumoniae including capsular polysaccharide protein-binding vaccines and 23-valent capsular polysaccharide vaccines.However,these vaccines have some disadvantages including limited serotype coverage,T-cell dependent,and cause capsular serotype replacement.Therefore,developing a new and more ideal pneumococcal vaccine has become an important goal.SPY1,a live attenuated vaccine for S.pneumoniae,was a D39 mutant strain of Streptococcus pneumoniae 2.We previously reported that SPY1 has significantly reduced amounts of capsular and low virulence and it can effectively protect mice against pneumococcal infection.SPY1 also offer better protection effect than currently available vaccines,and therefore has potential for further development.However,it has some disadvantages including the weak immunogenicity,thermal instability requires the cold chain for storage and transport,and antolysis.Biomineralization technology is the process that generating inorganic minerals under the control of biological macromolecules.It links organic soft substances with inorganic hard substances,and thus can protect organic substances.Therefore,we envisaged that replacement lyt A N-acetylmuramoyl L-alanine amidase domain with the genes of biomimetic nucleating peptides(BNPs with a large number of acidic amino acids that can chelate calcium ions in an alkaline environment and regulate the biomineralization process)in SPY1.Therefore,an non-autolysis SPY1 strain(SPY1?lytA-BNP)was constructed,and the bacteria could spontaneously form a calcium phosphate mineral shell in the high-calcium ion environment,and the immunogenicity of the live attenuated S.pneumoniae vaccine was improved.In this study,NW,W6,PA44 and N6 be selected as biomimetic nucleating peptides.And biomimetic nucleating peptides gene fragments were constructed with a lytA homologous arm were transformed into SPY1 replace lytA amidase domain by homologous recombination.The SacB gene and the chloramphenicol-resistance gene(Chl)were used as negative selection marker,the objective strain that does not contain any other functional protein genes except the biomimetic nucleating peptides gene was screened.The construction of the target strain was detected by PCR and its growth characteristics were examined.Thus,SPY1?lytA-NW,SPY1?lytA-N6,SPY1?lytA-PA44 and SPY1?lytA-W6 strains were constructed.At the same time,the SPY1 lytA amidase domain was replaced by a biomimetic nucleating peptides gene with a chloramphenicol resistance gene,results that SPY1?lytA-NW4,SPY1?lytA-N64,SPY1?lytA-PA444 and SPY1?lytA-W64 strains were constructed too.In situ mineralizationg of these bacterial.However,the results of electron microscopy showed that the calcium phosphate shell could not be formed on the surface of bacteria.Western blot analysis found that biomimetic nucleating peptides have no expressed on the cell wall,which may lead to the failure of bacteria mineralizationg.Therefore,the surface protein choline-binding protein A(CbpA)of S.pneumoniae was selected as a new biomimetic nucleating peptides insertion site.Redesign and synthesis the biomimetic nucleating peptides gene fragment and a primer.The targrt strain was screened by negative screening method.PCR was used to detect whether the bacteria were successfully constructed.The strain was cultured in a calcium-rich environment and titrated with different concentrations of phosphate to form a calcium phosphate mineralized shell on the surface.The mineralization of the vaccine strain was detected by scanning electron microscopy and direct fluorescent reaction.As a result,mineralization was unsuccessful.The mineralization strategy was changed,and the dialysis method was used to perform the mineralization process.But the bacteria still cannot been mineralized.The Streptococcus pneumoniae heat shock protein DanJ as a protein vaccine has been widely studied.Zeta potential showed that Dan J present a net negative surface charge.The Dan J protein was biomineralized by simplest addition of calcium ion and phosphorus ion,and a mineralization shell was well formed on the surface.The zeta potentials of proteins were compared with the constructed bacteria,and it was found that the surface charges of the two are comparable,but the particle sizes are quite different.Therefore,we speculated that because of the large particle size of the constructed bacteria,even have the biomimetic nucleating peptides were inserted into the bacterial surface proteins,the average acid amino acid content on the average area was not enough to support the full mineralization of the bacteria,resulting in incomplete bacterial mineralization.Some mineralized shells are easily detached and the like,eventually making it difficult for bacteria to form a complete mineralized shell.