Improvement Of Protein Vaccine Stability By Biomineralizaion

Objective:Vaccine is the most economical and effective method to prevent the spread of infectious diseases.Its main components are various attenuated or inactivated biologically active substances such as viruses,bacteria,toxoids and proteins.They are extremely sensitive to temperature,so the production and transportation of vaccines requires strict cryopreservation,which greatly increases the cost of vaccines and limits the widespread use of vaccines.Streptococcus pneumonia（S.pn）is the main pathogen of community-acquired pneumonia,which can also cause sinusitis,otitis media,and meningitis,etc.Vaccines are the main means to control the infections.Protein vaccines have become a hot spot of vaccine research because of its high conservation,and it can effectively activate B and T cell immunity.Unfortunately,they are highly heat-sensitive and readily denatured by even slight changes in the environmental temperature.Therefore,improving protein stability is of great significance for the application of protein vaccines.Therefore,improving the stability of proteins is of great significance for the application of protein vaccines.This study intends to use the biomimetic mineralization technology to prepare a Streptococcus pneumoniae protein vaccine based on the protein pneumolytic toxin PLY and heat shock protein DnaJ.It is coated with a layer of calcium phosphate shell through biomineralization to improve its stability,and explore whether this method has wide application value in protein products.Methods:Proteins WT-PLY,ΔA146PLY,DnaJ,and proteins containing biomineralized peptides WT-PLY-PA44,ΔA146PLY-PA44,DnaJ-PA44 were expressed and purified by molecular cloning technology,and biomineralized in a weakly alkaline solution rich in Ca²⁺and PO₄^3-;to analyze the size and surface composition of mineralized particles by TEM,EDS,etc.;to analyze the thermal stability of mineralized vaccines by proteinase K degradation experiments,hemolysis experiments,etc.;to evaluate the immune protective effects of mineralized vaccines by cell experiments and animal experiments.Results:The recombinant proteins WT-PLY,ΔA146PLY,DnaJ and their corresponding containing biomineralized peptides proteins WT-PLY-PA44,ΔA146PLY-PA44,DnaJ-PA44 were successfully prepared,and the purity was above 90%.Whether it is the proteins WT-PLY,ΔA146PLY,and DnaJ those are not linked to the mineralized peptide,or the proteins WT-PLY-PA44,ΔA146PLY-PA44,and DnaJ-PA44 those are linked to the mineralized peptide,the proteins change their originally clear and transparent liquid situation and become mineralized protein suspension with milky white.And preliminarily constructed mineralized particles with a relatively uniform density:WT-PLY@CaP,ΔA146PLY@CaP,and DnaJ@CaP,with sizes of 123.01±2.49 nm,115.09±2.43 nm,respectively and 85.09±5.02 nm;otherwise,WT-PLY-PA44@CaP,ΔA146PLY-PA44@CaP and DnaJ-PA44@CaP,whose sizes are 90.12±2.73 nm,108.21±3.43 nm and 95.02±2.74 nm,respectively.The results of proteinase K degradation experiments show that the unmineralized protein basically degrades with time,but the mineralized protein still has more residues;similarly,from the perspective of degradation rate,the rate of protein degradation after mineralization is less than that of unmineralized protein,suggesting that mineralized protein has a higher proteinase K tolerance than unmineralized protein.The hemolysis test results of WT-PLY and WT-PLY-PA44 show that mineralized protein can retain its hemolytic activity at 45°C for 24 h,and maintain its hemolytic activity at 55°C for 10 min;without mineralization,protein can only retain its hemolytic activity at 45°C for 30minutes,and maintain its hemolytic activity at 55°C for 3 minutes,which suggests that the thermal stability of mineralized protein at higher temperatures is higher than that of unmineralized protein.The results of cell detection experiments showed that the levels of IL-6 and TNF-αstimulated from the antigen-presenting cells by mineralized nanoparticles in vitro were significantly higher than those of unmineralized proteins;Compared with the non-mineralized protein immunization group,mice were induced to produce more highly specific antibodies.After nasal attack,the bacterial load was significantly lower than that of the non-mineralized protein immunization group.The level of inflammatory factors IL-6 and TNF-α,was also significantly lower than that of the unmineralized protein group.Conclusion:In this study,the biomineralization of vaccine proteins was carried out through a relatively simple biomineralization technology.Regardless of whether the protein is connected to the mineralized peptide or not,the protein can form nanoparticles with a CaP mineralized shell,but the protein connected to the mineralized peptide has higher efficiency.The resulting nanoparticles have a certain resistance to proteinase K degradation and high temperature tolerance,these results provide a valuable experimental basis for the preparation of stable protein formulations.