Preparation Of Ferritin Nanoparticle With Enterococcal Antigen And Study On Its Immunogenicity

Enterococci are conditional pathogens that do not infect organisms under normal circumstances.Because the transfer of plasmid,genes and pathogenicity between various organisms,enterococci can cause increasing infection of enterococci in mammals such as humans and pig.The treatment and prevention of enterococci is even more difficult.Setting the value of enterococcal infection is a critical step.Collagen adhesin ace promotes the adhesion of enterococci and damages the body.Therefore,studying the antigenic epitope vaccine of collagen adhesin can be the key to prevent and treat enterococci.However,the immunogenicity of epitope vaccines is generally low,the addition of vaccine adjuvants is required to enhance immunogenicity and increase antibody levels.Ferritin can not only function as a vaccine adjuvant but also as an antigen presentation system.At different p H values,ferritin can be assembled and disassembled automatically.It can use antigens attached to the surface of protein subunits to increase the specific antibody level of antigens.Experiment I in order to prepare ferritin nano cages,we first dissected healthy pigs raised by our group,extracted total RNA from fresh hearts by TRIzol method,reverse transcribed RNA into c DNA by reverse transcription kit,and used Primer 5.0designed a pair of specific primers for pig-derived ferritin H subunit,amplification the H subunit(546bp).The H subunit and the PMD18-T vector were ligated,transformed,and the plasmid was extracted.After sequencing,it was connected to the prokaryotic expression vector p ET-32 a.A large number of proteins were expressed successfully inducing proteins that were consistent with the expected molecular weight of the desired protein.The ferritin subunits were soluble proteins.The protein was purified by nickel column,SDS-PAGE and Western Blot proved that the ferritin subunits had good immunogenicity.The protein was concentrated by ultrafiltration,the samples were prepared,and the characterization of ferritin was observed by transmission electron microscope imaging.The results show that the ferritin subunits can self-assemble into nano cages under physiological conditions and have a hollow structure,similar to that of natural ferritin.We can conclude that iron-deficiency nucleus ferritin can be prepared by prokaryotic expression,with simple operation and high yield.It lays the foundation for ferritin as a vaccine adjuvant and carrier.Experiment 2 in order to prepare ferritin nano-cartilage carrying antigen epitopes,the amino acid sequence of collagen adhesin was first predicted by bioinformatics techniques,and the predicted six antigenic epitopes were synthesized in the prokaryotic expression vector p ET-.32 a,named p ET-32a-ace1,p ET-32a-ace2,p ET-32a-ace3,p ET-32a-ace4,p ET-32a-ace5,p ET-32a-ace6,and a laboratory-contained recombinant plasmid p ET-32a-ace,design a pair of new specific primers,amplify the ferritin H subunit,insert the target gene into the prokaryotic expression vector,transform,extract the plasmid,sequence,express the fusion protein,prepare the sample,and observe under the transmission electron microscope.Characterization of the fusion protein,the results are five fusions of p ET-32a-ace1-H,p ET-32a-ace3-H,p ET-32a-ace5-H,p ET-32a-ace6-H,and p ET-32a-ace-H.The protein is a soluble protein,and both p ET-32a-ace2-H and p ET-32a-ace4-H are inclusion bodies.Due to the nature of the protein itself,only soluble proteins were used.The results were shown for the five proteins SDS-PAGE and Western Blot.Proteins have strong immunogenicity,electron microscopy results:p ET-32a-ace1-H,p ET-32a-ace3-H,p ET-32a-ace5-H and p ET-32a-ace6-H is a ferritin nano-cage with a hollow structure and a diameter similar to that of native ferritin;p ET-32a-ace-H can also form a protein nano-cage,However,without the hollow structure,the diameter of the nano cage is much larger than that of natural ferritin.We can tell that ferritin is still able to self-assemble into ferritin after it is linked to an epitope,but because of the different lengths of antigenic sequences,the structure of nanocages is different.Since the antigen sequence of p ET-32a-ace-H is larger than the H subunit sequence,some antigenic proteins are enclosed in the hollow structure,and no hollow structure is observed by transmission electron microscopy.The preparation of five ferritin cages carrying antigenic epitopes provided the vaccine for the next animal experiment.In experiment 3,specific antibody levels in rabbits were determined by indirect ELISA.Firstly,a large number of five antigenic epitope vaccines of experiment 2were prepared,and rabbits were immunized.At the same time,rabbits of the control group were established and immunized with ordinary collagen adhesin.Ear veins were collected weekly and the antibody titer reached the heart after ELISA.Serum was separated and the immunogenicity of the six proteins was known to be based on ELISAresults:p ET-32a-ace5-H >p ET-32a-ace-H>p ET-32a-ace >p ET-32a-ace6-H>p E T-32a-ace3-H >p ET-32a-ace1-H.The antibody levels of p ET-32a-ace5-H andp ET-32a-ace-H were significantly higher than those of the p ET-32a-ace control group(p<0.01);the antibody level of p ET-32a-ace6-H was comparable to that of the control group Levels were similar;the antibody levels of p ET-32a-ace3-H and p ET-32a-ace1-H were significantly lower than those of the p ET-32a-ace control group,and it was concluded that ferritin could significantly enhance the immunogenicity of the antigen and increase the The level of antibody immunity,the best antigenic epitope vaccine is p ET-32a-ace5-H.In summary,the ferritin nanocages with antigenic epitopes can cause high levels and rapid immune responses.Ferritin,as a carrier and adjuvant,can significantly enhance the immunogenicity of epitopic proteins for the development of Enterococcus faecalis.The vaccine provided the theoretical basis and experimental basis.