Modification Of Aggregation Key Sites Of Recombinant Flagellin Protein KFD1 And Its Immunogenicities

Mucosa is the main way for pathogens to invade the human body.Mucosal immunity helps protect the body from pathogen invasion and it is the first defense mechanism against various infectious pathogens.Mucosal vaccines can help the body to establish an effective mucosal immune response and prevent pathogen invasion and infection in the initial stage.The recombinant subunit mucosal vaccine has the advantages of simple design principle,low production cost,clear immunogen,few side effects and high safety.However,adjuvants are needed to induce high-efficiency mucosal and systemic immune responses.Studies have shown that flagellin has good mucosal immune adjuvant activity,and can be expressed in fusion with antigen.However,flagellin has strong immunogenicity,and may therefore interfere with the immune effect of the vaccine target antigen.Recombinant flagellin KFD1 lacking the main immunogenic regions(D2,D3 regions)of flagellin retains TLR5 pathway activation activity and mucosal adjuvant activity,but tends to accumulate by itself.This thesis aims to modify KFD1 from the two aspects of reducing aggregation,increasing soluble expression,and reducing its own immunogenicity while retaining good adjuvant activity.First of all,based on the protein spatial structure prediction and crystal structure information,we selected a number of key sites that may potentially affect aggregation,and carried out mutation modifications.The soluble protein expression ratio is used to indicate its aggregation characteristics,and the structure and activity of the mutant protein are tested.The study found three mutation sites that significantly affect the self-aggregation characteristics of flagellin.Among them,prokaryotic expression of soluble target protein of M3 mutation induced accounted for the highest expression,but its ability to bind to TLR5 decreased significantly;M7 mutation can significantly enhance the stability of KFD1,and reduce its aggregation.On the other hand,we used online prediction tools to predict the T and B cell epitopes of KFD1,and used the epitope core site mutations for experimental verification and screening.The results of the study showed that the mutation modification for T and B cell epitopes significantly improved the soluble expression of recombinant protein.The modified protein of B cell epitope showed a reduction of about 40% in immunogenicity in the in vitro antibody binding experiment,while the mutant protein,there was no significant change in the TLR5 activating activity and adjuvant activity.Finally,we used the respiratory syncytial virus(RSV)P protein as the model antigen and designed three fusion-expressed proteins—P-KFD1-M7-Mutant,P-KFD1-B-Mutant,and P-KFD1-T-Mutant.The results of detecting antibodies and T cell responses after immunizing mice with three fusion proteins showed that the mutation modification for aggregation and B cell epitopes significantly improved the soluble expression and protein stability of the recombinant protein;KFD1-T-Mutant,modified for T cell epitopes,significantly reduced the titers of KFD1-specific antibodies.In conclusion,the recombinant protein engineering strategies adopted in this study achieved the goal of maintaining the adjuvant activity of KFD1,reducing its aggregation characteristics and its own immunogenicity.