Amphoteric Ionic Peptide-based Modification Of Pro-myosin Lymphatic-targeted Immune Tolerance Therapy Study

Food allergy(FA)is an increasingly severe public health problem.In recent years,its prevalence has been on the rise and has dramatically affected consumers’ quality of life.Allergen-specific immunotherapy(AIT)is the only treatment that can effectively modify the natural course of allergic diseases.However,at this stage,AIT has significant problems,such as the number of treatments,time consumption,and poor patient compliance,which limit its practical application.Studies have shown that lymphatic targeted delivery can reduce the number and duration of AIT injections,enhance the protective immune response,and significantly improve the efficacy of AIT.However,the lack of effective pathways for lymphatic delivery is mainly limited by the complicated synthesis process of traditional delivery vectors and the difficulty of mass production.In this paper,we apply the recombinant expression modification strategy of glutamate-lysine(EK)to pro-myosin(TM)to investigate whether EKylation modification can make TM lymphatic targeting and thus enhance its AIT efficacy in order to develop new ideas for future lymphatic delivery of protein drugs and novel AIT applications.To this end,this paper focuses on the following aspects of research.1.Construction of TM-EK recombinant protein based on amphiphilic peptide interface modification strategyUsing TM as a model protein for food allergy,the recombinant TM and TM-EK proteins were prepared by coupling amphiphilic poly-EK peptide with TM using recombinant expression technology and characterized accordingly;the effect of EKchemical modification on the immunogenicity of TM was analyzed in vitro and cellular levels using related immunological experiments.The results showed that the recombinantly expressed TM had a molecular weight of 34882.8 Da and TM-EK had a molecular weight of 42601.5 Da,and there was no fragment loss during the induced expression process;the EKylation strategy could reduce the aggregation of the protein,and at the same time significantly decreased the Ig E binding ability of TM.2.Study on the lymphatic targeting effect of TM-EK recombinant proteinNear-infrared fluorescence imaging was used to investigate amphoteric polyEKylation modification’s effect on improving the proteins’ lymphatic targeting by using the fluorescent dye Cy7 to label TM and TM-EK proteins.It was shown that 1 h after subcutaneous injection into the dorsal surface of the mouse foot,fluorescence signals of both TM and TM-EK were observed in both popliteal and sciatic lymph nodes,and the fluorescence signal of TM-EK was significantly more substantial than that of TM;similarly,ex vivo imaging analysis of popliteal lymph nodes showed that the fluorescence signal of TM-EK was also more vital than that of TM.To this end,EKylation modifications of proteins have been shown to enable lymphatic targeting.3.Study on immune tolerance therapy based on lymphoid-targeted TM-EK recombinant proteinThe BALB/c mouse allergy model was constructed,and the recombinant TM-EK protein with lymphatic targeting characteristics was applied to the AIT of food allergy.The study found that compared with the TM treatment group,TM-EK-based AIT significantly reduced the level of TM-specific Ig E in the serum of mice while upregulating the proportion of Th1 and Treg cells in the spleen and the expression of related cytokines,maintaining the steady-state balance of Th2/Th1 in mice,effectively alleviating food hypersensitivity,and causing no damage to the main organs and tissues of mice,providing a new idea for the development of safe and effective new AIT.