Effects Of Site-Directed Mutagensis And Peptide Aptamers On Major Allergens From Mud Crab

Shellfish,including crabs,is one of the eight major sources of food allergens proposed by the Food and Agriculture Organization.The food allergy caused by shellfish is usually life-long and has severe effects on the life of patients.In this study,the major allergens from mud crab?Scylla paramamosain?were known as tropomyosin?TM?and arginine kinase?AK?,the epitopes of them were studied in depth to reduce the allergenicity by using site-directed mutagenesis and peptide aptamers.The hypoallergenic proteins are expected to improve the immunotherapy for seafood allergic patients.Basing on the disulfide bonds and critical amino acids of conformational epitopes in TM and AK,the site-directed mutagenesis was performed to construct the recombinant expression vector pET-28a-mTM/mAK,the recombinants were induced and expressed in E.coli BL21?DE3?.Two soluble mutant TM?mTM?were generated,named mTM1(TM/_{R90A-E164A-Y267A})and mTM2(TM/_{N132A-M171A-Y267A}),respectively.Three inclusion bodies mutant AK?mAK?were generated,named mAK1(AK/_C201A),mAK2(AK/_K33A-T174A)and mAK3(AK/_T174A-C201A),respectively.Analyzed by Western blot using rabbit TM/AK polyclonal IgG antibody,the recombinant products were confirmed as target proteins.The results of circular dichroism spectra and surface hydrophobicity analysis showed that,compared with recombinant TM?rTM?,the secondary structures of mTMs were not changed with a typical?-helix structure and the tertiary structures of them were obviously altered.The IgE binding activity of mTM1 reduced significantly by18.1%,while mTM2 was not changed.Compared with recombinant AK?rAK?,the secondary and tertiary structures of mAKs were changed obviously,especially for mAK3,the?-helix content was 22.7%less.Moreover,the IgE binding activity of mAK1,2 and 3 were decreased by 54.2%,40.1%and 71.4%,respectively.The dendritic cells?DCs?phagocytic model and BALB/c mice sensitization model were established to further analyze the mutant protein in vitro and in vivo.The results showed that there was no significant change in the phagocytic capacity of DCs for rTM and mTMs.Among the AK group,the phagocytic capacity of DCs for mAK1 and mAK3 were obvious higher than that of rAK and mAK2.The specific IgG antibody level of experimental mice groups were significantly increased comparing with PBS group,there was also a significant increase of IL-4cytokines in spleen lymphocytes.Analyzing the linear epitopes and protease cutting sites of TM and AK,peptide aptamers were screened that could specifically bind to linear epitopes of TM and AK.Twelve peptide aptamers of TM were detected by IgG antibody,the peptide aptamers could significantly inhibit immunoreactivity of rTM and mTMs,the inhibition rates of peptide aptamers 5 on rTM,mTM1and mTM2 were 29.7%,30.2%and 31.1%,respectively.The results of AK IgG and IgE inhibition ELISA showed that,five peptide aptamers of AK could significantly inhibit immunoreactivity of rAK and mAKs.The IgG inhibition rates of peptide aptamers 3 were 30.6%,25.8%,27.6%and 23.5%on rAK,mAK1,mAK2 and mAK3,respectively.The IgE inhibition rates of peptide aptamers 3 were 28.3%,25.2%,26.5%and 21.7%on rAK,mAK1,mAK2 and mAK3,respectively.In summary,TM and AK were modified in order to reduce allergenicity and obtain peptide aptamer,which could reduce the allergenicity of shellfish major allergens,and to be applied in the immunotherapy of seafood allergy diseases.