Molecular Cloning And Epitopes Analysis Offilamin C From Scylla Paramamosain

The consumption of crab aquatic products is increasing year by year,and the allergic reactions caused by it are widely concerned.Filamin C(FLN c)is a new allergen in the muscle of Scylla paramamosain.The gene sequence,physical and chemical properties,and epitope of the allergen are not clear.Therefore,the study of FLN c gene sequence and antigen epitope is helpful to solve the problem of diagnosis of allergic diseases caused by the cross-reaction of crustaceans FLN c.In this study,FLN c was used as the research object,and the gene sequence of FLN c was obtained by nested polymerase chain reaction and rapid DNA amplification technology.The open reading frame of the gene was 2544 bp,encoding 847 amino acid residues,the nucleotide sequence accession number is MK747241.1,filling the gap in the FLN c gene sequence of crustacean aquatic products.The results of bioinformatics analysis showed that FLN c contains 9 domains,and its tertiary structure is folded like an immunoglobulin,with the characteristics of a typical filamin family;the results of phylogenetic tree analysis showed that the filamin family is highly evolved conservative.In addition,the epitope obtained by phage panning in the early stage of this laboratory was compared with S.paramamosain FLN c gene sequence,and 22 epitope peptides were matched;based on three bioinformatics online analyses and software predictions,FLN c 28 epitopes.The comparison analysis finally determined 11 epitopes of S.paramamosain FLN c.Further,FLN c was expressed in vitro to obtain purified recombinant FLN c(rFLN c).Compared with natural FLN c(nFLN c),it was found that the tertiary structure of rFLN c changed significantly;immunoglobulin E(IgE)binding capacity analysis showed that rFLN c was more easily recognized by the sera of patients with crustaceans than nFLN c.It is suggested that rFLN c can be used as a candidate allergen for the component resolved diagnosis of crab.Based on the analysis of domain composition and tertiary structure,FLN c was expressed in segments,and purified recombinant proteins rFLN c1(amino acid sequence 1-335 region)and rFLN c2(amino acid sequence 336-531 region),rFLN c3(amino acid sequence 532-847 region)with maltose binding protein tag.Immune binding activity analysis showed that the distribution of FLN c epitopes showed that rFLN c2 had the strongest immunological binding ability,and the 6 identified epitopes were distributed in this region,suggesting that the amino acid sequence 336-531 region is for FLN c Dominant regions of the epitope.The specific antibodies and serological analysis results of FLN c in 8 aquatic products of shellfish showed that FLN c was present in 8 aquatic products,and rFLN c could obviously inhibit FLN c and crustaceans in 8 aquatic products.The serum IgE binding capacity of allergic patients showed that different species of FLN c have the same or similar epitopes,and there is cross-reactivity in aquatic products.In summary,this study molecularly cloned and recombinantly expressed the FLN c of S.paramamosain,segmented expression to clarify FLN c epitope dominant regions,and analyzed the cross-reactivity of 8 shellfish allergens FLN c.These results It is of guiding significance to improve the gene sequence information of the crustacean FLN c and the diagnosis and treatment of crab allergic diseases.