| Staphylococcal enterotoxin (SE) is a group of extracellular toxic proteins with single polypeptide structure which can cause serious food poisoning in human. At present, SE can be divided into20genotypes, and the detectable rate of different serotypes relates on the isolated place of strain and host sources. So it is more practical significance to establish a assay for detecting the regional advantage SE serotype. In our previous studies, we found that SEA and SEG produced by Staphylococcus aureus, which isolated from raw milk samples in Hefei, Anhui Province, were major serotypes. The ELISA of detecting SEA has been specified in GB, but there is no immunological method to detect SEG by now. The general idea of the study is to establish icELISA which bases on the multi-epitope peptide designand and prepared with immunodominant linear B-cell epitopes of SEA and SEG, and can simultaneously detect both of SE.First of all, the advantage linear B-cell epitopes of SEA and SEG were selected and identified with epitope prediction followed by concrete experimental verification. The secondary structure, flexible regions, surface accessibility, hydrophilicity and antigenic index of the SEA and SEG proteins were comprehensive analyzed using the DNAStar Protean program, and six potential epitopes were screened. To verify the prediction, the potential epitopes were artificially synthesized and detected by peptide-enzyme linked immunosorbent assay (ELISA). The results showed that the potential epitope peptides at amino acid positions156to163,166to173and211to218were indeed immunodominant linear B-cell epitopes of the SEA protein, while the potential epitope peptides at amino acid positions37to46,120to127and141to149were indeed immunodominant linear B-cell epitopes of the SEG protein.Secondly, according to the sequence of the advantage B-cell linear epitopes in SEA and SEG proteins, and the design principles of tandem, a multi-epitope tandem was optimally designed. The six advantage B-cell linear epitopes were tandemly arranged by AAY (Ala-Ala-Tyr) spacer in different ways, then the practicality of the designed series were analyzed by the DNAstar Protean program. The results showed the optimized connection order of epitopes was P156-AAY-P166-AAY-P211-AAY-P37-AAY-P120-AAY-P141, in which each epitope maintained its independent antigenicity and the better antigen index analyzed by DNAstar Protean. Then the best series of the multi-epitope peptide was transformed into gene sequence in which the restriction enzyme sites and the termination codon were added at both ends, and the rare codons were also be replaced, then a tandemly arranged multi-epitope gene named AG gene with318bp was designed and cloned into the plaimid pUC57.Then, in order to establish indirect competitive ELISA (icELISA), a large number of multi-epitope peptide AG and special antibody were prepared. The recombinant expression plasmid named pET-32a-AG was constructed by gene recombination technology and transformed into E-coli Rosetta to be mass-induced expression, then the recombinant protein His-6EPIS was purified by Ni-Charged Resin affinity chromatography. It was found that the concentration of purified protein AG was1.7mg/ml and the purity reached to95%by SDS-PAGE analysis. The titer of rabbit anti-AG antisera was1:327680detected by indirect ELISA at28days post-immunization and1:16detected by agar gel immunodiffusion test, After saturated ammonium sulfate precipitation and purified by Protein G affinity chromatography, the titer of anti-SE-AG antisera wasl:819200detected by indirect ELISA, the concentration was lmg/mL, the purity can reach95%, which could fully meet the requirements of the immunoassay reagent.Finally, an icELISA based on multi-epitope peptide AG was established. icELISA was carried out with recombinant multi-epitope peptide AG and natural enterotoxin as competitive antigen respectively. Two standard curves were drew using the common logarithm of competitive antigen concentration as the abscissa and the competition inhibition rate of series concentration of antigen (A/Ao)%as the ordinate. The results showed that the linear equations of2standard curves were good fitness, proved that the icELISA based on multi-epitope peptide AG could be applied to detect SEA and (or) SEG synchronously. The test of sensitivity and repeatability showed that the minimum detection limit was5.068ng/ml, and the intra-and inter-assay coefficients of variation were less than15%, which indicated that the icELISA has good sensitivity and repeatability.In summary, the icELISA based on multi-epitope peptide AG is a fast (about4h), specific and sensitive detect methods which can detect type A and G Staphylococcal enterotoxin simultaneously. |
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