猪流行性腹泻病毒S蛋白多表位抗原免疫原性分析

Immunogenicity analysis of porcine epidemic diarrhea virus S protein with multiple epitopes

在猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)经典毒株CV777毒株S基因基础上,选取S抗原优势表位EP(499~638 aa、748~755 aa、764~771 aa和1368~1374 aa)和超抗原金黄色葡萄球菌肠毒素A(SEA)基因,优化后通过编码柔性连接肽(GGGGS)密码子进行连接,分别构建重组质粒pET-28a-S、pET-28a-S-SEA和pET-28a-S-EP-SEA。表达纯化的重组蛋白经SDS-PAGE和Western blot鉴定后免疫小鼠,以评价重组蛋白的免疫原性。结果显示,重组质粒经IPTG诱导后获得表达,SDS-PAGE分析显示其相对分子质量分别为21,48,54 kDa,与理论值相符。Western blot结果显示,重组蛋白与PEDV阳性血清具有良好的反应原性。纯化后的重组蛋白免疫小鼠,产生了较强的体液免疫和细胞免疫,其中免疫重组蛋白S-EP-SEA产生特异性IgG高于免疫重组蛋白S和S-SEA,细胞因子IFN-γ、IL-2和IL-4水平也高于其他2组。结果表明,本试验制备的重组蛋白S-EP-SE具有良好的免疫原性,为猪流行性腹泻多表位疫苗研制提供了理论依据。

The dominant epitopes EP(499-638 aa,748-755 aa,764-771 aa and 1368-1374 aa)in the S gene of PEDV strain CV777 were linked with S gene and the superantigen staphylococcal enterotoxin A(SEA)gene by sequence encoding a flexible peptide linker(GGGGS)to generate the recombinant plasmid pET28 a-S-EP-SEA,pET28 a-S,pET28 a-S-SEA,respectively.The recombinant proteins were expressed by IPTG induction,verified by SDS-PAGE and Western blot,and used to immunize mice to evaluate their immunogenicity.The results showed that the recombinant proteins were expressed in BL21 after IPTG induction and their relative molecular masses were about 21,48 and 54 kDa,respectively,which were consistent with the theoretical values.Western blot assay showed that these recombinant proteins had good reactivity with PEDV positive serum.Furthermore,mice injected with the purified recombinant protein generated the strong humoral and cellular immune response,mice injected with the purified S-EP-SEA generated a higher specific IgG level against S-EP-SEA protein than other recombinant proteins.The levels of IFN-γ,IL-2,and IL-4 were also higher in mice injected with S-EP-SEA protein than those mice administered with S and S-SEA proteins.Taken together,the above results suggest that the recombinant protein S-EP-SEA has good immunogenicity,which provides a theoretical basis for the development of PED multi-epitope vaccine.