猪流行性腹泻病毒S基因与SEA基因融合表达及免疫原性评价

Fusion Expression and immunogenicity evaluation of PEDV Spilke Gene and SEA Gene

试验旨在构建pET-28a-S-SEA融合表达质粒,并评价S-SEA蛋白的免疫原性。根据大肠杆菌密码子偏嗜性,对我国猪流行性腹泻病毒(PEDV)流行毒株(GenBank:LT906620.1)的S基因与金黄色葡萄球菌肠毒素A(SEA)基因(GenBank:MH053151.1)进行优化,通过柔性连接肽(GGGGS)连接后,克隆至表达载体pET-28a,得到pET-28a-S-SEA重组质粒,转化至宿主菌BL21(DE3)中诱导表达,经SDS-PAGE和Western Blot检测后,对最佳表达条件进行摸索。在此基础上,将亲和层析后的重组蛋白,免疫6周龄的雌性BALB/c小鼠,对免疫小鼠的血清特异性抗体水平和淋巴细胞增殖指数进行检测。结果成功构建pET-28a-S-SEA质粒,且在大肠杆菌中获得高效表达;小鼠试验结果表明,纯化的S-SEA蛋白能够诱导小鼠产生特异性免疫反应,促进淋巴细胞增殖,具有良好的免疫原性,为进一步研究猪流行性腹泻亚单位疫苗提供帮助。

The aim of the trial was to construct recombinant plasmid pET-28 a-S-SEA and evaluate its immunogenicity. Based on thepublished sequence of Spilke gene of Chinese PEDV popular strain(GenBank:LT906620.1) and SEA gene(GenBank: MH053151.1) on GenBank, the whole gene Spilke protein and SEA gene was optimized, inked by Flexible linker(GGGGS), ynthesized and ligated into pET-28 a plasmid according to E.coli codon bias without changing the amino acid sequence. The prokaryotic expression recombinant plasmid pET-28-S-SEA was constructed and transformed into E. coli BL21(DE3), induced and expression. Identified the protein by SDS-PAGE and Western blot and its expression conditions were optimized. To evaluate the immunogenicity of the protein, the mice were immunized with the S-SEAprotein that was obtained after affinity chromatography, and the titerof the antibody in mice serum was evaluated and splenocytes proliferation was determined. Results:The prokaryotic expression plasmid pET-28 a-S-SEA was constructed successfully and S-SEA protein could be expressed by the plasmid inEscherichia Coli. The Specific antibody levels in serum result and Thespleen lymphocyte proliferation assay in immunized mice serum indicated tha the purified S-SEA protein could inducespecific immune response in mice, and significant proliferationof antigen-sensitized lymphocytes, and with better immunogenicity, which will help further study the subunit vaccine that can effectively prevent and treat PEDV.