破伤风毒素T细胞表位P2对猪轮状病毒△VP8^＊蛋白免疫原性的增强作用

The enhance function of tetanus toxin T-cell epitope P2 to the immune response of porcine rotavirus △VP8*

仔猪轮状病毒腹泻严重危害养猪业，为了开发亚单位疫苗，本课题组前期构建了猪轮状病毒SB-1A株截短的VP8^＊蛋白（△VP8^＊，64～223位氨基酸）原核表达载体pET28a—SB1A△VP8^＊。为了进一步提高亚单位疫苗的免疫原性，将破伤风毒素的通用T细胞表位P2引入重组亚单位蛋白，构建重组载体pET28a—P2-SB-IA△VP8^＊。P2-S13-1A△VP8^＊和S13-1A△VP8^＊在大肠杆菌中表达并纯化。重组蛋白与氢氧化铝佐剂混合后经肌肉注射免疫Balb／c小鼠，利用间接ELISA法检测免疫小鼠的血清抗体水平。结果显示：P2-S13-1A△VP8^＊和S昏1A△VP8^＊在大肠杆菌中得到了高水平、可溶性表达；重组蛋白P2-SB-1A△VP8^＊诱导的血清抗体水平显著高于SB-1A△VP8^＊，说明P2可以增强SB-1A△VP8^＊的免疫原性，为日后开发猪轮状病毒亚单位疫苗奠定了基础。

Porcine rotavirus is a major cause of piglet diarrhea,resulting in serious burden to animal husbandry. In our previous research,the truncated VP8＊ protein （△VP8＊ ,64-223 aa ） prokaryotic expression vector pET28a-SB-1A/△VP8＊ of porcine rotavirus SB-1A strain was constructed. In an attempt to improve the immunogenicity of subunit vaccine protein, universal tetanus toxin T-cell epitope P2 was introduced to the upstream of truncated gene, constructed recombinant vector pET28a-P2-SB-1A △VP8＊ ,followed by the induction of P2-SBqA △VP8＊ recombinant protein in Escherichia coli and purification of recombinant protein P2-SB-1A △VP8＊ , as well as SB-1A △VP8＊. Balb/c mice were immunized intramuscularly with recombinant proteins formulated with aluminum hydroxide. The levels of sera IgG was measured by indirect enzyme-linked immunosorbent assay （ELISA） upon the optimization. The results showed that the two recombinant proteins were soluble and high yield. The serum antibody levels induced by recombinant protein P2- SB-1A △VP8＊ were extremely significantly higher than that by SB-1A △VP8,indicating that P2 can enhance the immunogenicity of SB-1A △VP8＊ and P2-SB-1A △VP8＊ recombinant protein will be potential of the porcine rotavlrus subunit vaccine candidate.