重组轮状病毒多抗原表位的克隆、表达及植物表达载体的构建

Cloning of Recombinant Rotavirus Multi-epitope and Construction of Plant Expression Vector

为了研制基于表位的轮状病毒亚单位疫苗,用PCR的方法得到RV(Rotavirus RV)VP4的抗原表位编码序列,并人工合成了RV VP7的3个抗原表位编码序列,通用辅助性T淋巴细胞表位(PADRE)氨基酸序列和破伤风毒素(Tetanus toxin,TT)上的一个T细胞激活位点[1,2],各位点间以非极性的甘氨酸和脯氨酸分隔以保持各表位的相对独立性,使用搭桥法PCR(Over lapextension PCR)、酶切、连接等基因工程的方法将各个表位片段串联拼接在一起,组合成一条轮状病毒(RV)多表位抗原基因,将其转入原核载体质粒pTHioHis B,在大肠杆菌DH5a克隆,并在BL21中表达,表达蛋白经SDS-PAGE分析,相对分子量与预期结果相符;重组蛋白经轮状病毒快速检测试剂盒检测有抗原性。将此多表位抗原基因插入高效植物表达载体pE1802,为研制重组轮状病毒抗原表位亚单位疫苗、转基因植物口服疫苗做了前期准备。

To study recombinant rotavirus epitope-based vaccine , design and synthesis the multiepitope gene fragment of rotavirus. In this experiment , we obtained a epitope from RV VP4 gene by PCR,at the same time, synthesize the epitopes from RV VP7 gene ,auniversial helper T lymphocyte epitope and an activated site of tetanus toxin, these epitopes are comparted by Glycin and Proline to keep independent, these epitope fragments were linked by over lapextension PCR, enzyme digestion and ligation,and formed a recombinant RV multi-epitope antigen gene, which was inserted into the prokaryotic expression vector pTHio His 13. the recombinant pTHio-RE expressed in E. coli 13L21. The relative molecular mass （Mr）of protein pTHio -RE corresponding to Mr planed in SDS-PACE. The immunogenicity of recombinant pTHio-RE confirmed by.