【摘要】 目的制备E7_49-57表位嵌合病毒样颗粒,并进行免疫原性分析。方法利用分子模拟软件Discovery Studio预测HPV16 L1的E7_49-57最佳嵌合位点,将E7_49-57表位嵌入预测位点。构建pET28a-16L1-E7_49-57重组质粒,于大肠杆菌中诱导表达HPV16L1-E7_49-57蛋白并利用镍柱进行亲和层析纯化。于体外重组VLPs后,进行动态光散射粒径和透射电镜分析。用E7_49-57嵌合VLPs免疫小鼠,假病毒中和试验检测免疫血清中和抗体滴度。流式多因子法检测Th1和Th2型细胞因子水平。结果 HI loop区355/356为最佳表位嵌合位点。正确表达了HPV16 L1-E7_49-57蛋白并组装E7_49-57嵌合VLPs。E7_49-57嵌合VLPs免疫小鼠3次后,小鼠血清中和抗体滴度log10平均值达到4.23,略低于野生型VLPs（log10平均值为4.45）。但是,相对于野生型VLPs,E7_49-57嵌合VLPs诱导产生Th1型细胞因子（INF-γ, IL-2, TNF-α）的水平显著提高。结论本研究制备的E7_49-57嵌合VLPs能刺激机体同时产生较强的体液和细胞免疫应答,可能兼具预防和治疗双重功效,为宫颈癌治疗性疫苗的研制奠定基础。更多还原

【Abstract】 To prepare E7_49-57 epitope chimeric VLPs(cVLPs-E7_49-57)and analyze its immunogenicity, the E7_49-57 epitope was inserted into the optimal chimeric site on HPV16 L1 which was predicted by the molecular simulation software of Discovery Studio. Then the recombinant plasmid pET28 a-16 L1-E7_49-57 was constructed, the HPV16 L1-E7_49-57 protein was induced to express in E. coli and purified by affinity chromatography using a nickel column. After in-vitro recombination, cVLPs-E7_49-57 was confirmed by dynamic light scattering and electron microscopy. Mice were immunized with cVLPs-E7_49-57, and the neutralizing antibody titer of immune serum was detected by a pseudoviral neutralization test. The levels of Th1 and Th2 cytokines were detected by flow multifactor technology. The results showed that the optimal chimeric site was HI loop（355/356）, HPV16 L1-E7_49-57 protein was expressed and cVLPs-E7_49-57 was assembled successfully. After 3 times of immunizing, the average log10 of serum neutralizing antibody titer reached 4.23, which was slightly lower than the wild-type VLPs（the average log10 was 4.45）. However, compared with wild-type VLPs, cVLPs-E7_49-57 could induce significantly higher levels of Th1-type cytokines（INF-γ, IL-2, TNF-α）. In conclusion, the cVLPs-E7_49-57 could stimulate the body to produce strong humoral and cellular immune response simultaneously, which might have both preventive and therapeutic effects. It lays the foundation for the development of therapeutic vaccines against cervical cancer.更多还原