人乳头瘤病毒11型L2NE7E6融合蛋白的原核表达及其诱发的T细胞免疫反应

目的:在原核表达系统中表达人乳头瘤病毒11型(HPV11)L2NE7E6融合蛋白,纯化蛋白后免疫小鼠,检测其诱发的T细胞免疫水平,并筛选HPV11 E6、E7特异的T细胞表位肽。方法:用重叠PCR方法构建HPV11L2NE7E6融合基因并插入原核表达质粒,在大肠杆菌中经IPTG诱导表达融合蛋白L2NE7E6,用SDS-PAGE和West-ern印迹鉴定融合蛋白的表达。Q柱纯化蛋白后免疫C57BL/6小鼠,分别用覆盖HPV11 E6和E7蛋白序列的肽库,采用酶联免疫斑点法(ELISPOT)检测其诱发的E7、E6特异的T细胞免疫反应,并筛选E7、E6特异的T细胞表位肽。结果:在原核表达系统中有效表达了HPV11 L2NE7E6融合蛋白,蛋白纯化后免疫C57BL/6小鼠,分别能检测到针对HPV11 E6、E7肽库刺激产生的特异性T细胞免疫反应。经肽池筛选到1条强的E6 T细胞表位肽E6aa41-55(AEI-YAYAYKNLKVVW);而E7只筛选到2条弱的T细胞表位肽,分别为E7aa53-67(QILTCCCGCDSNVRL)和E7aa73-87(DGDIRQLQDLLLGTL)。结论:HPV11 L2NE7E6融合蛋白能诱发小鼠产生E6、E7特异性细胞免疫反应,可作为尖锐湿疣免疫治疗候选疫苗。

Prokaryotic Expression and its Cellular Immune Response of Hu-man Papillomavirus Type 11 L2NE7E6 Fusion Protein

Objective： To express fusion protein L2NE7E6 of human papillomavirus type 11（HPV11） in Esche-richia coli, and study it＇s T cellular immune response in vaccinated C57BL/6 mice. Methods： HPV11 L2NE7E6 fusion gene was constructed by overlap extension PCR, then it was inserted into pET9a vector, and the fusion pro-tein L2NE7E6 was expressed in host strain E.coli BL21（DE3＋） harboring pET9aHPV11L2NE7E6 by induction with IPTG, and identified by using SDS-PAGE and Western blotting analysis. L2NE7E6 protein purified with Q column was inoculated to C57BL/6 mice and it＇s cell-mediated immunogenicity was assessed by IFN-γ enzyme-linked immunospot（ELISPOT） with overlapping synthetic peptides covering full length of the amino acid sequence of E6 and E7 proteins, the epitope mapping assay was performed to screen immune dominant epitope of E6 and E7. Results： HPV11 L2NE7E6 fusion protein was expressed effectively in E.coli, and the purified fusion protein L2NE7E6 could significantly elicit HPV11 E7 and E6 specific T-cell immune responses in vaccinated C57BL/6 mice. Epitope mapping showed that the dominant epitopes for HPV11 E6 were peptide E6aa41-55（AEIYAY-AYKNLKVVW）, and for E7 only two weaker epitopes were verified, which were E7aa53-67（QILTCCCGCDSN-VRL） and E7aa73-87（DGDIRQLQDLLLGTL）. Conclusion： The purified fusion protein HPV11 L2NE7E6 could in-duce strong specific T cellular immune response in vaccinated mice. It could be used as a candidate for further study genital wart immune therapeutic.