弓形虫优势表位融合于HPV16L1“N端”的融合体可显著提高表位免疫反应性

Enhanced epitope immunoreactivity of the dominant epitope of Toxoplasma gondii fused at the “N terminus” of HPV16L1

摘要:

弓形虫和人乳头瘤病毒 (HPV) 具有共同的免疫保护人群，选择HPV16型晚期结构蛋白1 (HPV16L1) 为载体，携带弓形虫多表位 (RSepitope) 以期提高表位免疫原性同时可实现共免疫效应。文中分别以构建的融合体RSepitope-HPV16L1 (RSepitope融合于HPV16L1“N”端)以及HPV16L1-RSepitope (RSepitope融合于HPV16L1“C”端)，脂质体转染COS-7细胞后，RSepitope-HPV16L1融合体形式可以在转染细胞中得到有效转录和翻译，分别可检测到相应的RSepitope和HPV16L1的mRNA和蛋白，但HPV16L1-RSepitope融合体形式在转染细胞中检测不到目标抗原的mRNA和蛋白。融合体采用“DNA初免-蛋白质加强免疫”的方案免疫BALB/c小鼠，酶联免疫吸附试验 (ELISA) 和酶联免疫斑点试验 (ELISPOT) 分别检测到RSepitope-HPV16L1免疫鼠血清中显著升高的体液和细胞免疫反应 (即最高水平的RSepitope特异性抗体IgG和IFN-γ)，且比较单独RSepitope免疫组 (不与HPV16L1融合)，产生的是显著升高的Th1和Th2免疫反应类型，提示HPV16L1作为表位载体的优势效应。而HPV16L1-RSepitope融合体形式体内也未能诱导有效的免疫反应。以上研究提示，HPV16L1“N”端可能是较为合适的表位融合位置，融合体表位特异性免疫学效应显著提高，研究结果为提高表位疫苗的免疫原性提供了一个合理的载体融合策略。

Abstract:

For improving epitope immunogenicity and achieving the co-immunization, late protein 1 (L1) of HPV type 16 (HPV16L1) was selected as the vector to carry the dominant epitope of Toxoplasma gondii because of the shared common population between Toxoplasma gondii and human papillomavirus (HPV). RSepitope-HPV16L1 (RSepitope fused at the “N-terminus” of HPV16L1) and HPV16L1-RSepitope (RSepitope fused at the “C-terminus” of HPV16L1) chimeras were constructed. After transfection of COS-7 cells with the recombinants, Western blot, RT-PCR, and immunofluorescence experiments confirmed that RSepitope-HPV16L1 could successfully express the corresponding mRNA and protein of RSepitope and HPV16L1, but the HPV16L1-RSepitope construct could not. A “prime-boost” immunization program was applied in mice to further evaluate the immune response elicited by the constructs, and the RSepitope-HPV16L1 immunization group produced the most significantly increased humoral and cellular immune responses (the highest RSepitope-specific IgG antibody level and the highest IFN-γ production, respectively), in which both elevated Th1 and Th2 immune responses were obtained. Moreover, the advantage of HPV16L1 as an epitope carrier was remarkable for RSepitope-HPV16L1, which induced a more prominent immunological response than RSepitope alone (without fusion with HPV16L1). Our research indicated that the N-terminus of HPV16L1 could be a better insertion site for enhancing target epitope immunogenicity, and our study offers a design for epitope vaccine of reasonable combination.