rBCG-HPV16 E6-E7疫苗的构建和免疫原性研究

Construction and immunocompetent study on the vaccine for rBCG-HPV16 E6-E7

目的:构建以BCG为载体的rBCG HPV16E6E7疫苗,研究rBCG HPV16E6E7重组表达体对小鼠免疫功能的影响,初步评价此疫苗对HPV相关肿瘤(宫颈癌)细胞的免疫治疗作用。方法:电穿孔法将pIJ702HPV16E6E7分泌型穿梭表达质粒导入BCG,构成rBCG HPV16E6E7疫苗。Westernblot法检测E7蛋白的表达。于C57BL/6小鼠皮下接种rBCG HPV16E6E7,取其脾细胞与E7蛋白在体外共同培育,MTT法检测T淋巴细胞增殖活性,51Cr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性(cytotoxicTlymphocyte,CTL),ELISA法检测免疫鼠血清中抗E7特异性抗体和细胞因子的表达水平。结果:rBCG HPV16E6E7重组疫苗能特异地表达HPV16E7蛋白,将该疫苗免疫小鼠后,可刺激机体产生细胞因子,如IL2、IFNγ及抗E7抗体的含量明显升高(P<0.01);脾淋巴细胞在体外受到E7蛋白的再次刺激后,淋巴细胞增殖性反应以及CTL活性明显增强(P<0.01)。结论:rBCG HPV16E6E7能增强小鼠的细胞和体液免疫反应,可作为HPV16治疗性疫苗。

OBJECTIVE： To establish the vaccination of rBCG-HPV16 E6-E7, and evaluate the specific CTL effect of CD8 ^＋ T lymphocyte induced by the naked plasmid and the potency of the vaccine. METHODS： Recombinant of pIJ702-HPV16 E6-E7 was transfected into BCG by the electroporation method. The expression of HPV16 E6-E7 in the transfected BCG was assayed by Western blotting, rBCG-HPV16 E6-E7 were vaccinated to mice, and the activities of cytotoxic T lymphocytes （CTL） isolated from immunized mice were tested in vitro with 51^Cr specific release assay. The production of cytokines and antibodies were detected by ELISA method. RESULTS： E7 gene was expressed in BCG steadily. Secretions of cytokines IL-2, IFN-γ and antibodies increased by injection of rBCG-HPV16 E6-E7 vaccinations. The CTLs activity and anti-E7 specific antibody production from immunized mice were efficaciously enhanced by rBCG-HPV16 E6-E7 plasmid. CONCLUSION： rBCG- HPV16 E6-E7 vaccines can induce special immunological responses for HPV16.