人乳头瘤病毒L2蛋白的病毒样颗粒疫苗研究  

Study of a virus-like particle vaccine containing N-terminal epitopes of human papilloma virus L2 protein

作　　者：蒋蓉 李军强 杨鸣鸣 朱涛 宇学锋 邵忠琦(指导) 

机构地区：[1]天津康希诺生物技术有限公司,天津市呼吸道细菌重组及结合疫苗企业重点实验室,天津300457

出　　处：《中国免疫学杂志》2016年第3期366-371,共6页Chinese Journal of Immunology

摘　　要：目的:构建乙肝病毒核心抗原(HBcAg)与人乳头瘤病毒(HPV)L2抗原的融合蛋白,在大肠杆菌中重组表达形成病毒样颗粒结构;通过小鼠模型检测HBc-L2融合蛋白的免疫原性,并研究免疫后获得的小鼠血清对HPV假病毒的中和效力。方法:通过DNA合成构建16型人乳头瘤病毒L2基因片段与HBcAg基因的融合基因,将其克隆至表达载体p ET9a并在大肠杆菌中进行HBc-L2融合蛋白表达;将经纯化、鉴定后的融合蛋白免疫BALB/c小鼠,用间接ELISA方法检测小鼠血清中针对L2抗原的抗体效价,并分别研究小鼠血清对16型和18型HPV假病毒的中和效力。结果:HBc-L2融合基因经大肠杆菌系统表达形成可溶性蛋白,经硫酸铵沉淀和CL-4B凝胶分离纯化后获得纯度>80%的HBc-L2蛋白;分子筛高效液相色谱-多角度激光光散射(SEC-MALS)联用技术和透射电子显微镜的分析结果表明,HBc-L2融合蛋白在表达过程中自动组装形成稳定的病毒样颗粒;将纯化后的HBc-L2蛋白免疫BALB/c小鼠可获得针对L2抗原的高滴度抗体,且小鼠血清具有中和16和18型两种假病毒的中和抗体活性。结论:HBc-L2病毒样颗粒可以有效地增强L2抗原的免疫原性,并可刺激机体产生针对多型HPV的免疫保护力,是一个具有潜力的新型广谱HPV疫苗。Objective： To prepare a virus-like particle（ VLP）,containing Hepatitis B virus core antigen（ HBcAg） and N-terminal peptides of the L2 protein of human papilloma virus（ HPV）,and investigate the immunogenicity of the VLP in mice and the protection against different strains of HPV. Methods： A fusion gene was synthesized to insert a DNA fragment,coding for the N-terminal epitopes of the L2 protein of HPV16,into the HBcAg coding sequence; HBc-L2 fusion protein was highly expressed in E. coli using the p ET9 a and BL21（ DE3） expression system; the purified fusion protein was used to immunize BALB/c mice and antibody titers against the L2 epitopes in mouse sera were determined by indirect ELISA; the levels of neutralizing antibodies against both HPV16 and 18 were also analyzed. Results： HBc-L2 fusion protein was expressed in E. coli and purified,with the purity 80%,by ammonium sulfate precipitation and CL-4B gel filtration; analysis of the purified fusion protein,using size exclusion chromatography with multi-angle laser light scattering detection（ SEC-MALS） and electron microscope,revealed that HBc-L2 was assembled into a stable VLP structure automatically following its expression; immunization of BALB/c mice with the purified VLPs resulted in high antibody titers in mouse sera against the L2 epitopes; furthermore,it was demonstrated that the sera from the immunized mice had neutralization activities against both HPV16 and HPV18. Conclusion： The immunogenicity of the L2 epitopes was highly enhanced by the construction of HBc-L2 fusion protein and the formation of the VLP structure; the fusion protein was also capable of inducing protections against different serotypes of HPV,therefore,it could be a potential HPV vaccine with a broad coverage and low production cost.

关 键 词：人乳头瘤病毒 L2抗原 乙肝病毒核心抗原 病毒样颗粒 免疫原性 中和抗体 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]