基于戊肝病毒的嵌合病毒样颗粒对人乳头瘤病毒16型肿瘤免疫治疗作用的研究

Study on the Effect of Chimeric Virus-like Particles Based on Hepatitis E Virus on Human Papillomavirus Type 16 Tumor Immunotherapy

目的 研究基于戊肝病毒(hepatitis E virus,HEV)的嵌合病毒样颗粒(virus-like particles,VLPs)对人乳头瘤病毒16型(human papillomavirus type 16,HPV 16)肿瘤免疫治疗作用。方法 将HPV 16 E7插入HEV的p239蛋白形成重组嵌合蛋白p239-HPV16E7。所构建的重组蛋白经大肠杆菌表达、纯化、复性后,通过电镜和动态光散射对所得蛋白颗粒大小形态进行表征。将蛋白颗粒免疫C57B/L小鼠,通过流式细胞技术与酶联免疫斑点免疫试验检测脾淋巴细胞特异性免疫细胞分化情况;并且利用TC-1肿瘤细胞在C57B/L小鼠中构建肿瘤模型,以此评价蛋白颗粒在小鼠体内的抗肿瘤免疫效果。结果 体外复性后的嵌合蛋白在电镜下观察到颗粒结构,粒径大小为22.80 nm。所得蛋白颗粒在C57B/L小鼠体内诱导产生良好的特异性细胞免疫反应。与对照组相比,试验组脾淋巴细胞的CD3^(+)/CD4^(+)、CD3^(+)/CD8^(+)比例均有明显差异(P<0.05),且分泌IFN-γ干扰素的效应T细胞显著增加。同时,所得蛋白颗粒能有效抑制TC-1荷瘤小鼠体内肿瘤细胞的生长,在实验周期内小鼠未出现死亡,而对照组小鼠体内肿瘤快速生长,且6周后全部死亡。结论 原核表达的嵌合蛋白p239-HPV16 E7形成病毒样颗粒并有效诱导针对HPV 16的抗肿瘤免疫。

OBJECTIVE To study the immunotherapeutic effect of chimeric virus-like particles(VLPs) based on hepatitis E virus(HEV) against human papillomavirus type 16(HPV 16) tumor.METHODS HPV16 E7 was inserted into the p239 protein of HEV to form the recombinant chimeric protein p239-HPV16 E7.The constructed recombinant protein was expressed by Escherichia coli,purified,and then refolded,and the protein was detected by electron microscopy and dynamic light scattering to confirm size and shape.Then,the C57B/L mice were immunized with the protein grain,and the lymphocyte differentiation of mouse spleen was detected by flow cytometry and enzyme-linked immune spot immunoassay;in addition,TC-1 tumor cells were used to construct tumor models in C57B/L mice to evaluate the anti-tumor immune effect of protein particles in mice.RESULTS After refold in vitro,the structure of chimeric protein was observed under electron microscopy,and the size of particle was 22.80 nm.The obtained protein particles induced favorable specific cellular immune response in C57B/L mice.Compared with the control group,the proportions of CD3^(+)/CD4^(+) and CD~3+/CD8^(+) in spleen lymphocytes of experimental groups were significantly different(P<0.05),and effector T cells secreting IFN-γ interferon were also increased remarkably.At the same time,the obtained protein particles could effectively inhibit the growth of tumor cells in TC-1 tumor-bearing mice,and the mice did not die during the experimental period,while the tumors in the control mice grew rapidly and all died after 6 weeks.CONCLUSION Chimeric protein p239-HPV16E7 which was expressed in prokaryotes can form virus-like particles and effectively induce anti-tumor immunity against HPV16.