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【目的】利用大肠杆菌表达系统制备人乳头瘤病毒11型病毒样颗粒(HPV11VLPs),并对其免疫原性和所诱导中和抗体的型交叉反应性进行研究。【方法】在大肠杆菌ER2566中非融合表达HPV11-L1蛋白,并通过离子交换层析,疏水相互作用层析其进行纯化。纯化后的HPV11-L1经体外组装形成病毒样颗粒,通过动态光散射,透射电镜检测其形态,并通过多种HPV型别假病毒中和实验评价HPV11VLPs的免疫原性及型交叉反应性。【结果】HPV11-L1蛋白在大肠杆菌中可以以可溶形式表达。经过硫酸铵沉淀、离子交换和疏水相互作用色谱纯化,目的蛋白纯度能够达到95%以上。纯化的HPV11-L1蛋白去除还原剂DTT后,能够在体外自发组装成为直径约50nm、与天然病毒颗粒形态高度相似的VLPs。动物实验结果显示,该病毒样颗粒在小鼠体内的中和抗体半数有效剂量(ED50)为0.031μg,在小鼠体内可诱导高达106的中和抗体滴度,这些中和抗体与HPV6有较好的交叉反应,与HPV18有一定的交叉反应,而与HPV16没有明显的交叉反应,这一结果与分子进化树的分析相一致。【结论】本研究利用原核表达系统获得了具有较高免疫原性的HPV11VLPs,为HPV11预防性疫苗的研制奠定了良好的基础。
【Objective】 Human papillomavirus type 11 virus-like particles (HPV11VLPs) were prepared by using E.coli expression system and the immunogenicity and the type cross-reactivity of the induced neutralizing antibodies were studied. 【Method】 HPV11-L1 protein was expressed in Escherichia coli ER2566 non-fusion and purified by ion exchange chromatography and hydrophobic interaction chromatography. The purified HPV11-L1 was assembled in vitro to form virus-like particles, and the morphology of the HPV11-L1 was detected by dynamic light scattering and transmission electron microscopy. Immunogenicity and cross-reactivity of HPV11 VLPs were evaluated by neutralizing HPV type VVPs. 【Result】 HPV11-L1 protein can be expressed in soluble form in E. coli. After ammonium sulfate precipitation, ion exchange and hydrophobic interaction chromatography purification, the purity of the target protein can reach more than 95%. The purified HPV11-L1 protein, after removing the reducing agent DTT, can spontaneously assemble in vitro into VLPs with approximately 50 nm in diameter and highly similar to the native virus particle morphology. Animal experiments showed that the virus-like particles in mice in vivo neutralizing antibody half effective dose (ED50) 0.031μg, in mice can induce up to 106 neutralizing antibody titers, these neutralizing antibodies and HPV6 have more Good cross-reactivity, cross-reaction with HPV18, but no significant cross-reaction with HPV16, which is consistent with the analysis of molecular phylogenetic tree. 【Conclusion】 In this study, HPV11 VLPs with high immunogenicity were obtained by using prokaryotic expression system, which laid a good foundation for the development of HPV11 preventive vaccine.