大肠杆菌来源的人乳头瘤病毒11型病毒样颗粒的制备及其免疫原性

Expression,purification and immunogenicity of human papillomavirus type 11 virus-like particles from Escherichia coli

【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] To produce human papillomavirus type 11 virus-like particles （HPV11 VLPs） from Escherichia coli and to investigate its immunogenicity and type cross neutralization nature. [ Methods ] We expressed the major capsid protein of HPV11 （HPV11-L1） in Escherichia coli ER2566 in non fusion fashion and purified by amino sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography, sequentially. Then we removed the reductant DTr to have the purified HPV11-L1 self-assemble into VLPs in vitro. We investigated the morphology of these VLPs with dynamic light scattering and transmission electron microscopy. We assayed the immunogenicity of the resultant HPV11 VLPs by vaccinations on mice and evaluated by HPV6/ll/16/18 pseudovirion neutralization cell models. [Results] We expressed HPV11-L1 in Escherichia coli with two forms, soluble and inclusion body. The soluble HPV11 L1 with over 95 % purity can self assemble to VLPs in high efficiency. Morphologically, these VLPs were globular, homogeneous and with a diameter of - 50 nm, which is quite .similar with native HPVll virions. The half effective dosage （ED50） of HPVll VLPs is 0.031 μg, and the maximum titer of neutralizing antibody elicited is averaged to 106. The cross neutralization activity （against HPV6/16/18） of the anti-HPV11 serum was found to have exact correlation to the inter-type homology in amino acid alignment. [ Conclusion] We can provide HPV11 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV11.