Xie Minghui, Li Shaowei, Shen Wentong, Li Zhongyi, Zhuang Yudi, Mo Xiaobing, Gu Ying, Wu Ting, Zhang Jun, Xia Ningshao
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Science, Xiamen University, Xiamen 361005, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Jul;25(7):1082-7.
Here, we presented a method to bacterially express the major structural protein L1 of Human Papillomavirus type 18 (HPV18) as soluble form. We found that the purified L1 could self-assemble to virus-like particles (VLPs). Further, we investigated the immunogenicity and the induced level of neutralizing antibody using these VLPs. First, the genome of HPV18 was cloned from a patient in Xiamen. It was used as template for PCR amplification of HPV18 L1 gene. The resultant DNA fragment was inserted into expression vector pTrxFus and expressed in Escherichia coli GI724. Second, L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography; and the purified L1 was subjected to self-assembly to form VLPs with the removal of premixed reductant DTT. Finally, the size and morphology of these VLPs was investigated by Dynamic Light Scattering and Transmission Electronic Microscopy as 29.34 nm in hydrated radius and globular particles similar with native HPV18. The half effective dosage (ED50) and maximum level of neutralizing antibody elicitation were measured by vaccinations on mice, rabbit and goat using pseudovirus neutralization cell model. The results showed that the ED50 of HPV18 VLPs is 0.006 microg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 10(7). As a conclusion, we can provide HPV18 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV18.
在此，我们展示了一种在细菌中表达人乳头瘤病毒18型（HPV18）主要结构蛋白L1的方法，使其呈可溶形式。我们发现纯化后的L1能够自组装成病毒样颗粒（VLPs）。此外，我们使用这些VLPs研究了其免疫原性和诱导产生的中和抗体水平。首先，从厦门的一名患者身上克隆出HPV18的基因组。将其用作模板进行HPV18 L1基因的PCR扩增。所得DNA片段被插入表达载体pTrxFus并在大肠杆菌GI724中表达。其次，通过硫酸铵沉淀、离子交换色谱和疏水相互作用色谱法纯化L1蛋白；纯化后的L1在去除预混的还原剂二硫苏糖醇（DTT）后进行自组装以形成VLPs。最后，通过动态光散射和透射电子显微镜研究这些VLPs的大小和形态，其水合半径为29.34 nm，呈与天然HPV18相似的球状颗粒。使用假病毒中和细胞模型通过对小鼠、兔子和山羊进行疫苗接种来测量中和抗体诱导的半数有效剂量（ED50）和最高水平。结果显示，HPV18 VLPs在小鼠中的ED50为0.006微克，在兔子和山羊中诱导产生的中和抗体最高效价可达10⁷。综上所述，我们能够从原核表达系统提供具有高免疫原性的HPV18 VLPs，这可能为HPV18预防性疫苗的研发开辟一条新途径。
