摘要
利用大肠杆菌表达系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,经过纯化和重组装过程获得HPV18病毒样颗粒(VLPs),研究其免疫原性和诱发中和抗体生成的水平。首先,提取HPV18的基因组DNA,通过PCR扩增获得HPV18 L1基因片段,将其插入pTrxFus表达载体,在大肠杆菌中可溶性表达HPV18 L1蛋白;其次,通过硫酸铵沉淀、离子交换层析和疏水相互作用层析获得高纯度的HPV18 L1蛋白,而后透析去除预先加入的还原剂DTT,使HPV18 L1蛋白自发组装成VLPs;最后,通过动态光散射技术和透射电子显微镜鉴定HPV18 VLPs的大小和形态,利用假病毒细胞中和实验评价HPV18 VLPs在实验动物体内的免疫原性和中和抗体生成水平。结果表明,HPV18L1蛋白可以在大肠杆菌表达系统中以可溶形式表达,经过纯化的HPV18 L1蛋白可以自发组装成为半径约为29.34nm、与HPV病毒外观相似的VLP。该VLPs在小鼠体内的中和抗体半数有效剂量为0.006μg,在兔及山羊体内诱导中和抗体滴度高达107。总之,本研究利用原核表达系统可简便高效地获得具有高度免疫原性的HPV18 VLPs,为HPV18预防性疫苗的开发奠定了基础,具有重要的应用意义。
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 HPVI8. 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 μg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 107. 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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第7期1082-1087,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目(No.30600106)
国家高技术研究发展计划重点项目(863计划)(No.2006AA020905)
国家工程中心技术研究建设项目(No.2005DC105006)资助~~
