摘要
利用PCR技术从HPV16阳性阴道分泌物标本中获得HPV16 L1基因片段,并将其插入表达载体pTO-T7中,构建重组表达质粒pTO-T7-HPV16-L1;以该重组质粒转化大肠杆菌ER2566并表达HPV16 L1蛋白;所表达的HPV16 L1蛋白经过硫酸铵沉淀、离子交换层析和疏水相互作用层析等纯化步骤后,HPV16 L1纯度达到98%以上,并可在体外装配为直径50nm的病毒样颗粒;动物免疫原性研究结果显示,该病毒样颗粒可诱导高滴度的针对HPV16的中和抗体。上述研究结果表明通过大肠杆菌表达系统制备的HPV16病毒样颗粒具有纯度高,与天然病毒颗粒形态高度相似的特点,并具有高度免疫原性,可以应用于HPV16病毒样颗粒的结构功能研究及HPV16疫苗研发等领域。
HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-TT-HPV16-L1. Then, the pTO-TT- HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.
出处
《病毒学报》
CAS
CSCD
北大核心
2009年第4期245-250,共6页
Chinese Journal of Virology
基金
国家自然科学基金(30600106
30500092)
国家工程中心技术研究建设项目(2005DC105006)
863计划重点项目(2006AA020905)
关键词
人乳头瘤病毒16型
大肠杆菌表达系统
纯化
病毒样颗粒
免疫原性
Human papillomavirus type 16
Escherichia coli expression system
Purification
Virus-like particle
Immunogenicity
