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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。
The HPV16 L1 gene fragment was obtained from HPV16-positive vaginal secretions by PCR and inserted into the expression vector pTO-T7 to construct the recombinant expression plasmid pTO-T7-HPV16-L1. The recombinant plasmid was transformed into E. coli ER2566 and expressed After HPV16 L1 protein was purified by ammonium sulfate precipitation, ion exchange chromatography and hydrophobic interaction chromatography, the purity of HPV16 L1 reached over 98% and could be assembled in vitro into virus-like particles of 50 nm in diameter Animal immunogenicity results show that the virus-like particles can induce high titer of neutralizing antibodies against HPV16. The above results show that the HPV16 virus-like particles prepared by the E. coli expression system have the characteristics of high purity and highly similar to the natural virus particle morphology and are highly immunogenic and can be applied to the structural and functional studies of HPV16 virus-like particles and HPV16 vaccine R & D and other fields.