人乳头状瘤病毒6型类病毒颗粒的制备及其中和抗体的检测

Production of human papillomavirus type 6 virus-like particles and detection of its neutralizing anti- bodies

目的利用大肠杆菌表达系统制备人乳头状瘤病毒6型（human papillomavims type6，HPV-6）类病毒颗粒（virus-like particles，VfJP）并研究其免疫原性。方法在大肠杆菌ER2566中表达HPV-6L1蛋白，并以硫酸铵沉淀、离子交换色谱、疏水相互作用色谱等手段对其进行纯化。纯化后的HPV-6L1经体外组装形成VLP后以动态光散射，透射电镜对其形态进行检测，并以假病毒中和实验评价HPV-6L1VLP在实验动物体内所诱导的抗HPV-6／11中和抗体水平。结果HPV-6L1蛋白在大肠杆菌中以可溶形式表达，经过纯化后的HPV-6L1蛋白可以在体外组装为半径25rim左右的VLP。该VLP可以在山羊及兔体内诱导高滴度的HPV-6／11中和抗体。结论大肠杆菌表达系统可以简便高效制备具有免疫原性的HPV-6VLP，可以用于HPV-6疫苗的研究。

Objective To produce human papillomavirus type 6 （HPV-6）virus-like particles with Escherichia coli expression system and study its immunogenicity. Methods HPV-6 L1 gene was inserted in- to prokaryotic expression vector pTO-T7 and then expressed in Escherichia coli ER2566. The HPV-6 L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interac- tion chromatography. Then the purified HPV-6 L1 self-assembled into virus-like particle after removing 1,4- dithiothreitol （DTF）. The morphology of the virus-like particles was investigated with dynamic light scatter and transmission electron microscopy, and the immunogenicity was determined with in vitro pseudovirions neutralization assay. Results HPV-6 L1 was expressed in soluble form in Escherichia coll. Following the removal of DTI＇, purified HPV-6 L1 protein could assemble into virus-like particles as 25 nm in the radius. And the animal immunization test showed HPV-6 virus-like particles can elite high titer neutralizing antibod- ies. Conclusion The bacterially expressed HPV-6 L1 VLP is highly immunogenicity and easy to produce. And it can be good candidate of HPV-6 vaccine.