戊型肝炎病毒基因Ⅳ型病毒样颗粒的表达和抗原性分析

Expression and antigenic analysis of recombinant ORF2 from hepatitis E virus genotype 4

目的表达戊型肝炎病毒(HEV)基因Ⅳ型(G4)结构蛋白ORF2 截短的基因片段,观察病毒样颗粒(VLP)的装配情况,对纯化的VLP进行抗原性分析.方法为了制备HEV病毒样颗粒,将结构蛋白ORF2的N末端111个氨基酸残基截掉,然后将112至660共549个氨基酸残基的基因与杆状病毒基因重组,在昆虫细胞Tn5表达.首先用聚丙烯酰胺凝胶电泳和Western Blotting检测基因是否正确表达,再用超速离心检测是否形成病毒样颗粒,最后用免疫电镜和ELISA分析病毒样颗粒的抗原特异性.结果含有截掉了N末端111个氨基酸残基的HEV G4的ORF2基因的重组杆状病毒感染昆虫细胞Tn5,除了可以表达最初的产物(相对分子质量为58×103的蛋白分子)外,也产生了一些可能经过宿主细胞酶类处理的蛋白分子,相对分子质量分别为57×103、56×103和54×103,并分泌到培养液中.经超速离心和密度梯度离心,电镜观察发现只有54×103的蛋白分子可以自行组装成VLP,颗粒直径为23～24 nm,浮密度为1.285 g/cm3,与HEV G1比较,G4 VLP具有相同的大小和浮密度,但表达量低.G4 VLP与HEV病人血清可以发生抗原抗体反应,用G4 VLP作抗原可以检测HEV的G1、G3、G4型特异性IgM和IgG抗体.结论该研究证实截短的HEV G4的ORF2结构蛋白基因可以在重组杆状病毒表达系统高效表达.形成的VLP具有HEV病毒颗粒类似的抗原活性,可用于制备抗HEV诊断试剂和开发预防用疫苗.

Objective To express the structure protein ORF2 from hepatitis E virus (HEV) genotype Ⅳ(G4) and to produce HEV-like particles and analyze its antigenicity. Methods The structure protein ORF2, with its N terminus truncated (amino acid residues 112-660), was expressed in insect Tn5 cell lines by recombinant baculovirus. Results In addition to the primary translation product with a molecular mass of 58×10 3, a series of further processed protein molecule with molecular weight of 57×10 3, 56×10 3 and 54×10 3 were generated and release into culture media. Electronic microscope (EM) observation and gradient density analysis revealed that 54×10 3 protein self-assembled to form viral-like particle (VLP). The buoyant density of VLP in CsCl was 1.285 g/cm 3 and their diameter was 23-24 nm. The antigenicity of VLP was tested by Western blot and immunoelectron microscope (IEM). The VLP as antigen was able to detect anti-HEV IgG and IgM with ELISA. Conclusion The VLP was yielded efficiently by baculovirus expressing system. The particles possess antigenicity similar to that of authentic HEV particles. It may be applied as a sensitive and practical method for anti-HEV antibody detection and a candidate for HEV vaccine.