HEV ORF2截短融合蛋白的原核表达及其免疫原性

Prokaryotic expression and immunogenicity of HEV ORF2 truncated fusion protein

目的原核表达3型戊型肝炎病毒(hepatitis E virus,HEV)第2个开放阅读框(open reading frame 2,ORF2)截短融合蛋白(HEV3-179-Fe),并检测其免疫原性。方法分别扩增HEV3-179和Fe蛋白基因片段,经Overlap PCR法连接并扩增,克隆至载体pET-28a,构建重组表达质粒pET-28a-HEV179-Fe,将其转化感受态E.coli BL21(DE3),取阳性菌株,IPTG诱导表达融合蛋白HEV3-179-Fe,进行10%SDS-PAGE分析。目的蛋白经镍离子金属鳌合亲和层析纯化后,加入硫酸铵进行病毒样颗粒(virus-like particles,VLPs)重构,置电镜下观察。HEV3-179-Fe蛋白VLPs与氢氧化铝佐剂吸附后免疫小鼠,ELISA法测定效价,以评价HEV ORF2截短融合蛋白的免疫原性。结果重组表达质粒pET-28a-HEV3-179-Fe构建正确。目的蛋白HEV3-179-Fe相对分子质量约40000,主要以包涵体形式存在,表达量均达35%以上,纯度约95%,可与鼠抗HEV 3多抗发生特异性反应。硫酸铵重构后于电镜下可见直径约20 nm的VLPs,其免疫小鼠的血清效价达1∶4800000以上。结论原核表达了HEV3-179-Fe,且在小鼠体内具有较好的免疫原性。本实验为以HEV ORF2-179蛋白为基础的VLPs基因工程疫苗的研发奠定了基础。

Objective To express the truncated fusion protein HEV3-179-Fe of open reading frame 2(ORF2)of hepatitis E virus type 3(HEV3)in prokaryotic cells and determine its immunogenicity.Methods HEV3-179 and Fe gene fragments were amplified separately then linked and amplified by overlap PCR,and inserted into vector pET-28 a.The constructed recombinant plasmid pET-28 a-HEV179-Fe was transformed to competent E.coli BL21(DE3),and the positive colonies were screened and induced with IPTG.The expressed fusion protein HEV3-179-Fe was analyzed by SDS-PAGE,purified by Ni-Agarose affinity chromatography,added with ammonium sulphate for reconstruction of virus-like particles(VLPs),and observed by electron microscopy.The HEV3-179-Fe protein was adsorbed to aluminium hydroxide adjuvant and immunized to mice,100μL for each,at weeks 0,2 and 4 respectively.The serum samples of mice were collected at week5 after the first immunization and determined for titer by ELISA to evoluate the immunognicity of truncated HEV ORF2 fusion protein.Results Colony PCR and sequencing confirmed that recombinant plasmid pET-28 a-HEV3-179-Fe was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 40000,mainly existed in a form of inclusion body and contained more than 35%of total somatic protein,which reached a purity of about 95%after purification and showed specific reaction with mouse polyclonal antibody against HEV3.VLPs at diameters of about 20 nm were observed under electron microscope after reconstruction with ammonium sulphate.The antibody titer in sera of mice immunized reached more than 1∶4800000.Conclusion The HEV3-179-Fe were expressed in prokaryotic cells,which showed good immunogenicity.It laid a foundation of development of recombinant VLP vaccine based on HEV ORF2-179 protein.