截短型水痘-带状疱疹病毒糖蛋白E的原核表达及免疫原性评价

Prokaryotic expression and immunogenicity of truncated varicella-zoster virus glycoprotein E

目的原核表达截短型水痘-带状疱疹病毒(varicella-zoster virus,VZV)糖蛋白E(glycoprotein E,gE),并评价其免疫原性。方法通过GenBank获取VZV gE的第25~537位氨基酸(gE25-537)相应的核酸序列,经密码子优化,合成质粒pUC-VZV-gE25-537-His;以其为模板扩增目的片段,克隆至载体pET-21b,构建重组表达质粒pET-21b-VZV-gE25-537-His,转化感受态E.coli BL21(DE3),经IPTG诱导表达,包涵体经Urea溶解后,进行镍离子层析柱纯化。将纯化蛋白与弗氏佐剂混合,经皮下多点免疫小鼠,共免疫3次,末次免疫2周,经尾部静脉采血,分离血清,检测血清效价和中和效价。结果重组表达质粒pET-21b-VZV-gE25-537-His经双酶切及测序鉴定证明构建正确。目的蛋白相对分子质量约62000,主要以包涵体形式表达,纯化后纯度达85%,且可与鼠抗gE单克隆抗体发生特异性结合。免疫3次后,小鼠血清效价可达1∶54954,中和效价为1∶21.75。结论原核表达了VZV gE,该蛋白具有良好的免疫原性,有望成为制备带状疱疹疫苗的候选抗原。

Objective To express varicella-zoster virus(VZV)glycoprotein E(gE)in prokaryotic cells and evaluate its immunogenicity.Methods The nucleic acid sequence of amino acids 25~537 of VZV surface glycoprotein E(gE25-537)was obtained from GenBank,based on which plasmid pUC-VZV-gE25-537-His was synthesized by codon optimization and used as a template for PCR.The amplified target fragment was cloned into vector pET-21 b,and the constructed recombinant plasmid pET-21 b-VZV-gE25-537-His was transformed to competent E.coli BL21(DE3)and induced with IPTG.The inclusion body was dissolved with urea,and the target protein was purified by nickel affinity chromatography.The purified protein was mixed with Freund adjuvant,with which mice were immunized s.c.in several sites for 3 times.Serum samples were collected 2 weeks after the last immunization and determined for neutralizing antibody titer.Results Restriction and sequencing proved that recombinant plasmid pET-21 b-VZV-gE25-537-His was constructed correctly.The target protein,with a relative molecular mass of about 62000,mainly existed in a form of inclusion body,which reach a purity of 85%after purification and showed a specific binding to mouse monoclonal antibody against gE.The serum antibody titer of mice after immunization for 3 times reached 1∶54954,while the neutralizing antibody titer was 1∶21.75.Conclusion Truncated VZV-gE protein was expressed in prokaryotic cells,which showed good immunogenicity in mice after purification and might be used as a candidate antigen for preparation of herpes zoster vaccine.