去N端信号肽的水痘-带状疱疹病毒糖蛋白E的原核可溶性表达及其免疫原性评价

水痘-带状疱疹病毒(varicella zoster virus，VZV)糖蛋白E(glycoprotein E，gE)是VZV亚单位疫苗的主要候选蛋白，但目前原核表达系统制备的gE蛋白以包涵体形式为主，可溶性差。本研究采用去除第1～30氨基酸序列的VZV gE胞外域基因，将其与原核表达载体pET32a连接，并转化至感受态细胞BL21(DE3)中。使用异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-thiogalactoside，IPTG)诱导表达，His-tag柱纯化重组gE蛋白，蛋白质印迹法(Western blot，WB)检测其特异性。用该重组gE蛋白免疫BALB/c小鼠制备多克隆抗体，酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)和间接免疫荧光法检测多克隆抗体效价及特异性。结果显示，BL21/pET32a-VZV gE工程菌可以表达可溶性重组gE蛋白，纯化后纯度约为90%。WB鉴定该重组蛋白具有良好的免疫反应性。ELISA检测显示小鼠抗VZV gE多克隆抗体效价＞1∶10 000，间接免疫荧光实验结果显示该抗体特异性较高。结果表明，本研究在原核表达系统中成功表达可溶性重组VZV gE蛋白，同时该蛋白具有较强的免疫原性，这为VZV gE亚单位疫苗的研制和大规模生产奠定了基础。

Soluble prokaryotic expression of varicella-zoster virus glycoprotein E without N-terminal signal peptide and its immunogenicity evaluation

Varicella zoster virus (VZV) glycoprotein E (gE) is the main candidate protein for VZV subunit vaccine. Unfortunately, the expression of gE in prokaryotic expression was mainly found in the inclusion body. In this study, a deletion of N-terminal 30 amino acids of gE was constructed on pET32a, expressed in BL21 (DE3). The soluble gE was purified and used to make polyclonal antibodies in BALB/c mice. The titer and specificity of polyclonal antibodies were determined by enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence. The results showed that the soluble recombinant gE produced by BL21/pET32a-VZV gE system is a practical way for vaccine development.