Objective: To perform prokaryotic expression and preliminary characterization of the recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis.
Methods: The recombinant poly-epitope vaccine EgG1Y162-2 (4) against Echinococcus granulosus based on the linker GSGGSG was subjected to structural three-dimensional (3D) modeling using immunoinformatics to analyze the structural changes and evaluate the antigenicity of the vaccine. The pET30a-EgG1Y162-2 (4) recombinant plasmid was generated using double digestion with EcoR I and Sal I, and then transformed into competent cells. Following protein induction with isopropyl-β-D-thiogalactoside (IPTG), the prokaryotic expression proteins were characterized using Western blotting, and the antigenicity of the recombinant protein was analyzed using sera from cystic echinococcosis patients and health volunteers.
Results: The four EgG1Y162-2 proteins coupled by the 3D structure of the recombinant vaccine EgG1Y162-2 (4) presented independent and effective expression and good antigenicity. The highest protein expression was detected in the supernatant following induction of the recombinant plasmid pET30a-EgG1Y162-2 (4) by 0.2 mmol/L IPTG at 37 °C for 4 h, and a pure protein component was seen following elution with 60 mmol/L imidazole. Western blotting analysis of the recombinant multiepitope protein HIS-EgG1Y162-2 (4) showed a band at approximately 39 kDa, and this band was recognized by sera from cystic echinococcosis patients.
Conclusions: A recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis has been successfully constructed, which provides a preliminary basis for researches on recombinant multi-epitope vaccine against cystic echinococcosis.
［摘要］ 目的 对细粒棘球蚴病重组多表位疫苗 EgG1Y162-2 (4) 进行原核表达并初步鉴定。方法 利用免疫信息学方 法对基于接头序列 “GSGGSG” 的细粒棘球蚴病重组多表位疫苗 EgG1Y162-2 (4) 进行三维结构建模, 分析其结构变化并评 估该疫苗抗原性。通过 EcoR I和Sal I 双酶切构建 pET30a-EgG1Y162-2 (4) 重组质粒, 将重组质粒转化入感受态细胞, 利 用异丙基-β-D-硫代半乳糖苷 (isopropyl-β-D-thiogalactoside, IPTG) 诱导蛋白表达, Western blotting 鉴定原核表达蛋白产物, 并应用细粒棘球蚴病患者及健康人血清分析重组蛋白抗原性。结果 构建的重组疫苗 EgG1Y162-2 (4) 三维结构串联的 4 个 EgG1Y162-2 蛋白均能独立、有效表达且具有良好抗原性。重组质粒 pET30a-EgG1Y162-2 (4) 在 0.2 mmol/L IPTG 37 °C 诱导 4 h 条件下, 上清中目的蛋白表达量最高, 使用 60 mmol/L 咪唑洗脱的蛋白成分较纯。Western blotting 分析发现, 重组多表位蛋白 HIS-EgG1Y162-2 (4) 在约 39 kDa 位置处出现条带, 且该蛋白可被细粒棘球蚴病患者血清识别。结论 成 功构建了细粒棘球蚴重组疫苗 EgG1Y162-2 (4), 为研究细粒棘球蚴病重组多表位疫苗奠定了基础。.
Keywords: Characterization; Cystic echinococcosis; EgG1Y162-2 (4); Linker sequence; Multi-epitope vaccine; Prokaryotic expression.