摘要
目的用原核表达系统表达水痘-带状疱疹病毒(varicella-zoster virus,VZV)糖蛋白(glycoprotein,g)E胞外区,并初步评价其免疫效果。方法将VZVgE胞外区经密码子优化后,克隆至原核表达载体pET-48b中,测序鉴定后,转化进感受态细胞BL21(DE3)中,用异丙基硫代β-D-半乳糖苷(isopropyl β-D-thiogalactoside,IPTG)诱导其在大肠杆菌中表达。采用SDS-PAGE电泳,通过免疫印迹试验(western blot,WB)鉴定其特异性。用Ni2^+-NTA柱对目的蛋白进行纯化并复性后,免疫Balb/c小鼠,用ELISA法检测小鼠血清的特异性及效价。结果以包涵体的形式成功表达了VZVgE胞外区,纯化复性后纯度约为90%。WB鉴定其具有良好的免疫反应性。免疫小鼠后可产生与商品化水痘减毒活疫苗水平相当的特异性抗体。结论原核表达系统成功表达VZV gE并评价其诱导机体特异性免疫反应的能力,为VZV疫苗的研发提供科学依据。
Objective To express extracellular domain of varicella-zoster virus (VZV) glycoprotein E (gE) in Escherichia coli and to evaluate its immunization efficacy. Methods The extracellular domain of VZV gE was codon optimized and cloned into the prokaryotic expression vector pET-48b. After sequencing identification, the vector was transformed into competent cell BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce the expression in E. coli. SDS-PAGE electrophoresis and western blot (WB) were used to identify the specificity. The target protein was purified and renatured by Ni^2+-NTA column and was used to immunize Balb/c mice. ELISA was used to detect the specificity and titer of serum. Results The extracellular domain of VZV gE was successfully expressed as inclusion bodies, and the purity was about 90% after purification and renaturation. WB indicated that VZV gE had good immunoreactivity. After immunizing mice, specific antibodies can be produced at the same level as the commercial live attenuated varicella vaccine. Conclusions The VZV gE protein was expressed in prokaryotic expression system successfully and its ability of inducing specific immune responses was confirmed. The study provides scientific basis for the development of VZV vaccine.
作者
高燕
陈琪
任梦
陈虹
岳文涛
Gao Yan;Chen Qi;Ren Meng;Chen Hong;Yue Wentao(Central Laboratory, Beijing Obstetrics and Gynecology Hospital of Capital Medical University, Beijing 100123, China;Department of Pharmacy, Second People's Hospital of Guilin, Guilin 541001, China)
出处
《国际病毒学杂志》
2019年第2期108-111,共4页
International Journal of Virology
基金
国家自然科学基金(81672838)
北京市医院管理局临床技术创新项目(XMLX201705)。
关键词
水痘-带状疱疹病毒
糖蛋白E
原核表达
免疫评价
Varicella-zoster virus
Glycoprotein E
Prokaryotic expression
Immunological evaluation
