摘要
目的:对裂谷热病毒截短型Gc蛋白进行真核表达与纯化,并鉴定其免疫反应性。方法:在截短型Gc蛋白的基因5'和3'端分别添加tPA信号肽和StrepⅡ-tag,再将其克隆到真核表达载体pCAGGs中,转染Expi293F细胞进行真核表达;用StrepTrap亲和层析柱纯化,ELISA检测该蛋白与特异抗体的免疫反应性。结果:截短型Gc蛋白在Ex⁃pi293F细胞培养上清中获得表达,经纯化后得到的Gc蛋白浓度为0.7 mg/mL;该蛋白与针对Gc蛋白特异的单克隆抗体特异性结合。结论:截短型Gc蛋白的表达与纯化为裂谷热病毒疫苗和中和抗体研究奠定了良好的基础。
Objective:To express Rift Valley fever virus(RVFV)truncated Gc protein in eukaryotes,further purify it and identify its immunoreactivity.Methods:The tPA signal peptide and StrepⅡ-tag were added at the 5'and 3'ends of the truncated Gc protein gene,respectively.Then,the truncated Gc protein gene was cloned into the eukaryotic expression vector pCAGGs and transfected into the Expi293F suspension cells.The product was purified via StrepTrap affinity column.Finally,the immunoreactivity of the purified protein was identified by the specific monoclonal antibody through the ELISA.Results:The truncated Gc protein was expressed in the supernatant of the Expi293F suspension cells and further purified,and the concentration of purified protein was 0.7 mg/mL.The purified protein can bind anti-Gc monoclonal antibody.Conclusion:Successful expression and purification of the truncated Gc protein lay a strong foundation for developing of the vaccine and neutralizing antibody against RVFV.
作者
郝勐
张胜男
李建民
夏咸柱
HAO Meng;ZHANG Sheng-Nan;LI Jian-Min;XIA Xian-Zhu(Comparative Medicine Center,Institute of Laboratory Animal Sciences,Peking Union Medical College&Chinese Academy of Medical Sciences,Beijing 100021,China;Beijing Institute of Biotechnology,Beijing 100071,China;College of Wildlife and Protected Areas,Northeast Forestry University,Harbin 150040,China;Institute of Military Veteri⁃nary Medicine,Changchun 130000,China)
出处
《生物技术通讯》
CAS
2019年第5期604-608,635,共6页
Letters in Biotechnology
基金
军委后勤保障部重大项目（2012AA02A302）
