Prokaryotic expression and antigenic character analysis of enterovirus EV-D68 surface structure protein VP2 and VP3
Haiting Long, Jing Kong, Huiwen Zheng, Bingxiang Li, Han Zhang, Heng Li, Yubin Liu, Haitao Fan, Li Yu, Guoyang Liao, Ming Sun, Longding Liu
Abstract
ObjectiveVP2 and VP3 surface structural proteins of EV-D68 were expressed in prokaryotic system, and the affinity and binding charactors of two proteins were analyzed to investigate their capacity of binding to memory B cells.
MethodsThe VP2 and VP3 genes of EV-D68 were amplified by PCR and inserted into prokaryotic expression vector pGEX-5X-1.Rosetta(DE3)pLysS, transformed by the constructed recombinant expression plasmids pGEX-5X-1-VP2 and pGEX-5X-1-VP3, were induced by IPTG.The expressed recombinant protein in the form of inclusion bodies were extracted, followed by washing, dialyzing and renaturing.The recombinant protein was then identified by SDS-PAGE electrophoresis and Western blotting.The activity and affinity of VP2 and VP3 were evaluated by competitive ELISA.The binding capacity of VP2 and VP3 to the memory B cells was detected by flow cytometry.
ResultsThe recombinant expression plasmids pGEX-5X-1-VP2 and pGEX-5X-1-VP3 were successfully constructed and verified by double restriction enzyme digestion and sequencing.We then obtained both VP2 and VP3 proteins.The purity of VP2 and VP3 proteins were 80% and 75%, respectively.Both proteins showed antigenic specificity.VP2 showed a medium affinity with affinity constant of 2.7 × 107 M-1, and the VP3 showed a low affinity with affinity constant of 3.25 × 106 M-1.Specific memory B cells in spleen after viral challenge to mice can be detected by flow cytometry.
ConclusionVP2 and VP3 proteins of EV-D68 were obtained in prokaryotic system with favorable antigen specificity.These proteins can specifically bind to memory B cells with high affinity and binding capacity.These findings offer the targets for sorting EV-68-specific memory B cellsand provide insight for further investigation in the neutralizing epitopes of VP2 and VP3 and their broad-spectrum neutralizing antibodies.
Key words:
Enterovirus D68; VP2 protein; VP3 protein; Prokaryotic expression
Contributor Information
Haiting Long
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Jing Kong
Department of Medical Laboratory and Prophylactic Medicine, Medical College of KunMing Vniversity, Kunming 650214, China
Huiwen Zheng
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Bingxiang Li
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Han Zhang
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Heng Li
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Yubin Liu
Department of Infectious diseases, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China
Haitao Fan
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Li Yu
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
The Eighth Section of Biological Production, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Guoyang Liao
Liao Guoyang Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Ming Sun
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
Longding Liu
Respiratory Virus Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, China
