Expression and immunogenicity analysis of CyHV-2 ORF4 protein in baculovirus expression system
MENG Shaodong1，2, JIANG Xinyu2, GE Heng2, HU Guangyao2, YANG Dazuo1, ZHANG Jialin2*, LI Qiang2
1.Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs,Dalian Ocean University, Dalian 116023, China; 2.College of Marine and Biology Engineering, Yancheng Institute of Technology, Yancheng 224051, China
Abstract: To understand the function of open reading frame 4 (ORF4) protein of Cyprinid herpesvirus 2 (CyHV-2), ORF4 protein and its immunogenicity were subsequently identified using baculovirus expression system. CyHV-2 ORF4 gene was amplified by PCR, and the recombinant plasmid pFastBac HTA-ORF4 was constructed. The plasmid was transformed into competent Escherichiacoli DH10 Bac to obtain the recombinant Bacmid-ORF4 which was transfected into the Sf9 cells using liposome transfection technique. The recombinant protein was identified by indirect immunofluorescence assay (IFA) and Western blot, after purified by His affinity chromatography, its immunogenicity was analyzed by mice immunological test. Results showed that the Sf9 cells turned round, and had large nuclei at 72 h post transfection. IFA showed that significant green fluorescence was observed in the cells infected by recombinant baculovirus. Furthermore, Western blot revealed that the purified protein reacted specifically with anti-His tag monoclonal antibody. Mouse immunologic trial showed that the recombinant ORF4 protein induced specific immune reaction in mice, suggesting that the produced protein had good immunogenicity. The findings will provide material for the function analysis of CyHV-2 ORF4 protein in the future.
2021, Vol. 36 
