Prokaryotic Expression and Polyclonal Antibody Preparation of Carp edema virus P4a Protein
FANG Zhen-Zhen2, YANG Yuan-Yuan2, YU Hong3, SUN Xue-Liang2,*, SHI Hong-Yue2, CHEN Cheng-Xun2, JI Yan-Bin2, LI Ying-Ying1,*
1 Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of Aquatic Animal Immunology and Sustainable Aquaculture/Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380,PR China; 2 Tianjin Key Laboratory of Aqua-Ecology and Aquaculture/College of Fisheries, Tianjin Agriculture University, Tianjin 300384, China; 3 Tianjin Fishery Research Institute, Tianjin 300202, China
Abstract：Carp edema virus (CEV) is a DNA virus popular in recent years, which seriously endangers the healthy development of koi carp (Cyprinus carpiohaematopterus) and carp (Cyprinus carpio) culture. In this study, specific primers were designed and the full genome sequence of CEV P4a was amplified. The pET-32a-P4a recombinant plasmid was constructed using prokaryotic expression system. The recombinant protein P4a was obtained after induction by isopropyl β-D-thiogalactopyranoside (IPTG). After SDS-PAGE identification, purification and renaturation, the His-tagged fusion protein was obtained, and immunized New Zealand white rabbits (Oryctolagus cuniculus) were prepared to prepare polyclonal antibodies. The concentration of the antibody was detected by bicinchoninic acid assay (BCA) method and identified by Western blot. The construction of the pET-32a-P4a prokaryotic expression vector was successfully verified through experimental validation. The NCBI Blast analysis showed that the homology of the P4a vector sequence and the theoretical sequence of P4a protein was 99%. The concentration of the antibody was 2.05 mg/mL by BCA method. SDS-PAGE and Western blot showed that the antibody has good immunogenicity. In summary, the polyclonal antibody against CEV P4a protein was successfully prepared in this experiment, which provides data support for the immunological detection and vaccine development of CEV.
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