Longjun Guo1, Yuehua Lu, Liping Huang, Yanwu Wei, Changming Liu. 1. Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin, PR China.
Abstract
OBJECTIVE: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic syndrome in pigs. The capsid (Cap) protein encoded by ORF2 is the main structural protein involved in the host immune protective response to PCV2. It is therefore important to map the antigenic epitopes of the PCV2 Cap protein. METHODS: In this study, 5 monoclonal antibodies (mAbs) against the recombinant PCV2 Cap protein, expressed by the baculovirus system in Sf21 insect cells, were generated. The antigenic epitope recognized by these mAbs was located in the Cap A protein by Western blot analysis using 4 overlapping minifragments covering the Cap protein expressed in Escherichia coli. To locate the epitope more accurately, 3 sets of overlapping peptides were synthesized. RESULTS: The results demonstrated that 4 of the 5 mAbs recognized the same core epitope ((26)RPWLVHPRHRY(36)) located in the nuclear localization signal (NLS) region at the N terminus of the Cap protein. The other mAb (1D2) reacted with the recombinant Cap protein only, indicating that it recognizes a potential conformational epitope. This mAb demonstrated a neutralizing effect on PCV2. CONCLUSION: This is the first study to identify an antigenic epitope in the NLS region of the PCV2 Cap protein using mAbs. The results of this study will facilitate future investigations into the mechanism and function of nuclear localization of this protein.
OBJECTIVE:Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic syndrome in pigs. The capsid (Cap) protein encoded by ORF2 is the main structural protein involved in the host immune protective response to PCV2. It is therefore important to map the antigenic epitopes of the PCV2 Cap protein. METHODS: In this study, 5 monoclonal antibodies (mAbs) against the recombinant PCV2 Cap protein, expressed by the baculovirus system in Sf21 insect cells, were generated. The antigenic epitope recognized by these mAbs was located in the Cap A protein by Western blot analysis using 4 overlapping minifragments covering the Cap protein expressed in Escherichia coli. To locate the epitope more accurately, 3 sets of overlapping peptides were synthesized. RESULTS: The results demonstrated that 4 of the 5 mAbs recognized the same core epitope ((26)RPWLVHPRHRY(36)) located in the nuclear localization signal (NLS) region at the N terminus of the Cap protein. The other mAb (1D2) reacted with the recombinant Cap protein only, indicating that it recognizes a potential conformational epitope. This mAb demonstrated a neutralizing effect on PCV2. CONCLUSION: This is the first study to identify an antigenic epitope in the NLS region of the PCV2 Cap protein using mAbs. The results of this study will facilitate future investigations into the mechanism and function of nuclear localization of this protein.