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Characterization of monoclonal antibodies that recognize the amino- and carboxy-terminal epitopes of the pseudorabies virus UL42 protein

Author:
Du, Wenjuan, Wang, Yiping, Huang, Liping, Wei, Yanwu, Chen, Dongjie, Sun, Jianhui, Wu, Hongli, Feng, Li, Liu, Changming
Source:
Applied microbiology and biotechnology 2016 v.100 no.1 pp. 181-192
ISSN:
0175-7598
Subject:
DNA-directed DNA polymerase, Suid herpesvirus 1, Western blotting, amino acids, enzyme-linked immunosorbent assay, epitopes, humoral immunity, mice, monoclonal antibodies, peptides, sequence analysis, virus replication
Abstract:
The pseudorabies virus (PRV) UL42 protein, known as the DNA polymerase processivity factor, is an essential protein required for viral replication. The in vitro function of UL42 has been characterized; however, there is little information concerning the linear B cell epitopes of UL42 that are recognized during humoral immune responses. We generated and characterized six UL42-reactive monoclonal antibodies (mAbs) from mice that had been immunized with a recombinant form of UL42. Through western blotting analysis, we identified two regions of UL42 (amino acids 39–148 and 302–384) that reacted with these mAbs. We then synthesized a panel of UL42-derived peptides spanning the two regions and screened the six mAbs. We were able to identify three linear epitopes (¹¹⁶SGGVLDALK¹²⁴, ³⁵⁴KRPAAPR³⁶⁰, and ³⁶⁰RMYTPIAK³⁶⁷) by enzyme-linked immunosorbent assays. The ¹¹⁶SGGVLDALK¹²⁴ epitope was located at the amino-terminus, while the other two epitopes were at the carboxy-terminus. Using these mAbs, we found that UL42 localized to the nucleus during viral replication and could be immunoprecipitated from PRV-infected PK-15 cells. We also established a UL42 mAb-based immunoperoxidase monolayer assay for the determination of PRV titers. Sequence analysis showed that the linear epitopes of UL42 were highly conserved among PRV strains. Taken together, our results indicate that the six generated mAbs could be useful tools for investigating the structure and function of UL42 during viral replication. In addition, these mAbs could be applied to diagnostic and therapeutic approaches for the effective control of PRV infections.
Agid:
4778016