Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections. IMPORTANCE The continuous variation of CSFV and PRV poses a serious threat to vaccination strategies. However, it is expensive to eliminate CSFV in the swine industry in China through a phase-out policy, and the existing CSFV vaccine C-Strain cannot distinguish infected from vaccinated individuals. In addition, the mutation of PRV in 2011 is also an urgent need to develop new PRV vaccine strains. In this study, we constructed a recombinant PRV strain expressing for the first time the full-length E2 protein, mice immunized with the recombinant PRV strain produced high titer antibodies against both PRV and CSFV E2 protein and showed that it could protect against both PRV variant challenge and CSFV fever response in rabbit models. It can be used to prevent two diseases with one injection.Copyright © 2023. Published by Elsevier B.V.
