| Porcine pseudorabies (PR) is an acute infectious disease of domestic animals and wildlife caused by pseudorabies virus (PRV). It was characterized by high fever, extreme itching, and encephalomyelitis. This disease often occurs in the populations of pigs, and causes significant economic losses in the pig industry around the world. But now there is no effective drug treatment for PR, so vaccination is the main measures to control the incidence and prevalence of the disease. In this study, a gE gene deletion mutant and a gE/TK double genes deletion mutant without reporter gene was constructed from a PRV isolate strain JSZ strain using a Cre-loxP system, and the growth characteristic and immune response of the recombinant pseudorabies virus were investigated. This study provides a platform for the development of the recombinant attenuated PRV vaccine and its vectored vaccine.1Construction of gE gene deletion mutant and gE/TK double genes deletion mutantThe gE and TK genes were PCR amplified from PRV-JSZ strain as recombinant arms, enhanced green fluorescent protein EGFP was chosen as a reporter gene, and then the transfer vectors pSKgERL-GFP and pSKTKRL-GFP with loxP sites were constructed by insertion of the EGFP gene into gE gene and TK gene. Genomic DNA of PRV-JSZ was extracted by proteinase K and phenol treatment, and10μg of the genomic DNA was co-transfected with1μg of the transfer plasmid pSKgERL-GFP into PK15cells using calcium phosphate. When obvious cytopathic effect was observed, the infected cells containing recombinant pseudorabies virus were collected. Virus was serially diluted and inoculated onto monolayers of PK15cells. The recombinant virus rPRV-gE-/GFP expressing EGFP was purified by plaque purification under fluorescence microscope.10μg of the genomic DNA of rPRV-gE-/GFP was mixed to2.5U Cre recombinase and the mixture was incubated at37℃for1h; DNA was extracted by phenol from the mixture, and the calcium phosphate-DNA precipitate was transfected into PK15cells. Subsequently, a gE gene deletion mutant without the reporter gene EGFP was obtained under fluorescence microscope and purified by plaque purification in PK15cells. Sequence analysis of PCR production of gE gene from rPRV-gE-showed that a883-bp segment of gE gene was deleted. The genomic DNA of rPRV-gE-was further co-transfected with the transfer plasmid pSKTKRL-GFP into PK15cells, and the cells were harvested when80%cytopathic effect was observed. Subsequently, the recombinant virus rPRV-gE-/TK-/GFP was obtained following plaque screening. The genomic DNA of rPRV-gE-/TK-/GFP treated by Cre recombinase was transfected into PK15cells, and a gE/TK double gene deletion mutant without reporter gene EGFP was obtained by plaque purification. The deletion of498bp segment in TK gene of rPRV-gE-/TK-was confirmed by PCR.2Growth Characteristic and immune response of deletion mutantThe titers of PRV-JSZ, rPRV-gE-, and rPRV-gE-/TK-in PK15cells were10-4932TCID50/0.1mL,10-4.944TCID50/0.1mL and10-4444TCID50/0.1mL, respectively, indicating that the gE mutant had a similar growth speed and titer in PK15cells when compared with its parental strain, but the growth speed of the gE/TK mutant in PK15cells was relatively slower when compared with its parental strain. The LD50of the wild type strain, the gE mutant and the gE/TK mutant for mice were3.16×103TCID50,5.01×104TCID50and more than1×105TCID50, respectively. Compared with the parental strain, the virulence of gE mutant in mice was decreased, while gE/TK mutant showed a significant attenuation in virulence. Mice immunized with104TCID50recombinant viruses showed no clinical symptom and growth retardation. Antibodies against PRV were detected by ELISA. Antibodies were induced by rPRV-gE-/TK-at21day post-immunization, while no antibody was detected in the mouse of rPRV-gE-group. All mice were challenged with the virulent PRV-JSZ, the protection rate was80%for each deletion mutant. In conclusion, the rPRV-gE-/TK-was safe and could protect mice against virulent PRV challenge. |
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