Construction Of GE And TK Genes Deletion Mutant Of Pseudorabies Virus And Recombinant Pseudorabies Virus Expressing Of Porcine Epidemic Diarrhea Virus S1 Gene

Pseudorabies(PR)and porcine epidemic diarrhea(PED)are two important infectious diseases,which have caused huge economic losses in the pig industry because of their prevalence in China in recent years.The marked vaccine that could be used to differentiate a vaccinated animal from the infected animal(DIVA)is one of the effective strategies to control and eradicate Pseudorabies virus(PRV)infection.Classical gene deleted vaccines had played an important role in the prevention and control of PR.However,due to the emergence of PRV mutants,immunization failure against PRV appeared in pigs from time to time in the past few years.The prevalence of PED also increases the need of vaccine.Since most of current vaccines are-attenuated live vaccines,there are potential safe problems.With the development and application of PRV vector,the co-expression of foreign genes has become possible,and the combination vaccines have been widely concerned.S gene is the main protective antigen of PEDV,which is the preferred target protein for the vaccine development.Therefore,in this study,the TK and gE genes deleted strain rPRV-TX-gE-/TK-of PR mutant was constructed by homologous recombination,and then the S1 gene of PEDV was inserted into the rPRV-TX-gE-/TK-to carry out the recombinant virus co-expressing PEDV S1 protein.This study will provide vaccine candidates for the prevention and control of these two kinds of swine infectious diseases.1.Construction of the gE and TK genes double deletion mutant of the PRV strainThe pSKTKRL-GFP transferring plasmid vector with green fluorescent protein marker EGFP and genomic DNA of rPRV-TX-gE-were co-transfected into PK15 cells in a proportion of 10:1 by using calcium phosphate precipitation,viruses were harvested when 80%cell cytopathy were observed in the cells.The recombinant virus rPRV-TX-gE-/TK-GFP with reporter gene was purified by plaque purification method.After reported gene was knockout by using recombinant Cre,the recombinant virus without reporter gene was obtained after plaque purification.The sequence analysis of TK gene form the recombinant virus showed that there was a loxP residue in deletion TK gene,indicating a successful construction.The recombinant virus was named as rPRV-TX-gE-/TK-.2.Growth characteristic and immune efficacy of rPRV-TX-gE/TK-mutantThe mutant strain was replicated in PK15 cells,and the results of the growth curve showed that the rPRV-TX-gE-mutant in PK15 cells was slightly higher than its parental strain PRV-TX,whereas,the growth curve of the rPRV-TX-gE-TK-in PK15 cells was slightly lower than its parental strain PRV-TX,there was no significant difference between the two strains,indicating that gE/TK double genes deletion had no influence on virus replication.The results of LDso determination showed that LD50 of PRV-TX,rPRV-TX-gE-and rPRV-TX-gE-TK-in BALB/c mice were 3.5×103TCID50?3.1×103TCID50 and more than 1×106TCID50,respectively,indicating that further deletion of the TK gene based on the gE gene deletion of mutant significantly reduce the virulence of the virus.BALB/c mice were inoculated by intramuscular injection at the dose of 104 TCID50 of the rPRV-TX-gE-/TK-or Bartha-K61 respectively,and the serum were collected at 7 d,14 d and 21 d.Antibodies against the variant strain PRV-TX or the classical strain PRV-SZ were detected by indirect ELISA method.Fourteen days or twenty-one days after vaccination,the ELISA antibodies titers against the variant strain PRV-TX or the classical strain PRV-SZ in mice vaccinated with rPRV-TX-gE-/TK-were more higher than those of the control group.The ELISA antibodies titers against the classical strain PRV-SZ in mice vaccinated with Bartha K61 were more higher than those of the control group,but the ELISA antibodies titers against the variant strain PRV-TX in mice vaccinated with Bartha K61 were much low.After 21 day post immunization,the mice were challenged by the variant strain PRV-TX or the classical strain PRV-SZ at the dose of 104 TCID50.During a seven-day observation,the protection rates of rPRV-TX-gE-/TK-and Bartha K61 were 100%and 0 after challenged with the variant strain PRV-TX,and 20%and 70%after challenged with the classical strain PRV-SZ.During a fourteen-day observation,the protection rate of rPRV-TX-gE-/TK-was reduced to 30%after challenged with the variant strain PRV-TX.These results suggested that the rPRV-TX-gE-/TK-could provide good protection against the lethal challenge of variant strain within 7 days,while it could provide partial protection against classic strain challenge.However,the Bartha-K61 vaccine could provide good protection against the classic strain challenge rather than the variant strain challenge.3.Construction of recombinant pseudorabies virus co-expressing the S1 gene of PEDVPrimers were designed based on the S gene sequence of PEDV published by GenBank.The S1 gene of PEDV with a length of 2376 bp was amplified by PCR,and was cloned into the transfer vector pSKTKRL-GFP containing the upstream and downstream homologous arms of TK gene from PRV to construct the transfer plasmid pSKTKRL-PEDV-S 1.The recombinant plasmid pSKTKRL-PEDV-S 1 and the genomic DNA from the double deletion strain rPRV-TX-gE-/TK--GFP were co-transfected into PK15 cells by using calcium phosphate precipitation,and the recombinant pseudorabies virus rPRV-TX-gE-/TK-S 1 co-expressing PEDV S1 protein was purified by reverse screening of EGFP-negative plaques.The results showed that the successful insertion of S1 gene into the genome of the recombinant virus rPRV-TX-gE-/TK--S 1 was confirmed by PCR,and expression of S1 gene in the recombinant virus rPRV-TX-gE-/TK--S1 was confirmed by indirect immunofluorescence assay(IFA)and Western-blot.The LD50 of Bartha-K61 was 3.5×104TCID50,while that of rPRV-TX-gE-/TK' and rPRV-TX-gE-/TK--S1 were?1?106 TCID50 in BALB/c mice,indicating that the safety of parental virus was not affected by the insertion of the foreign gene S1.