| Classical swine fever(CSF)is a highly contagious disease caused by classical swine fever virus(CSF virus,CSFV),which is characterized by high fever retention,anorexia,widespread hemorrhage,diarrhea and conjunctivitis.Currently,CSF mostly presents an atypical form in pig farms due to the widespread vaccination of the lapinized attenuated vaccine against classical swine fever in China,and the incidence tends to increase and it can be co-infected with other diseases such as porcine reproductive,respiratory syndrome and pseudorabies.Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by the porcine reproductive and respiratory syndrome virus(PRRS virus),which mainly leads to reproductive disorders in sows and respiratory symptoms in piglets.There are many types of PRRSV such as classical strains,highly pathogenic strains,NADC30-like and NADC34-like in pig farms in China,and NADC30-like is gradually becoming an epidemic strain,which makes the prevention and control of PRRS more difficult.Pseudorabies(PR)is an acute infectious disease of pigs caused by pseudorabies virus(PRV),which causes a large number of newborn piglets death,sows abortion and male pig infertility.In late 2011,a variant strain of PRV with stronger virulence emerged in China’s pig population,leading to outbreaks of PR in large-scale pig farms that had been immunized with pseudorabies vaccine in many provinces in China,which seriously hindered the sustainable development of China’s pig industry.At present,vaccination is still the most effective means to prevent,control and purify CSF,PRRS and PR.Thus,it is essential to develop the safe and effective multiple vaccines for the prevention and purification of these three diseases.The CSFV E2 protein is a major antigenic protein that can effectively induce protective immune responses.Four antigenic structural domains exist at the N-terminal end of the E2 protein: B(amino acids 1~83),C(amino acids 1~110),D(amino acids 86~110),and A(amino acids 86~176),which are the main targets for the development of novel vaccines.In order to obtain a recombinant pseudorabies virus expressing the B/C/D/A four epitope regions of CSFV E2 protein,in the present study,the fragment containing four epitope regions B/C/D/A of CSFV E2 protein was inserted into the Bam H I site of the p G-EGFP recombinant plasmid.The resulting recombinant plasmid p G-E2AD-EGFP was transfected into ST cells that had been inoculated with PRV variant three-gene deletion strain r PRV NY-g E-/g I-/TK-.The transfection solution was harvested,and then the resulting recombinant virus r PRV-E2AD-EGFP was purified by screening green fluorescence plaques.r PRV-E2 AD was obtained by knocking out the EGFP+ gene using gene-editing technology termed clustered regularly interspaced palindromic repeats(CRISPR)/associated(Cas9)system.The results of m RNA level and protein level showed that A-D epitope region of r PRV-E2 AD could be expressed in ST cells.The GP5 protein harbors the major neutralizing epitope and induces the body to produce neutralizing antibodies,and it is a popular target for the development of novel PRRSV vaccines.To obtain a recombinant pseudorabies virus expressing CSFV E2 AD and PRRSV GP5,the NADC30-like PRRSV GP5 gene was inserted into the eukaryotic expression vector p BApo-EF1a_Pur_DNA via Bam H I and Hind III digestion sites to generate the eukaryotic expression plasmid p BA-GP5/NA.The expression cassette containing the homologous arms and GP5 gene was amplified from p BA-GP5/NA and cloned into the linear p G-E2AD-EGFP vector by homologous recombinase to produce the recombinant plasmid p G-E2AD-GP5/NA-EGFP.The recombinant virus r PRV-E2AD-GP5/NA-EGFP was rescued by transfection,homologous recombination with the parent strain and plaque purification.The recombinant virus r PRV-E2AD-GP5/NA was obtained by knocking out the fluorescent tag and purified by plaque.r PRV-E2AD-GP5/NA was then verified by RT-PCR,Western blot and indirect immunofluorescence assay,showing that the GP5 gene of recombinant virus r PRV-E2AD-GP5/NA could be expressed in ST cells.Two recombinant virus strains(r PRV-E2 AD and r PRV-E2AD-GP5/NA)were tested for culture characteristics,growth characteristics,genetic stability,safety and immune potency in mice.The results showed that the two recombinant viruses had similar growth characteristics on ST,IPEC-J2,PK-15 and Vero cells,with the highest TCID50 on ST cells at 106.625/0.1 m L and 106.5/0.1 m L,respectively.Two recombinant viruses had similar physicochemical properties to the parental strain.The one-step growth curve test showed that the two recombinant viruses did not change significantly compared with the parental strain and had good genetic stability,which indicated that the insertion of exogenous genes did not affect the proliferation characteristics of the parental strain.Safety tests demonstrated that the two recombinant viruses did not show any symptoms and fed normally within two weeks after infection,which tentatively indicated that the two recombinant viruses were safe for mice and could induce detectable ELISA and neutralizing antibodies against PRV and ELISA antibodies against E2 AD protein of CSFV in mice,while no antibodies against PRRSV GP5 protein were detected.After the virulent PRV challenge assay,the two recombinant virus groups and the parental strain group showed individual mouse death,but PRV was not detected in both dead and surviving mice,indicating that the mice were protected against the strong PRV NY strain.In conclusion,the recombinant viruses r PRV-E2 AD and r PRV-E2AD-GP5/NA were successfully obtained and could express CSFV E2 AD protein and PRRSV GP5 protein with no significant changes in biological properties.The mouse test showed that the corresponding antibodies to PRV and CSFV E2 AD proteins were induced,but not to PRRSV GP5 protein,and the reason needs to be further investigated.This study provides technical support for the development of a live vector vaccine for PRV and the next improvement of the recombinant virus. |
