Construction And Immunogenicity Evaluation Of Recombinant Pseudorabies Virus GI/gE Deletion Strain With Double GB Gene

As a common second-class animal disease,pseudorabies(PR)is widely prevalent in the world,which seriously restricts the development of swine breeding industry in the world.Because of the pathogen,pseudorabies virus(PRV)has invisible infection and neurotropic properties,it has been difficult to get rid of.Drawing on the case of pseudorabies removal in Europe and the United States,China introduced the Hungarian Bartha-K61 inactivated vaccine for the pseudorabies clearance program in the 1980s.Pseudorabies is effectively controlled in China,but since the end of 2011,the rabies epidemic has been erupted in many domestic Bartha vaccine immunized farms.The pathogen is a pseudorabies virus mutant,which is more pathogenic and presents a new round of pseudorabies epidemic trends.The currently applied PRV vaccine has not been completely resistant to the new epidemic strain of pseudorabies virus,and a large number of scholars have begun to conduct vaccine research on new pseudorabies virus strains.In this study,the g I/g E deletion strain r PRV-AH-g I^-/g E^-/EGFP⁺labeled with enhanced green fluorescent gene(EGFP)was constructed in the laboratory,the principle of homologous recombination was used to insert the g B exogenous expression cassette sequence into the g I/g E deletion site in the genome of r PRV-AH-g I^-/g E^-/EGFP⁺.The recombinant virus r PRV-AH-g I^-/g E^-/g B⁺was screened which efficiently expresses the g B gene,and evaluated its immunogenicity in Kunming mice.The main research contents include the following aspects:1.Construction and biological characterization of recombinant virus r PRV-AH-g I^-/g E^-/g B⁺The new domestic PRV strain PRV-AH-China-2013(hereinafter referred to as PRV-AH)was isolated in the early stage of the laboratory,and the g I/g E deletion strains r PRV-AH-g I^-/g E^-and r PRV-AH-g I^-/g E^-/EGFP⁺were constructed.Based on this,the g B coding region sequence was amplified and the His tag sequence was tagged at its 3'end,and further cloned into the mammalian eukaryotic expression plasmid p CDNA3.0 multiple cloning site.The g B exogenous expression cassette sequence,CMV-g B-SV40 poly A.CMV-g B-SV40 poly A was amplified and cloned into the pre-established g I/g E homology arm plasmid p MD-LA-RA to obtain the recombinant plasmid p MD-LA-g B-RA.The plasmid was transfected into BHK-21 cells by lipofection,and BHK-21 cells were infected by the virus r PRV-AH-g I^-/g E^-/EGFP⁺.After sufficient homologous recombination in BHK-21 cells,combined with plaque purification and limiting dilution method,the recombinant virus r PRV-AH-g I^-/g E^-/g B⁺was obtained.PCR sequencing results showed that the g B exogenous expression cassette sequence was successfully inserted into the expected position.After 15 continuous passages,the g B exogenous expression cassette sequence was still stably inherited by the recombinant virus r PRV-AH-g I^-/g E^-/g B⁺,and no deletion mutation occurred.The growth kinetics of the recombinant virus r PRV-AH-g I^-/g E^-/g B⁺was compared with the related strains r PRV-AH-g I^-/g E^-/EGFP⁺and PRV-AH,and the growth kinetics of them were basically similar.There was no significant difference,and the highest toxicities were 10^-8.57/m L,10^-8.44/m L,and 10^-8.69/m L,respectively.Compared with the virus r PRV-AH-g I^-/g E^-,the real-time PCR results showed that the g B exogenous expression cassette sequence was highly efficiently transcribed on the r PRV-AH-g I^-/g E^-/g B⁺genome.At 24th hour post-infetion,the total m RNA expression level of g B on the r PRV-AH-g I^-/g E^-/g B⁺was significantly increased,reaching 29.24 times of g B m RNA expressed by r PRV-AH-g I^-/g E^-.Western Blot results showed that the g B-His fusion protein was highly expressed in BHK-21 cells with a size of 117 KD,which was consistent with the expected size.2.Evaluation of immune efficacy of inactivated vaccine r PRV-AH-g I^-/g E^-/g B⁺in Kunming miceAfter inactivating the virus with formaldehyde,the inactivated vaccine was prepared by mixing the aqueous adjuvant MONTANIDE GEL 01.The immunological titers of inactivated vaccine r PRV-AH-g I^-/g E^-/g B⁺,r PRV-AH-g I^-/g E^-and a commercial vaccine in mice were compared,to evaluate the application prospect of inactivated vaccine r PRV-AH-g I^-/g E^-/g B⁺.The immunization protocol was to select two different immunization doses of 10⁵TCID₅₀and 10⁶TCID₅₀.After 4 weeks of immunization for the first time,the immunization was boosted once,blood was collected regularly,and the serum neutralizing antibody level of the mice was determined.Four weeks after the second immunization,mice were challenged with the new epidemic strain PRV-AH to evaluate the immunoprotective potency of vaccines.The results of mouse neutralization experiments showed that the serum antibody level in the r PRV-AH-g I^-/g E^-/g B⁺immunized group increased rapidly after the second dose,and peaked at 3 weeks after the second dose.There was a significant difference between the 10⁵TCID₅₀and 10⁶TCID₅₀immunization groups,and 10⁶TCID₅₀was the preferred immunization dose for mice.In the 10⁶TCID₅₀immunization group,the serum antibody level of the r PRV-AH-g I^-/g E^-/g B⁺immunization group reached 1:81.85 three weeks after the second immunization,which was significantly higher than that of the r PRV-AH-g I^-/g E^-immunization group(1:53.24),The commercial vaccine immunization group reached to 1:77.87 four weeks after the second immunization,and the duration of immunization was longer.The immune level was equivalent to that of r PRV-AH-g I^-/g E^-/g B⁺immunization group.It was initially shown that an increase in the expression of the g B gene significantly increased the immunogenicity of the virus.The results of mouse challenge experiments showed that there was no death in mice in the 10⁶TCID₅₀immunization group,all of which could provide 100%protection rate to mice,completely resistant to PRV epidemic strains.In the 10⁵TCID₅₀immunization group,the protection ratio of r PRV-AH-g I^-/g E^-/g B⁺inactivated vaccine to mice was 75%,and the protection ratio of r PRV-AH-g I^-/g E^-inactivated vaccine to mice was 87.5%.In summary,r PRV-AH-g I^-/g E^-has good immunogenicity and has a highly effective immune effect against PRV strains.It can be used as a vaccine candidate for pseudorabies,and its immune titer is further evaluated in piglets.