Construction Of Recombinant Pseudorabies Virus Expressing Porcine Circovirus Type 2 Can Protein

Porcine circovirus type 2(PCV2)can cause a variety of pig disease syndromes,among which the most serious is postweaning multisystem wasting syndrome(PMWS).Capsid protein(Cap)of PCV2 is an important immune protective antigen,which can induce the production of specific antibodies against PCV2 in the body.And it is an ideal target antigen to develope genetic engineering vaccine.Pseudorabies(PR),also known as Aujeszky's disease(AD),which is caused by pseudorabies virus(PRV).And it is difficult to eradicate PR in pig farms once infected.PCV2 and PRV have caused great harm to swine industry in China.So far there has been no effective bivalent vaccine against both PCV2 and PRV.Therefore,building up a recombinant pseudorabies virus strains expressing PCV2 Cap protein is very important for the upgrading of the above disease control technology.In this study,the gI(US7)gene,as a non-essential site,was selected as the insertion site,and TK and gE gene deleted HD/c strains were used as the parents.CRISPR/Cas9 technology was used in combination with classic homologous recombination technology to rescue the recombinant virus.Finally,we obtained a strain of TK-/gE-/gI-/Cap+ recombinant pseudorabies virus.Verification of the rescued recombinant virus showed that the cap gene of PCV2 had been correctly inserted into the genome of HD/c strain.Compared with the parental strain,there was no significant difference in the one-step growth curve and the size of plaque between the recombinant strain and the parental PRV HD/c strain,indicating that the insertion of PCV2 cap gene did not affect the replication of the recombinant virus.By Western Blot and indirect immunofluorescence test to verify the Cap protein expression,results show that the cap genes successfully inserted into the HD/c genome,but it does not successfully express the Cap protein,delete EGFP expression cassette is not influence the Cap protein expression.Literature review speculated that the possible reason was that the promoter dependent on PRV itself was not enough to activate the expression of exogenous genes,so it was necessary to strengthen the promoter to ensure the stable expression of exogenous genes.In order to provide the detection tool for recombinant pseudorabies virus,monoclonal antibody against gB glycoprotein in the envelope of pseudorabies virus was prepared in this study.The gB gene was amplified using pseudoranrabies virus strain HD/c as a template and constructed into a prokaryotic expression vector of pET-28a,and the expression of gB protein was induced by IPTG.Then BALB/c mice were immunized with purified gB protein,and the spleen cells of the immunized mice were fused with myeloma cells of the same strain.Positive hybridoma cells were screened by subcloning,and 3 monoclonal hybridoma cells were obtained which could secrete anti-gB antibody stably.The characteristics of gB monoclonal antibody were identified,and the results showed that the prepared gB monoclonal antibody had good Western Blot and indirect immunofluorescence reactivity with the prokaryotic expression gB protein,eukaryotic expression gB protein,and gB protein in the virus infected cells.The epitope regions recognized by the three monoclonal antibody cell were located in the 63-124 aa region.