Construction Of Recombinant Pseudorabies Virus Expressing Cap Of Porcine Circovirus Type 3 And Preparation Of Monoclonal Antibodies Against GC?gG Of Pseudorabies Virus

Pseudorabies Virus(PRV)belongs to the genus Herpesvirus of the herpesvirus family.The natural host of the virus is pig,which can cause abortion of sows,stillbirth,and respiratory problems.Since 2011,pseudorabies has been re-emerging in China,causing huge economic losses to pig industry in China.And porcine circovirus type 3(PCV3)is an emerging pathogen of porcine viral infectious diseases that can produce dermatitis and nephropathy syndrome,reproductive disorders,and heart and multisystem inflammation in 2016.The viral capsid(Capsid,Cap)protein is the only structural protein that can stimulate animals to produce serum antibodies against PCV3,and is an ideal antigenic protein for the development of genetically engineered vaccines.So far,PRV and PCV3 co-infections are common in veterinary clinics,so the development of recombinant vaccine to prevent double infections of PRV and PCV3 is particularly urgent.In this study,the type ? PRV HD/c strain was used as the parent strain,and then the non-structural protein genes gC and gG were selected as target sites.Specific primers were designed to amplify the homologous arms flanking these two genes,and simultaneously construct fluorescent expression cassette expressing GFP and PCV3-Cap expression cassette with CMV promoter.Classical molecular biology methods were used to sequentially ligate specific gene fragments to the pUC18 vector.A homologous recombination transfer vector targeting two sites was constructed and verified by sequencing.The plasmid can stably express the PCV3 Cap protein at the cell level,providing an important molecular tool for the subsequent construction of a recombinant PRV expressing the PCV3 Cap.At the same time,using the type ? PRV HD/c genome of the laboratory as a template,the truncated sequences of the envelope protein gC and gG genes were amplified,and the main antigenic region of gC and gG were expressed prokaryotically by using the pET-28a(+)vector.The recombinant His-gC and His-gG proteins by denaturing purification were used as immunogens to immunize BALB/c mice and New Zealand white rabbits,respectively,so that we can obtain highly reactive polyclonal antibodies identified by WB and IFA.Meanwhile,spleen cells of the immunized mice and myeloma cells were taken for cell fusion,followed by subcloning screenings for four times using the limiting dilution method.Then,three monoclonal hybridoma cell lines capable of stably secreting antibody against the gC and gG protein of PRV were obtained,respectively.WB and IFA tests showed that the two kind of successfully prepared monoclonal antibodies reacted well with exogenous transfection samples and PRV-infected samples,and preliminary identification of antigenic epitopes was also performed.And the use of monoclonal antibodies against gC can be used for clinical detection of PRV vaccine strains and epidemic strain,and the preparation of monoclonal antibodies against PRV gC and gG protein also provides basic tools for further research of its biological function and identification of gene-deleted vaccine strains.