| Pseudorabies,also known as Aujeszky’s disease,is an infectious disease of swine caused by Pseudorabies virus.The main clinical symptoms are reproductive disorders of sows,abortion,mummified fetus,stillbirth or weak litter of pregnant sows.Piglets have high fever and nervous symptoms,and even die of exhaustion,with high mortality.The boars’ reproductive system is weakened.Due to the characteristics of fast spread,high mortality and wide range of infection,it is difficult to completely eradicate the disease,which seriously hinders the healthy development of pig industry.Therefore,the establishment of a serological diagnostic method for PR combined with vaccine immunization is the main means to control or even eradicate PR.Research have shown that PRV gE protein is a major glycoprotein and a major virulence protein encoded by gE gene.As gE protein is a non-essential protein for viral replication,the deletion of gE gene will not affect the immunogenicity of the virus.Therefore,gE gene deletion vaccine has been widely developed and used.Before 2011,China effectively controlled the epidemic of PR through the use of Bartha-K61 vaccine.However,in recent years,due to the emergence of PRV variant strains,Bartha-K61 vaccine can no longer provide fully effective protection,and PR breaks out again in many pig farms,seriously affecting the healthy development of pig industry.At present,immunity of gene deletion vaccine has become the main means to eradicate and purify PR,and enhancing the differential diagnosis of PRV-infected pigs has become the key to eradicate PR.Therefore,PRV gE monoclonal antibody was prepared and used to detect PRV-infected cells and tissues and identify gE antigen epitopes.This study lays a foundation for understanding the biological information of gE protein,establishing rapid and specific diagnostic methods for PR and immunoprophylaxis.At the same time,it also lays a good foundation for the development and production of vaccines,and contributes to the eradication and purification of PR.In this study,inactivated PRV HN-1201 was used as the immunogene-immunized BALB/c mice.The mice with good immune effect were screened by indirect enzyme linked immunosorbent assay.Spleen cells of immunized mice were fused with SP2/0 cells by cell fusion technique.Two monoclonal cell lines secreting gE antibody,named 1-F11-3 and 3-E11-3,were screened by immunoperoxidase monolayer cell assay and Western blot assay combined with cell subcloning technique.The number of chromosomes of established monoclonal cell lines was analyzed.The number of chromosomes of monoclonal cells was 95～105,which was equal to the sum of the number of chromosomes of SP2/0 cells and spleen cells.The subclasses of 1-F11-3 and 3-E11-3 antibodies were identified by ELISA as IgG2a and IgM,respectively,and the light chain is κ chain.The highest dilution of purified antibody for Western blot detection was 1:14000 and 1:12000 respectively,and the indirect immunofluorescence titer of 1-F11-3 was 2-14.The antibody could be used for Western blot and IFA detection with high sensitivity.The IFA test showed that the 1-F11-3 antibody did not cross-react with common swine pathogens,and did not react with gE gene deficient PRV vaccine strains,indicating strong specificity of the antibody.Immunohistochemical test confirmed that the monoclonal antibody could be used to detect virus infection in animal tissues.gE truncated eukaryotic expression plasmid was constructed by progressively truncated gE sequence.Using the monoclonal antibodies 1-F11-3 and 3-E11-3,by Western blot test detection PRV HN-1201 gE antigen epitope sequences are 64GDDRRAGFGSALASLR79 and 156PPEVPRLRRGPPIVTPERW S175 respectively.In conclusion,this study successfully prepared gE monoclonal antibody with good reactivity,which laid a foundation for the study of gE protein structure and function,epitope analysis,and the development of an immunological diagnostic kit for detecting gE antigen. |
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