| Marek's disease(MD),caused by avian herpesvirus,Marek's disease virus(MDV),is a highly tumorigenic herpesvirus disease,which can induce lethal T-cell lymphoma.It is also a natural virus-induced tumor model.Existing vaccines can prevent tumors caused by MDV,but cannot effectively prevent the spread or infection of MDV,which has led to the emergence of more and more virulent strains.Meq protein is related to the incubation period,transformation and evolution of MDV.The expression of Meq protein is the key to the transformation of T cells into lymphoma cells.At present,there are not only attenuated vaccine strains but continuously evolving wild strains in immunized chickens,and even vvMDV and vv+MDV that cannot be protected by vaccines,which brings difficulties to the prevention and treatment of MD.Because the avian leukemia virus can also cause tumors in infected chickens,which make difficult to distinguish them in histology.So it is necessary to establish an accurate and specific detection method to detect tumors caused by MDV.In this study,we used the baculovirus expression system to express the Meq oncoprotein,and then used the prepared anti-Meq monoclonal antibody to initially establish an immunohistochemical detection method for the diagnosis of lymphoma caused by MDV.1.Cloning and expression of Marek's virus meq geneIn this study,specific primers of meq gene are synthesized based on the data published in Genbank.The meq gene of Marek's disease virus RB1B strain was amplified by PCR.The purified PCR products were ligated into the pCAGGS and the pFastBacHTA and then we constructed the eukaryotic recombinant plasmid pCAGGS-Meq and the recombinant plasmid pFastBacHTA-Meq respectively.The correct pCAGGS-Meq plasmid was transfected into 293T cells and analyzed by IFA and Western blot.The results show that the pCAGGS-Meq can express in 293T cells.After that the recombinant pFastBacHTA-Meq plasmid was transposed into DH10Bac competent cells to obtain the recombinant shuttle plasmid rBac-MDV-Meq.The recombinant shuttle plasmid was transfected into Sf9 cells to obtain recombinant baculovirus rBacMDV-Meq.Then the virus-infected Sf9 cells were analyzed by IFA and Western blotting.The results showed that the Meq protein expressed by recombinant baculovirus is soluble and non-secreted in the nucleus,with a molecular weight of about 60 kDa.2.Preparation of monoclonal antibodies to Meq of Marek's Disease VirusIn this study,the Meq protein expressed by the recombinant baculovirus rBac-MDV-Meq was used as the immune antigen to immunize BALB/c mice aged 6-8 weeks.Immunization is performed every 10 days.After 3 times,the lymphocytes of the mice with high titer were fused with SP2/0 cells.Screened with 293T cells transfected by pCAGGS-Meq through IFA detection,four hybridoma cell lines that can secrete monoclonal antibodies against Meq were obtained.These hybridoma cell lines were named:MDV-Meq-1D6,MDV-Meq-1D10,MDV-Meq-6B3 and MDV-Meq-6D10.After verification by Western blot,it was proved that the four monoclonal antibodies can react specifically with the eukaryotic expressed protein.Subclass identification showed that 3 of the 4 monoclonal antibodies were the heavy chains were IgG1,the other one was IgG2a.The light chains of 4 monoclonal antibodies were all Kappa chains.The mice ascites of 4 hybridoma cells were prepared,the titer of the four monoclonal antibodies was about 1:3200 in IFA.3.Establishment of immunohistochemical method based on Meq protein Malik's diseaseThis study is based on the prepared monoclonal antibodies to meq protein of MD and optimizes multiple steps in the immunohistochemistry steps,such as antigen retrieval,monoclonal antibody dilution,DAB color development and hematoxylin counterstaining time.This method can detect Meq protein in heart and liver tumor tissues artificially infected by MDV RB1B.The staining shows that the antigen is mainly located in the nucleus,which provides a reference for the diagnosis and prevention of MDV in the future. |
PDF Full Text Request