Cloning And Expression Of Cyprinid Herpesvirus 3 Encoded Tumor Necrosis Factor Receptor Gene And Preparation Of Polyclonal Antibody

As a pleiotropic cytokine,tumor necrosis factor(TNF)can participate in the body’s immune response after interacting with specific tumor necrosis factor receptor(TNFR).Some viruses adopt strategies that encode cytokines and cytokine receptors similar to escape host immune surveillance and clearance.It has been confirmed that poxviruses and herpesviruses encode viral tumor necrosis factor receptor(vTNFR).Whole genome sequencing analysis confirmed cyprinid herpesvirus 3(CyHV-3)ORF4 and ORF12 can encode vTNFRs,but the role of vTNFR in viral immune escape remains to be studied.In this paper,the CyHV-3 vTNFRs gene was cloned and named vTNFR4 and vTNFR12,respectively,collectively called vTNFRs.Recombinant vTNFRs(rvTNFRs)protein was obtained through prokaryotic expression system,the effect of the protein on the expression regulation of host cell immune related factors and cell apoptosis was studied,and polyclonal antibodies were prepared.(1)CyHV-3 vTNFRs gene cloning and expressionCyHV-3 ORF4 and ORF12 gene sequences were amplified by PCR.The target sequence was cloned into the prokaryotic expression vector p ET-32a(+),transformed into Transetta(DE3)competent cells,and then induced by IPTG.Then,it was purified according to the operation of the Bio-Rad medium-high pressure chromatography system to obtain a recombinant ORF4 protein(rvTNFR4)with a size of about 53 k Da and a recombinant ORF12 protein(rvTNFR12)of 32 k Da,collectively called rvTNFRs.(2)Study on the activity of CyHV-3 vTNFRs proteinIn order to study the effect of CyHV-3 vTNFRs on the expression level of immune-related factors,the recombinant vTNFR(rvTNFR)protein was used to stimulate head kidney-derived macrophages from common carp,and the expression of immune-related factors was detected by fluorescent quantitative PCR.The results show that high concentrations(20ug/m L)of CyHV-3 rvTNFRs can up-regulate the expression of immune-related genes,especially interleukin-6(IL-6),interleukin-8(IL-8),interleukin-12(IL-12),Interleukin-1β(IL-1β),major histocompatibility complex I(MHC I)and CXC chemokine a(CXCa).And this regulation is usually the most significant at 12 or 24 hours after stimulation.In order to study whether rvTNFRs protein can promote the apoptosis,EPC cells were stimulated with rvTNFRs protein,and apoptosis was detected by light microscopy,Hoechst33342 staining and flow cytometry.Light microscopy and Hoechst33342 staining revealed that rvTNFRs can cause apoptosis in EPC cell.Flow cytometry analysis further confirmed this conclusion,and found that the apoptosis rate caused by vTNFR4 was higher than vTNFR12.(3)Preparation of Polyclonal Antibodies against CyHV-3 rvTNFRsThe obtained rvTNFRs was injected subcutaneously into mice according to the routine immunization procedure.After three immunizations,mouse serum was collected to obtain a mouse-derived polyclonal antibody.The specificity of polyclonal antibody serum was confirmed by Western Blot,and the titer of polyclonal antibody serum was initially determined by agar diffusion test.The titer of rvTNFR4 murine polyclonal antiserum is 1:2,and the titer of rvTNFR12 murine polyclonal antiserum is 1: 4.In this experiment,CyHV-3 vTNFRs gene was successfully cloned and expressed,and purified rvTNFRs protein was obtained.The research confirmed that CyHV-3 vTNFRs can up-regulate the expression of some immune-related factors and induce the apoptosis of EPC cells,providing a theoretical and experimental basis for further study the mechanism of CyHV-3 vTNFRs in the process of virus invasion to the host.In addition,the prepared vTNFRs protein polyclonal antibody also provides the basis for the establishment of ELISA,indirect immunofluorescence and other detection methods.