The Polyclonal Antibody Preparation Of Classical Swine Fever Virus NS5B And Swine Immune Response Factors TRIF, MYD88, MAVS And A Preliminary Exploration Of The Interaction Between Them

Classical Swine Fever (CSF) is an acute or chronic, fever and highly contagious infectious disease, which is caused by classical swine fever virus (CSFV). CSFV NS5B protein is a kind of non-structural protein with the RNA-dependent RNA polymerase (RdRp) activity. It is in charge of viral genome transcription and replication by binding to 3’untranslated region (UTR). We prepared anti-NS5B polyclonal antibody in order to further explore the interaction between CSFV NS5B and host immune response factors.CSFV could suppress the type Ⅰ interferon (IFN-Ⅰ) production and to escape the host immune system, result in a persistent infection. The induction of IFN-Ⅰ is regulated by Toll-like receptor (TLR) and RIG-Ⅰ-like receptor (RLR) signal transduction pathways. In the TLR signal pathways, adaptor molecules TIR domain-containing adapter inducing interferon-β (TRIF) and myeloid differentiation primary response gene 88 (MyD88) accept the signal of upstream acceptors and transfer the signal to the downstream molecules to cause a series of cascade reaction. And in the RLR signal pathways, adaptor molecule mitochondrial antiviral signaling protein (MAVS) activates the downstream moleculars transcription and expression. Currently, there is no anti-TRIF, MyD88 and MAVS antibodies for sail on the biological products market. Without these antibodies, it will be difficult to study CSFV infection and the interaction mechanism between CSFV and the host. So we prepared anti-TRIF, MyD88 and MAVS polyclonal antibodies in order to further explore the regulatory mechanism of TLR and RLR signal pathways during CSFV infection or laid the foundation for the research of immune escape mechanism of CSFV.In this study, the fragment of CSFV NS5B gene and host swine TRIF, MyD88 and MAVS gene ORF were amplified by RT-PCR. The prokaryotic expression vectors pET-NS5B(222), pET-TRIF(217), pET-MyD88(202) and pET-MAVS(220) were constructed. These prokaryotic expression vectors were transfected into an E.coli Rosetta (DE3) respectively to express the recombinant proteins via IPTG induction, then these recombinant proteins were purified by Ni-NTA affinity chromatography and other methods. The purified recombinant proteins were used to immunize the Kunming mouse to prepare polyclonal antibody. The reactivity and the specificity of these polyclonal antibodies were detected by Western Blot and indirect immunofluorescent assay (IFA). As the results, these polyclonal antibodies showed a high reactivity and specificity to the recombinant prokaryotic proteins in E.coli and to natural eukaryotic proteins in PK-15 cells with CSFV infection. The reactivity of the anti-CSFV NS5B polyclonal antibody with prokaryotic expressed CSFV NS5B was identified to reach a dilution of 1:8000, and other three polyclonal antibodies reached a higher dilution of 1:16000. The transient expressed NS5B protein in eukaryotic PK-15 cells with the eukaryotic vector pcDNA3.0-NS5BFL was detected by Western Blot and IFA. The best working dilution for IFA of the anti-CSFV NS5B polyclonal antibody was determined as 1:200。The best working dilution of the anti-CSFV NS5B, anti-TRIF, MyD88 and MAVS polyclonal antibodies reacted with their relevant proteins expressed in eukaryotic PK-15 cells with CSFV infection respectively were 1:2000,1:1000,1:500 and 1:1000.In this study, we dedected the change of the immune response factors TRIF、MyD88 and MAVS mRNAs and proteins under 1.0 MOI CSFV infection by quantitative real-time PCR (qPCR) and Western Blot. Taken together, we found that the TRIF mRNA were up-regulate little after CSFV infection, and the TRIF protein level did not show a obvious up-regulation. However, MAVS and MyD88 mRNA respectively showed a significant increase after 12 and 24 h of CSFV infection, the changes of protein level were consistent with their mRNAs. For these reasons, we speculate that the TLR and RLR signal pathways in PK-15 cells were activated by CSFV infection. The activated TLR signal pathway was mainly via the MyD88-independent signal transduction pathway.