| Innate immunity is the body’s first defense barrier.It recognizes various pathogen-associated molecular patterns（PAMPs）through a series of pattern recognition receptors（PRRs）,and then initiates the cellular innate immune response.Therefore,PRRs play an important role in innate immunity.PRRs mainly include Toll-like receptors（TLRs）,RIG-I-like receptors（RLRs）,NOD-like receptors（NLRs）,C-type lectin-like receptors（CLRs）and cytoplasmic DNA receptors（CDRs）.Among them,RIG-I-like receptors（RLRs）are cytoplasmic double-stranded RNA（dsRNA）recognition receptors,consisting of RIG-I,MDA5 and LGP2,which can mediate or affect type I interferon（IFN）responses thus initiates an antiviral,especially anti-RNA virus immune response.RIG-I and MDA5 have similar structures.RIG-I can preferentially recognize short double-stranded dsRNA（<300 bp）and dsRNA containing a triphosphate group at the 5’ end（5’-ppp-dsRNA）,while MDA5 can preferentially recognize long-chain dsRNA（>1000 bp）.Activated RIG-I and MDA5 recruit TRAF3 and TRAF6 to form a signaling complex by binding to mitochondrial antiviral signaling protein（MAVS）.The signaling complex activates the downstream protein kinases TBK1 and IKKs,which activate the transcription factors IRF3 and NF-κB,finally produce IFNs and pro-inflammatory cytokines,which plays an antiviral role.Compared with RIG-I and MDA5,LGP2 lacks the N-terminal tandem CARD signaling active region and cannot activate downstream signals,but LGP2 can regulate RIG-I and MDA5-mediated IFN signaling through stronger dsRNA binding ability,thereby affecting antiviral effect.RLRs also play a key role in recognizing and defending against animal virus infection,but the research on animal RLRs including RIG-I,MDA5 and LGP2 is limited,mainly due to the lack of specific detection antibody tools.Therefore,it is particularly important and urgent to develop specific monoclonal antibodies against RLR proteins of livestock and poultry.In this study,the prokaryotic system was used to express the porcine RLR LGP2 protein,and its specific monoclonal antibodies were developed by immunizing BALB/c mice,and the systemic characteristics and functions of the prepared porcine LGP2,RIG-I and MDA5 monoclonal antibodies were identified.The purpose was to provide research tools for the in-depth study of the molecular structure,tissue and cellular localization of porcine RLR proteins,and their interaction mechanism with viruses,and to lay the foundation for the establishment of fast and simple detection methods.The main research contents were as follows:1.Procine LGP2 gene cloning,expression and protein purificationTotal RNA was extracted from porcine macrophages（3D4/21）,and the encoding gene of porcine LGP2 was amplified by RT-PCR.The recombinant intermediate plasmid was obtained by ligating pLGP2 with the intermediate vector pENTR4-MCS-2HA.The above recombinant intermediate plasmid was site-specific recombination（LR）with the target eukaryotic expression vector pDSET47 to obtain the recombinant eukaryotic expression plasmid pDEST47-pLGP2-2HA.Western-blot（WB）was used to confirm the correct protein expression of the recombinant plasmid in transfected 293T cells,which provided experimental materials for later monoclonal antibody screening.At the same time,the above recombinant intermediate plasmid was site-specifically recombined（LR）with the target prokaryotic expression vector pDSET527 to obtain the recombinant prokaryotic expression plasmid pDEST527-pLGP2-2HA,which was transformed into DE3/BL21 competent Escherichia coli（E.coli）for prokaryotic expression and protein purification.The optimal condition for prokaryotic expression of pLGP2 was 1mM IPTG,induced at 37℃ for 6-9 h.WB analysis with tag antibodies HA and His showed that the molecular weight of the protein expressed by pLGP2 was 80 kD,which was in line as expectation,and the protein was mainly expressed in bacterial inclusion bodies.The pLGP2 protein in the inclusion bodies was purified by urea solubilization and concentration gradient dialysis.After Coomassie brilliant blue staining and comparison with the standard BSA protein,the purified protein of pLGP2 was obtained at a concentration of 1 mg/mL and a total of about 20 mg.2.Preparation of porcine LGP2 monoclonal antibodyBALB/c female mice aged 6-8 weeks were immunized with purified pLGP2 protein according to the optimal immunization program,and blood was collected from the tail vein and serum was separated before cell fusion.Serum antibody titers were determined by ELISA,and the immunized mice with the highest serum titers were selected for booster immunization and subsequent cell fusion experiments.The spleen cells of the immunized mice were fused with myeloma cells（SP2/0）to obtain hybridoma cells.The antibody titer in the supernatant of hybridoma cells was detected by ELISA,and the positive hybridoma cell lines with P/N value≥2.1 were screened based on the OD450 value.The reactivity of the supernatant of ELISA-positive hybridoma cells with the eukaryotic expression of pLGP2 was further detected by WB.The results showed that the supernatant of the two pLGP2-positive hybridoma cells and the sera of immunized mice could specifically recognize the pLGP2 protein,suggesting that the pLGP2 monoclonal antibodies were successfully prepared.LGP2 monoclonal antibody ascites was prepared by intraperitoneal inoculation of 2 strains of pLGP2-positive hybridoma cells with 1-3×106（0.3 mL）cells to provide research materials and tools for the study of pLGP2 protein-related functions.3.Identification and application of porcine RIG-I,MDA5 and LGP2 monoclonal antibodiesThe antibody subtype identification kit was used to detect the subtypes of the two pLGP2 monoclonal antibodies prepared above and the pRIG-I and pMDA5 monoclonal antibodies previously prepared by our laboratory.The results showed that the four monoclonal antibodies were all IgG2b subtype.WB results showed that pRIG-I,pMDA5 and pLGP2 mAbs could recognize the corresponding proteins in transfected cells and endogenous stimulated proteins,but there was no cross-recognition among them.The pcDNA-2HA recombinant expression plasmid of the N-terminal 2CARDs region and the C-terminal Δ2CARDs region of pRIG-I/pMDA5 was cloned and constructed.The above recombinant plasmids were transfected into 293T cells,and the reactivity of RIG-I and MDA5 monoclonal antibodies with the expressed proteins was detected by WB.The result showed that both RIG-I and MDA5 monoclonal antibodies recognized their C-termianlΔ2CARDs regions.The N-terminal helicase ATP-binding domain（L1）,intermediate helicase CTER domain（L2）and C-terminal CTR domain（L3）recombinant pcDNA-2HA expression plasmids of pLGP2 were transfected into 293T cells,respectively.The reactivity of L1,L2 and L3 showed that the two pLGP2 mAbs both recognized the N-terminal helicase ATP-binding domain（L1）.The human MDA5,RIG-I,LGP2 expression plasmids were transfected into 293T cells,respectively,and the reactivity of pRIG-I,pMDA5 and LGP2 mAbs was tested by WB.RIG-I and MDA5 mAbs have no cross-reactivity with human proteins;pLGP2 mAb（B9-2）has no cross-reactivity with human LGP2,while pLGP2 mAb（A1-3）has cross-reactivity with human LGP2.These results clearly showed that we obtained monoclonal antibodies highly specific to the three proteins of the porcine RLR family.The gene expression of pRIG-I,pMDA5 and pLGP2 in different tissues（heart,liver,spleen,lung,kidney,lymph node）of normal pigs and PRRSV-infected pigs were detected by real-time quantitative PCR.The results showed that compared with normal pigs,the expression levels of RLRs in other tissues except the heart were significantly increased in PRRSV-infected pigs,suggesting that RLRs played an important role in PRRSV infection.Different tissues of PRRSV-infected pigs were sliced,and immunohistochemical experiments were carried out using the prepared pRIG-I,pMDA5 and pLGP2 specific monoclonal antibodies.The results showed that pRIG-I,pMDA5 and pLGP2 were mainly expressed in the liver and kidney,followed by Lymph nodes and spleen,which are consistent with real-time PCR results. |
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