| The innate immune system plays a very important role in pathogen recognition and activation of protective immune response through the recognition of pathogen-associated molecular patterns(PAMPs)by its pattern recognition receptors(PRRs),which will influence and shape the subsequent adaptive immune response against microbial infections.Nucleic acids including RNA and DNA have been recognized as significant PAMPs especially for viruses.RNA viruses pose a serious challenge to human and animal health;therefore the study on immune biology of RNA receptors is critical to control viral infections.RNA receptors consist of TLR3,TLR7,TLR8,RIG-I,MDA5,NLRP3,NOD2 and a few other minorities.This study focuses on double-stranded RNA(dsRNA)recognition receptor TLR3 of porcine(pTLR3),which includes cloning and expression of pTLR3,identification of its signaling function;establishment of pTLR3-NF-kB signaling reporter cell line and application for screening of African swine fever virus(ASFV)regulatory genes;analysis of signal-to-function interplay between pTLR3 and pRIG-I/pMDA5;and finally the preparation of pTLR3 monoclonal antibody for downstream studies.The main research contents are as follows:1.Molecular cloning,protein expression and signal function of porcine TLR3The porcine TLR3 gene(pTLR3)was amplified by RT-PCR from the total RNA of porcine alveolar macrophages,and the purified PCR product from digestion of Sal I and Sma I was ligated with the vector pENTR4-MCS-2HA digested with Sal I and EcoR V to construct the intermediate shuttle plasmid pENTR4-pTLR3-2HA.Sequence analysis revealed that the full length of the porcine TLR3 open reading frame was 2737 bp.The pTLR3 was transferred into the pcDNA-DEST47 vector by LR site-specific recombination,and the eukaryotic expression plasmid pcDNA pTLR3 was constructed.After transfection of pcDNA pTLR3 into HEK293T cells,the correct protein expression of pTLR3 was determined by Western blotting.In the ISRE promoter luciferase reporter assay,porcine TLR3 was able to significantly mediate downstream transcription factor IRF3 activation by the ligand dsRNA analog HMW polyI:C stimulation,indicating that pTLR3 has signal function.2.Establishment of porcine TLR3-NF-?B signal stable reporter cell line and screening of African swine fever virus(ASFV)regulatory multi-gene family(MGF)proteinsThe pTLR3-293-NF-?B cell line stably expressing pTLR3 was obtained by transfecting pcDNA pTLR3 into the existing 293-NF-?B dual luciferase signal reporter cells.This stable cell line is capable of producing a highly efficient,specific and stable response to the dsRNA analog high molecular weight(HMW)polyI:C stimulation,therefore suitable for the screening of TLR3-NF-?B signaling pathway regulators.The above pTLR3-NF-?B together with the existing human TLR3/RIG-I-ISRE signaling reporter cell line were used to screen for the signaling regulators from the African swine fever virus(ASFV)genes.The ASFV plasmids were transfected into the reporter cells and stimulated with different stimulators.The dual luciferase assay was used to detect downstream reporter signals and screen for the cell signaling regulators.The results showed that ASFV multi-gene falily(MGF)regulatory proteins 12L,13L,UK,A276R,A528R can inhibit pTLR3-NF-KB and TLR3-ISRE ceil signaling pathways;12L and UK inhibit RIG-I/MDA5-ISRE signaling;while these ASFV proteins have no significant effect on IFN-ISRE signaling.ASFV structurally related proteins P30 and P54 significantly inhibited dTLR3-NF-kB activity,but had no significant effect on pTLR3-ISRE,RIG-I/MDA5-ISRE and IFN-ISRE signaling.Importantly,we found that the cytotoxicity of both P30 and P54 are very significant.P54 is an ASFV internal membrane protein,and its apoptotic function has been reported.The cytotoxic effect of P30 has not been reported,deserved for further investigation.3.Investigation of immune signaling relationship between porcine TLR3 and porcine MDA5/RIG-IThe gRNAs of pTLR3 were designed using CRISPR-CAS9 technology,the lentiviruses expressing the gRNAs were packaged,and used to infect PAM and PK15 cells.After puromycin selection,pTLR3 knockout stable cells,TLR3 KO PAM and TLR3 KO PK15 cells were obtained,the above cells were stimulated with HMW/LMW polyI:C,EMCV,VSV,SeV and downstream gene expression were examined.Quantitative(q)RT-PCR results showed that TLR3 knockdown enhanced the expression of downstream genes of RIG-I/MDA5 stimulated by LMW polyI:C,EMCV,VSV and SeV,suggesting that pTLR3 can inhibit pRIG-I/MDA5 signaling.At the same time,using the pRIG-I KO,pMDA5 KO PAM and PK15 cells constructed previously in our laboratory,the expression of HMW polyI:C-stimulated TLR3 downstream genes was weakened,suggesting that both pRIG-I and pMDA5 can enhance the pTLR3 signaling.On the other hand,different concentrations of pTLR3 together with pRIG-I or pMDA5 were co-transfected into 293T cells,and RT-qPCR and ELAM promoter experiments showed that pTLR3 can inhibit gene expression and NF-kB activity downstream of RIG-I or MDA5.Conversely,different concentrations of pRIG-I or pMDA5 together with pTLR3 were co-transfected into 293T cells.The results showed that both pRIG-I and pMDA5 enhanced gene expression and ISRE promoter activity downstream of pTLR3.MAYS is a signal adaptor protein downstream of RIG-I and MDA5.Different concentrations of pMAVS together with pTLR3 were co-transfected into 293T cells,which also showed that MAYS enhanced the ISRE promoter activity downstream of pTLR3.These results are consistent with the results of experiments on PAM and PK15 KO cells.Next,the effects of the molecular domains of pTLR3?pRIG-I and pMDA5 on other molecular signaling were further analyzed.The pcDNA eukaryotic expression plasmids of pTLR3 extracellular domain(pTLR3 ECD),deletion of extracellular domain(pTLR3 AECD),pRIG-I/MDA5 2CARDs deletion region(pRIG-I ?2CARDs,pMDA5 ?2CARDs)were constructed.Different concentrations of pTLR3ECD or pTLR3 ? ECD together with pRIG-I/MDA5 were co-transfected into 293T cells.The results of RT-qPCR and ELAM promoter assay showed that pTLR3 ECD can inhibit gene expression and NF-?B activation downstream of pRIG-I and pMDA5.Conversely,different concentrations of pRIG-I 2CARDs,pRIG-I A 2CARDs,pMDA5 2CARDs or pMDA5 ? 2CARDs together with pTLR3 were co-transfected into 293T cells.The results of RT-qPCR and ISRE promoter assay showed that both pRIG-I 2CARDs and pMDA5 2CARD can enhance the downstream gene expression and the activity of ISRE promoter of pTLR3.These results indicated that the full length of pTLR3 and its ECD can inhibit the NF-?B activity and downstream gene expression of pRIG-I/pMDA5,but the effect of pTLR3 ?ECD on pRIG-I/pMDA5 signaling is not significant.The full length of pRIG-I and pMDA5 and their 2CARDs enhanced the expression of pTLR3 downstream gene and ISRE activity,while pRIG-I?2CARDs and pMDA5 ?2CARDs had no effect on TLR3 activity.4.Preparation of porcine TLR3 monoclonal antibodyThe pENTR4-pTLR3 intermediate plasmid was specifically recombined with the pDest527-cDNA vector using the LR reagent to construct a prokaryotic pTLR3 expressing plasmid.After induction by IPTG,and the optimal condition for the highest expression level of inclusion body was determined:the concentration of IPTG was 1 mM,the temperature was 37?,and the time was 2 h.Under this condition,a large amount of cultured bacteria was used for protein purification,and the pTLR3 was purified from the inclusion body.BALB/c mice were immunized to prepare for monoclonal antibody production.The positive hybridoma cells were identified by ELISA to obtain a cell line capable of stably secreting pTLR3 antibody after three rounds of limiting dilution and identificaton.The secreted antibody has the titer up to 1:102400 in ELISA and can recognize endogeneous pTLR3 in Western-blotting. |
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