Expression And Antibody Preparation Of Innate Immune Receptors NLRP3 And MDA5 Genes In Chick

The pattern recognition receptors play central roles in sensing pathogens and trigger innate immunity,the first line of defense against invading pathogens.NLRP3?NACHT,LRR and PYD domains-containing protein 3?receptor and MDA5?melanoma differentiation associated gene 5?receptor are belong to NLRs?Nucleotide-binding oligomerization domain like receptors?family and RLRs?Retinoicacid inducible gene like receptors?family,respectively.They can be activated by viral RNA,then mediate the NF-?B signaling pathway and trigger intracellular antiviral immune response.Unlike mammalian NLRP3 or MDA5,molecular characterization and regulation mechanism?s?of chick NLRP3 or MDA5 protein are unclear.Here,we studied NLRP3?chNLRP3?and chicken MDA5?chMDA5?receptor by gene cloning,expression and preparation antibody.Our results can provide a better understanding of chick NLRP3 and MDA5 for further explore the mechanism?s?of these receptors in immune response.Firstly,we designed three pair of specific primers for cloning chNLRP3 and chMDA5 gene coding sequences by RT-PCR or SOE PCR,respectively.The sequences were inserted into the cloning vector pEASY-Blunt and then transformed into E.coli strain DH5?.The recombinant plasmids were identified by PCR and double restriction enzyme digestion.Sequence similarity and homology were analyzed with DNAstar software.The results showed that CDS length of chNLRP3 and chMDA5 receptor were 2205 bp and 3306 bp,respectively.Then,the recombination plasmids were constructed.The highest similarity percentage of chNLRP3 amino acid sequence in our study was 95.2% with Chinese yellow chicken NLRP3?KF318520?.Compared with fish NLRP3?XM₀1504447?,the percentage of similarity is 4.8%.The homology of chMDA5 with MDA5 from jungle fowl?AB₃71640?is higher than the others'.The similarity percentage between them is97.2%,but is lower than other to with MDA5 of the grass carp?JN₉86720?.Secondly,we predicted antigen epitopes of chNLRP3 and chMDA5 proteins using the DNAStar software.The primers were designed for PCR amplification based on the epitopes from N-terminal 1-200 amino acids?aa?of chNLRP3 and N-terminal1-255 aa of chMDA5.Then,the amplification sequences of PCR were inserted into the prokaryotic expression vector pET-32 a.Purified fusion proteins following expression induced by IPTG were injected into a rabbit and antiserum were prepared,respectively.The results showed that we amplified a 600 bp fragment of chNLRP3 and a 765 bp fragment of chMDA5,and constructed prokaryotic expression recombinant plasmid pET-32a/chNLRP3 and pET-32 a/MDA5.Anti-chNLRP3 antibody and anti-chMDA5 antibody were prepared and the specificity of them was identified by ELISA and western blot assay.Finally,we constructed the recombination plasmids following sub-cloning the receptor genes into the eukaryotic expression vector pmcherry-N1 or pEGFP-N1.Then,the control plasmids and the recombination plasmids were transfected into DF-1 cells with lipofectamine 3000 reagent.The fusion protein expression was detected at 48 hours after transfection by fluorescence microscope,respectively.The cell lysis suspensions were detected with anti-chNLRP3 antibody or anti-chMDA5 antibody by western blot method.Here,the results showed that the recombination plasmids chNLRP3/Cherry-N1 and chMDA5/GFP-N1 were constructed and transfected into DF-1 cells.The over expression of chNLRP3 protein?108.6 kDa?or chMDA5?137.2kDa?protein were detected with fluorescence microscope images and western blot assay.In summary,we successfully constructed the prokaryotic expression recombinant plasmids of chick NLRP3 and chick MDA5 gene by gene clone.We prepared the antibodies for chNLRP3 and chMDA5 by immuning rabbits with purified fusion proteins.Moreover,the eukaryotic expression recombinant plasmid for chNLRP3 and chMDA5 were constructed and transfected into DF-1 cells.The over expression of chNLRP3 or chMDA5 were detected with prepared antibodies by fluorescence microscope images and western blot assay.The results will help for further detection the mechanism?s?of sensing pathogens and triggering immune response of NLRP3 and MDA5 in chick.