Cloning,Molecular Characterization And Expcession Pattern Of Chicken Innate Immune Receptor NLRX1

The NLR,(Nucleotide-binding domain and leucine-rich repeat-containing receptors)family is not only involved in the identification of invasive pathogenic microorganisms,but also in the regulation of immune response.NLRX1(NLR family member X1)is one of important NLR,family members.In this study,a full-length cDNA sequence of chicken NLRX1(chNLRXl)was cloned at first.The molecular structure and biological function of chNLRX1 were studied through bioinformatics analysis,protein expression,cell localization,siRNA interference and inflammation induction.1.Cloning,prokaryotic expression and antibody preparation of chNLRXl geneComparative analysis of NLRX1 gene sequences of multiple species from NCBI,,the conservative sequence and 5' and 3' terminal sequence of chNLRXl were amplified by RT-PCR and RACE,respectively,using the total RNA reverse transcription cDNA of chicken lung tissue as template.The 3225 bp full-length DNA sequence(GenBank number:MH704638.1)and the longest open reading frame(ORF)were obtained by splicing.The homology analysis showed that chNLRXl had 53.2%-86.5%homology with NLRX1 gene of mammals,ducks and fish.The chNLRX1 gene predicted to encode 989 amino acids,containing relatively conserved NACHT and LRR domains.Molecular conformation analysis showed that chNLRX1 showed close to fish NLRX1 in spatial structure.The coding region(chNLRXl-ORF)and N-terminal 1-492 bp(chNLRX1-part)gene sequences of chNLRX1 were amplified by polymerase chain reaction(PCR)and inserted into the prokaryotic expression pET-32a(+)vector,then transfered into E.coli Rosseta competent cells.The chNLRX1-part/His fusion protein was obtained by IPTG induction.The chNLRX1-part/His fusion protein was purified and immunized New Zealand rabbits to prepare anti-chNLRX1 antibody.Western blot analysis showed that the antibody could specifically recognize chNLRX1 fusion protein.2.Eukaryotic expression and subcellular localization of chNLRX1The gene encoding chNLRX1 was amplified by PCR and inserted into eukaryotic expression vectors pmCherry-C1 and pEGFP-C 1.The recombinant eukaryotic expression plasmid chNLRX1 was constructed.The recombinant plasmid was transfected into HEK293T cells and chicken DF-1 cells mediated by liposome.The nuclei were stained and labeled with fluorescent dyes Hoechst 33342 and Mito Tracker Red.The chNLRXl was detected by fluorescence microscopy and Western blot chNLRX1 is mainly located in the mitochondrial region of the cytoplasm.This suggests that chNLRXl has a mitochondrial localization function similar to that of NLRX1 in other animals.3.Tissue distribution and induced expression of chNLRX1Total RNA was extracted from thymus,spleen,lung,liver,kidney and bone marrow of 15-day-old chickens and retrieved to prepare DNA.The relative quantitative RT-PCR method of SYBR Green I fluorescence was established to analyze the distribution of chNLRXl in tissues.The results showed that the expression of chNLRXl was relatively high in thymus and low in liver.Chicken DF-1 cells were treated with 10 ?g/mL LPS.The expression of chNLRX1 and inflammatory cytokines IL-6 and TNF-? in chicken DF-1 cells was detected by RT-qPCR at 0 to 24 h.The results showed that the expression of chNLRXl was significantly up-regulated at 2h and then down-regulated.The expression of IL-6 was down-regulated after 2 h,and the level of TNF-? was up-regulated until 24 h(compared with 8 h).Salmonella gallinarumin stimulates chicken HD 11 cells with cell-to-bacterial ratios of 1:50 and 1:100.The transcription levels of chNLRX1,NF-?B,TNF-?,IL-1?and IL-6 in HD11 cells were detected by RT-qPCR.The results showed that excessive bacteria stimulated HD 11 cells inhibited the transcriptional expression of chNLRXl.1:50 low-dose Salmonella increased the transcriptional level of chNLRXl in cells at 3 hours,then decreased,while 1:100 high-dose stimulation did not change chNLRXl until 5 hours.Inflammatory factors IL-1? changed significantly compared with IL-6 and TNF-?.IL-1? increased significantly at 5h of low-dose bacterial stimulation and 3-5h of high-dose bacterial stimulation.However,NF-?B was up-regulated only after 3h of low-dose bacterial stimulation.4.siRNA interference of chNLRX1 expressionThree siRNA interference sequences named sh-NLRX1-1,sh-NLRX1-2 and sh-NLRX1-3 were designed according to the transcript of chicken NLRX1 gene.The recombinant plasmid chNLRX1-GFP was transfected into HEK293T cells,and the cells were incubated with shRNA and sh-Ctrl mediated by adenovirus.After 36 hours,the interference effect was analyzed by microfluorescence and Western blot.The results showed that compared with sh-Ctrl interference,the fluorescence intensity of NLRX1 overexpression was significantly decreased in the three interference groups,especially sh-NLRX1-2.Western blot analysis showed that sh-NLRX1-2 had the best effect,and its interference efficiency reached 87%.In conclusion,this study cloned and analyzed the chicken innate immune receptor NLRX1 gene and its molecular characteristics for the first time.Anti-NLRX1 antibody was prepared by prokaryotic expression of NLRX1 protein.Using cell transfection and microfluorescence analysis,it was clear that chicken NLRX1 has mitochondrial localization function in eukaryotic cells.RT-qPCR was used to analyze the distribution of NLRX1 in six chicken tissues,which showed relatively high expression in thymus.LPS stimulation and Salmonella infection could significantly affect the expression of siRNA in chicken cells.Finally,siRNA interference mediated by adenovirus was screened.Our results will lay a foundation for further study on the biological function of chicken NLRX1.