Cloning Of TLR9 From Nibea Albiflora And Exploration Of Its Immune Related Mechanism

Nibea albiflora,as a relatively common commercial fish in the offshore waters of China,which is widely distributed in the Donghai Sea,the Huanghai Sea,the Bohai Sea and the Nanhai Sea.However,due to overfishing and severe damage to the marine environment,the wild resources of Nibea albiflora are gradually decreasing,and its breeding industry has gradually developed,but due to the deterioration of genetic traits and the breeding environment inevitably leads to the frequent occurrence of diseases in the farmed Nibea albiflora.Therefore,it revealing the anti-disease mechanism of the Nibea albiflora immune system is the key breakthrough point to solve the disease problem.Toll-like receptors(TLR)are type I transmembrane proteins,which are important pattern recognition receptors(PAMP recognization receptors,PRRs)for fish innate immunity,which can turn on immune cells by recognizing pathogenic substances that invade biological cells’response.TLR9 is an important member of the TLRs family.It can recognize the unmethylated cytosine-phosphate-guanine dinucleotide sequence(CpG DNA)in the genome of viruses and bacteria,and further recruit downstream MyD88 molecules to activate a series of transcription factors.Expression,thereby completing signal transduction and generating an immune response.Therefore,in this paper,the identification,phylogenetic analysis and related expression characteristics,subcellular co-localization and co-localization and interaction analysis of the yellow amberjack TLR9 and its downstream factor MyD88 were carried out.The main research results obtained are as follows:1.According to the transcriptome data of N.albiflora,the Toll-like receptor 9(named NaTLR9,GenBank accession number:MN125017.1)and myeloid differentiation factor MyD88(named NaMyD88,GenBank accession number:MN384261.1)were obtained.the full length of NaTLR9 was 3677 bp,its open reading frame was 3180 bp,encoding 1059 amino acids,the N-terminal contains a signal peptide(1-19aa)sequence,and the amino acid sequence contains 13 leucine Repeated enrichment region(LRRs),1 leucine enrichment region(LRR-TYP),1 leucine enrichment region(LRR-CT)and a TIR domain at the C-terminal;The full length of NaMyD88 was 1672 bp,its ORF region is 879 bp,and it encoded 292 amino acids.The N-terminal contains a death domain,a short connection domain in the middle,and the C-terminal contains a TIR domain.In the multiple sequence alignment,it was shown that the two molecules are highly conserved in special motifs and structural and functional regions.It was speculated that their immune functions should be similar in animals respectively.The phylogenetic tree analysis showed that the amino acid sequences of NaTLR9 and NaMyD88 were respectively.It had homology with TLR9 and MyD88 of other bony fishes,especially Miichthys miiuy and Larimichthys crocea.2.The analysis of the TIR domain and NaMyD88 of NaTLR9 showed that the NaTLR9-TIR-pEGFP fusion protein was mainly distributed in the cytoplasm of EPC cells.The NaMyD88-pDsRED fusion protein was expressed as red and enriched in the cytoplasm,the possibility of space interaction between them is inferred.Further,the interaction between NaTLR9 and NaMyD88 was verified by co-immunoprecipitation assay.The pCDNA3.1-FLAG-NaTLR9-TIR and pCMVMyc-NaMyD88-TIR plasmids were constructed and co-transfected into 293T cells,western blot was used to detect the recombinant myd88-TIR-Myc protein,and no protein bands were detected in the control group.thus,TLR9 and MyD88 can interact with each other through the TIR functional area to complete the signaling pathway.3.NaTLR9 and NaMyD88 had a wide range of tissue distribution characteristics.NaTLR9 and NaMyD88 could be detected in the spleen,liver,kidney,stomach,gills,and muscle,but the highest content of NaTLR9 in the kidney was 90 times that of muscle tissue,followed by the stomach,spleen,liver and gills.And NaMyD88 was also the highest expression in the kidney.Pseudomonas plecoglossicida and Poly(I:C)were used to stimulate N.albiflora.The test showed that NaTLR9 and NaMyD88 were expressed in the kidney tissue in a time-dependent manner.After P.plecoglossicida infection,the highest expression level at 2 h was 9.52 times that of the control group,and after being attacked by Poly(I:C),it peaked at 24 h(39.51 times higher than the control group).Similar to this,the expression trend of MyD88 was the same.It indicates that NaTLR9 might rely on NaMyD88 to play an important role in the immune signal pathway of N.albiflora against the invasion of foreign microorganisms.4.In vitro construction of recombinant proteins contained active functional domains of protein interaction efficiency,that was,the plasmids contained the functional domains(LRRs,TIR)of NaTLR9 were connected to the pET28a,and the functional domains of NaMyD88 linked to pET32a plasmids,respectively,and transformed into E.coli(BL21/DE3)was expressed,and the obtained target protein was expected molecular weight,it was purified by Ni-NAT Superflow kit.It was further sonicated crushed,separated and purified.then the supernatant and precipitate were obtained.SDS-PAGE detection showed that the expression products were inclusion bodies,which conformed to the predicted molecular weight,but the functional mechanism of the protein needed to be further explored.5.In order to verify whether pET28a-NaTLR9-LRRs recombinant protein and pET28a-NaTLR9-TIR recombinant protein had the ability to bind bacteria,this experiment selected three kinds of Gram-negative bacteria:Vibrio parahaemolyticus,Vibrio alginolyticus,and Vibrio harveyi were tested for bacterial and protein binding.The successfully constructed pET28a-NaTLR9-LRRs recombinant protein and pET28a-NaTLR9-TIR were expressed in E.coli BL21(DE3),and the inclusion body protein was isolated and purified,and then the protein was renaturation.The resulting protein and bacteria were lightly shaken at room temperature.After shaked for 1 hour,then washed and purified several times,and then performed Western blot to detect the result of protein bound to the bacteria,the NaTLR9-TIR recombinant protein and NaTLR9-LRR recombinant protein could be combined with V.parahaemolyticus,V.alginolyticus and V.harveyi.it indicated that NaTLR9 is a pattern recognition receptor.It could identify pathogenic microorganisms and play an important role in the innate immunity of organisms.The above experimental consequences showed that there was an interaction between NaTLR9 and NaMyD88,which proved that there might a TLR-NF-κB innate immune signaling pathway that depended on the MyD88 molecule in N.albiflora,which played an important role in antibacterial infections.Therefore,the results of this experiment would be in the future,it would allow a reference for studying the functional mechanism of natural immune molecules from the protein level,and at the same time,it would also afford theoretical guidance for revealing the healthy breeding of marine fish such as N.albiflora.