Cloning And Expression Of Toll-like Receptor 7 And 9 Gene In Squaliobarbus Cunrriculus

Barbel chub(Squaliobarbus curriculus)commonly known as wild grass carp,with grass carp are belong to the subfamily Leuciscus,squaliobarbus.Barbel chub has better resistance than grass carp anti GCRV reovirus invasion characteristics,Toll like receptors(TLRs)is the most important fish innate immune response pattern recognition receptors(PRRs).Toll like receptor 7(TLR7)and Toll like receptor(TLR9)gene,a major member of the TLR7 subfamily,play an important role in antiviral immune response.(1)The full-length sequence of TLR7 gene(ScTLR7)was cloned by RACE-PCR,and its structural characteristics were analyzed by bioinformatics method.Real-Time qPCR technique was used to analyze the expression of ScTLR7 immune response GCRV.The results showed that the full length cDNA of TLR7 gene was identified by GenBank accession number KY472228.The results showed that the full length of ScTLR7 cDNA was 3715 bp,including 3162 bp of open reading frame(ORF),encoding 1053 amino acid residues,5’-untranslated region(UTR)403 bp and 3’-UTR 150 bp.The relative molecular weight is 121.70 kD and the theoretical isoelectric point is 7.88.Through the I-TASSER forecasting barbel chub TLR7 protein tertiary structure showed that ScTLR7 protein was a typical horseshoe-shaped structure,including 28 α-helices and 26 β-sheets.The results of SMART domain prediction showed that ScTLR7 had typical structural features of TLRs,which consisted of N-terminal signal peptide,15 leucine domain(LRRs),transmembrane domain(TM)and Toll / interleukin-1 receptor Domain(TIR).The phylogenetic tree of TLR7 amino acids showed the closest relationship with ScTLR7 was megalobrama amblycephala.The phylogenetic tree of TLR7 amino acid showed that the amino acid sequence of TIR domain of TLR7 gene was highly conserved,but not found three conserved boxes Box1,Box2 and Box3 in the TIR with the ligand joint.TLR7 was expressed in 9 tissues of barbel chub,TLR7 expression in the kidney and spleen of barbel chub was the highest,the expression pattern of TLR7 in grass carp and barbel chub head kidney was similar to response to GCRV,and the relative expression of ScTLR7 and CiTLR7 reached the peak at 96 h.(2)The full-length cDNA of TLR9 gene was cloned by RACE-PCR,and its structural characteristics were analyzed by bioinformatics method.Real-Time qPCR technique was used to analyze the expression of ScTLR9 immune response GCRV.The results showed that the full length c DNA of TLR9 gene was identified by GenBank accession number KU955845.The results showed that the full length of ScTLR9 cDNA was 3687 bp,including the open reading frame(ORF)of 3180 bp,encoding 1059 amino acid residues,5’-untranslated region(UTR)128 bp and 3’-UTR 379 bp.The relative molecular weight of the deduced protein was 121.61 kD and the theoretical isoelectric point was 8.91.The tertiary structure of TLR9 protein was predicted by I-TASSER.The ScTLR9 protein was a typical horseshoe-shaped structure,including 22 α-helices and 12 β-sheets.The SMART domain prediction results show that ScTLR9 consists of N-terminal signal peptide,16 leucine domain(LRRs),transmembrane domain(TM)and Toll / interleukin-1 receptor domain(TIR)typical structural features of TLRs.The phylogenetic tree of TLR9 amino acid showed that the amino acid sequence of TIR domain of TLR9 was highly conserved and the TIR domain of ScTLR9 was highly conserved in the TLR9 TIR domain of different species.Contains three highly conserved regions: Box 1(YDAFVVFD),Box 2(LCLEDRDWLPG),and Box 3(FWNNL).TLR9 was expressed in 10 tissues of barbel chub.TLR9 expression in the kidney and middle kidney of barbel chub was the highest,and the expression level of TLR9 in barbel chub and grass carp after GCRV infection showed that the expression pattern of TLR9 in grass carp and barbel chub head kidney showed a significant difference.In response to GCRV,ScTLR9 had shorter response time than CiTLR9,and the response rate of ScTLR9 reached the peak value,and the relative expression level of ScTLR9 was much higher than grass carp.(3)By building the barbel chub toll-like receptors prokaryotic expression vector of ScTLR9,named pET-30a(+)TLR9.The recombinant plasmid pET-30a(+)TLR9 was transformed into BL21(DE3),Rosetta,BL21(DE3)plysS into the host protein of Toll-like receptor 9 gene of barbel chub.BL21(DE3)plysS was the most suitable host bacteria.By IPTG induction expression showed that the optimal conditions were as follows: 0.25 mM IPTG,4h,BL21(DE3)plysS.Through the analysis of the soluble of recombinant fusion protein showed that the target protein is soluble.By SDS-PAGE and Western blot analysis showed that ScTLR9 size is about 82 kD protein,consistent with expected results.The above results laid the foundation for the study of the function of Toll-like receptor 9 gene in barbel chub.