Cloning Of Some Genes In TLR And NF-?B Signaling Pathway And Their Immune Responsecharacterizations In Large Yellow Croaker

The infectious disease is very serious in the commercial culture of large yellow croaker,Larimichthys crocea.The pathogens of large yellow croaker are very complicate.Therefore,to better understand its immune recognition and activation mechanism is very important for the development of healthy culture industry of large yellow croaker.In the present study,the full length cDNA sequences of TLR1,TLR2,TLR21 in TLR family,p65,p52,c-rel,I?B in NF-?B family,IL-11 and IL-21 in IL family were cloned from large yellow croaker by RT-PCR and RACE-PCR.Their molecular structures are analyzed and the tissue expression and temporal expression profiles of these genes in spleen,head-kidney and liver after the stimulation with LPS,poly I:C and pathogenic bacteria V.Parahaemolyticus parahemolyticus were investigated.In addition,their temporal expression profiles of these genes in LCK cellline after the stimulation with LPS,poly I:C,PGN and Flagellin were also described.We also obtained the recombinant expressin protein of MyD88 which is the key adaptor molecule of TLR pathway,and investigated the proteins interacting with MyD88 using GST-pulldown Technology.Furthermore,IL-1?,IL-21 recombinant expression products were obtained in E.coli.The results are as follows:(1)The some genes in TLR familyThe full-length cDNA of LcTLR1 was 3216 bp,containing a 39 bp 5' untranslated region(UTR),a 768 bp 3' UTR and a 2409 bp open reading frame(ORF)which encoded 802 amino acids,containing LRR domain,the transmembrane region domain and TIR domain.A broad expression of LcTLR1 in most detected tissues,with the predominant expression in blood,heart,spleen and kidney and weak expression in skin,intestinal and stomach is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,LcTLR1 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS poly I:C PGN and Flagellin,LcTLR1 expression levels also showed up-regulation in LCK cellline(p<0.01).Poly I:C could not make LcTLR1 transcripts show up-regulation in the LCK cellline.The full-length cDNA of LcTLR2 was 2802 bp,containing a 135 bp 5' untranslated region(UTR),a 189 bp 3' UTR and a 2478 bp open reading frame(ORF)which encoded 816 amino acids,containing LRR domain,the transmembrane region domain and TIR domain.The LcTLR2 predominantly express in blood,heart,spleen,liver and kidney and weakly express in brain,skin,and gill.After injection with LPS poly I:C and V.parahemolyticus,LcTLR2 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS poly I:C PGN and Flagellin,LcTLR1 expression levels also showed up-regulation in LCK cellline(p<0.01).Poly I:C could not make LcTLR2 transcripts show up-regulation in the LCK cellline.The full-length cDNA of LcTLR21 was 3240 bp,containing a 97 bp 5' untranslated region(UTR),a 334 bp 3' UTR and a 2909 bp open reading frame(ORF)which encoded 937 amino acids,containing LRR domain,the transmembrane region domain and TIR domain.A broad expression of LcTLR21 in most detected tissues,with the predominant expression in spleen,kidney,liver and stomach and weak expression in brain is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,LcTLR21 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,LcTLR21 expression levels also showed up-regulation in LCK cellline(p<0.01).(2)Some genes in NF-?B familyThe full-length cDNA of Lcp65 was 2974 bp,containing a 27 bp 5' untranslated region(UTR),a 1051 bp 3' UTR and a 1896 bp open reading frame(ORF)which encoded 631 amino acids,containing RHD domain and IPT domain.A broad expression of Lcp65 in most detected tissues,with the predominant expression in intestinal,spleen,stomach and gill and weak expression in skin is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,Lcp65 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,Lcp65 expression levels also showed up-regulation in LCK cellline(p<0.01).The full-length cDNA of Lcp52 was 4083 bp,containing a 128 bp 5' untranslated region(UTR),a 1261 bp 3' UTR and a 2694 bp open reading frame(ORF)which encoded 898 amino acids,containing RHD domain,IPT domain,ANK domain and DEATH domain.A broad expression of Lcp52 in most detected tissues,with the predominant expression in intestinal,head-kidney,stomach and gill and weak expression in skin and liver is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,Lcp52 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,Lcp52 expression levels also showed up-regulation in LCK cellline(p<0.01).The full-length cDNA of Lcc-rel was 3465 bp,containing a 77 bp 5' untranslated region(UTR),a 1453 bp 3' UTR and a 1932 bp open reading frame(ORF)which encoded 644 amino acids,containing RHD domain and IPT domain.A broad expression of Lcc-rel in most detected tissues,with the predominant expression in intestinal,heart,spleen and gill and weak expression in skin and blood is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,Lcc-rel expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,Lcc-rel expression levels also showed up-regulation in LCK cellline(p<0.01).The full-length cDNA of LcI?B was 1407 bp,containing a 65 bp 5' untranslated region(UTR),a 415 bp 3' UTR and a 927 bp open reading frame(ORF)which encoded 309 amino acids,containing ANK domain.A broad expression of LcI?B in most detected tissues,with the predominant expression in intestinal,skin,liver and kidney and weak expression in gill and head-kidney is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,LcI?B expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,LcI?B expression levels also showed up-regulation in LCK cellline(p<0.01).(3)Some genes in IL familyThe full-length cDNA of LcIL-11 was 1106 bp,containing a 503 bp 3' untranslated region(UTR),and a 603 bp open reading frame(ORF)which encoded 200 amino acids,containing IL-11 domain.A broad expression of LcIL-11 in most detected tissues,with the predominant expression in skin,gill,brain,spleen and heart and weak expression in muscle and head-kidney is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,LcIL-11 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,LcIL-11 expression levels also showed up-regulation in LCK cellline(p<0.01).The full-length cDNA of LcIL-21 was 880 bp,containing a 66 bp 5' untranslated region(UTR),a 385 bp 3'and a 429 bp open reading frame(ORF)which encoded 142 amino acids,containing IL-15 domain.A broad expression of LcIL-21 in most detected tissues,with the predominant expression in skin,gill,intestinal,kidney and heart and weak expression in brain and liver is demonstrated.After injection with LPS poly I:C and V.parahemolyticus,LcIL-21 expression levels showed up-regulation in head-kidney,liver and spleen(p<0.01).After stimulation with LPS,poly I:C,PGN and Flagellin,LcIL-21 expression levels also showed up-regulation in LCK cellline(p<0.01).(4)Conducting the recombinant expressin of MyD88,the key adaptor molecule of TLR pathway,and study the proteins interacting with MyD88 using GST-pulldown Technology in vitro.The result shows that the interested protein may be the cytoplasmic dynein.(5)Using E.coli expression system,the IL-1? and IL-21 recombinant proteins were obtained purified.The recombinant protein of IL-1? was mainly in inclusion body.And the recombinant protein of IL-21 was predominant in supernant with a soluble expression product.