Characterization of shrimp integrin β and its function in immunomodulation by dsRNA-mediated gene...
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P-168. Proinflammatory function of HMGB1 in the freshwater mussel Cris- taria plicata Y. Li, C.G. Wen * , B.Q. Hu, D. Wu, Z.Y. Tao. School of Life Sciences and Food Engineering, Institute of Life Science, Nanchang University, Nanchang 330031, China Abstract The freshwater mussel Cristaria plicata, which is of great economical importance and known as “pearl bivalves” in the aquaculture industry of China, widely respectively, have certain economic value. HMGB1 is a kind of nuclear protein which has many functions in cells. The existing studies have shown that HMGB1 may be a late inflammatory factor in the extracellular. In the present study, the HMGB1 cDNA of the freshwater mussel Cristaria plicata was cloned using the RT-PCR tech- nique. The comparison of the deduced amino acid sequences with HMGB1 from other species showed that the CpHMGB1 had three functional areas, A box, B box and C box. While B box was considered to be the main area of the HMGB1 protein had a proinflammatory activity, the A box was a box B antagonist. The CpHMGB1 was 621 nucleotides (nt) in length and encoding 207 amino acid residues, and the molecular weight of the predicted HMGB1 was 24KDa with the calculated PI being 5.7. Further- more, the DNA products and vector pET-30a were digested by the Kpn I, EcoR I enzymes, and then ligated. The recombinant plasmids were transformed into the prokaryotic cell Rosetta-gami (DE3) and induced with IPTG when the OD600 being 0.65 at 37 C for 12h, and then was purified by using the native Ni2+ affinity chromatography. Finally, the purpose of pure protein obtained. The levels of SD mice serum TNF-a were raised after be injected by rCpHMGB1, the maximum secretion was observed at 4 h later, and as rCpHMGB1 concentration rises, the mea- surement of TNF-a increased. It showed that the rCpHMGB1 had pro- inflammation capacity in SD mice. * Corresponding author. E-mail address: [email protected] (C.G. Wen) P-157. Identification and expression analysis of two important molecules in TLR signal pathway: IRAK4 and TRAF6 Y. Li, X. Xiao, X. Li, A. Li * . Key Laboratory for Aquatic Products Safety of Ministry of Education/ State Key Laboratory of Biocontrol, The school of Life Sciences, Sun Yat- sen University, 135 Xingang West Street, Haizhu District, Guangzhou 510275, Guangdong Province, People's Republic of China Abstract TLR signal pathway plays a crucial role in host innate immune responses. Cryptocaryon irritans is a pathogenic ecto-parasite ciliate that infects salt- water fish causing heavy economic losses. However, the role of TLR signal pathway in host defense against C. irritans infection is poorly understood. In this paper, we firstly cloned two important signal transduction mole- cules of IRAK4 (EcIRAK4) and TRAF6 (EcTRAF6) from grouper (Epinephelus coioides). Full-length of IRAK4 was 1859 bp containing an ORF of 1395 bp encoding a putative polypeptide of 464 amino acid residues. EcIRAK4 has a conserved death domain (DD) and a serine/threonine/tyrosine protein kinase domain (STYKc). TRAF6 has an ORF of 1713 bp encoding 570 amino acids. The putative amino acids possess the characteristic motifs of other TRAF6, including a ring domain, two zinc fingers, a coiled-coil region, and a MATH domain. Phylogenetic analysis and multiple alignments indicated that EcIRAK4 and EcTRAF6 was respectively clustered into fish group and shared a 62–83% and 65–86% sequence identity with that of other fish species. Both genes were constitutively expressed in all tested tissues, with the highest expression level in the head kidney and blood, respec- tively. Post infection with C. irritans, we detected both genes’ expression in the local infection sites and system immune organs. Results demon- strated the both genes were significantly up-regulated in the skin, but slightly fluctuated in other three tissues at most time points, implying that parasite infection could regulate TLR signal pathway responses in local attack site. * Corresponding author. E-mail address: [email protected] (A. Li) P-355. Characterization of shrimp integrin b and its function in immunomo- dulation by dsRNA-mediated gene silencing Y.C. Lin 1 , J.C. Chen 1, * , W. Cheng 2 , C.H. Liu 2 , Y.H. Chang 1 . 1 Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan; 2 Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 912, Taiwan Abstract The mature protein (764 aa) of white shrimp Litopenaeus vannamei integrin b (LV-B) contains 1) an extracellular domain (ED) of 692 aa, 2) a transmembrane domain (TD) of 23 aa, and 3) a cytoplasmic domain (CD) of 49 aa. The cloned LV-B grouped together with crayfish Pacifastacus leniusculus integrin b (PL-B1), but was far away from vertebrate integrin b1, b3, b5, b6, b7, and b8. A Southern blot analysis indicated that the cloned LV- B was a single copy of genomic DNA. LV-B mRNA was highly expressed in haemocytes. LV-B was downregulated in shrimp 24 and 96 h after having received white spot syndrome virus (WSSV). LV-B expression by haemo- cytes of shrimp was higher in the postmoult stage, and lower in the pre- moult (D 2 /D 3 ) stage. LV-B expression was significantly higher by shrimp reared in 2.5& and 5& salinities. Shrimp injected with integrin b dsRNA showed gene silencing of integrin b after 36 h. LV-B-silenced shrimp showed decreased HC, GC (including semi-granular cell), total haemocyte count (THC), respiratory burst (RB), and lysozyme activity, but showed increased RB/HC, superoxide dismutase (SOD) activity/HC, and the phe- noloxidase (PO) activity/GC. LV-B-silenced shrimp showed upregulated expressions of lipopolysaccharide- and b-glucan-binding protein (LGBP), peroxinectin (PX), prophenoloxidase I (proPO I), proPO II, proPO-activating enzyme (ppA), a2-macroglobulin (a2-M), cytMnSOD, mtMnSOD, and heat shock protein 70 (HSP70). It was concluded that integrin b plays important roles in proPO activation, phagocytosis, and the antioxidant system for immunomodulation in shrimp. * Corresponding author. E-mail address: [email protected] (J.C. Chen) P-356. Immunosuppression and immunomodulation of white shrimp at low salinity and under starvation Y.C. Lin, J.C. Chen, Y.H. Kuo * , Y.H. Chang. Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan Abstract Survival, immune parameters, resistance against Vibrio alginolyticus, and gene expressions of white shrimp Litopenaeus vannamei juveniles which had been reared at different salinity levels were examined. The survival rates of shrimp reared at 2.5& and 5& were significantly lower than those of shrimp reared at 15&, 25&, and 35&. Shrimp reared at lower salinity levels showed lower hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, and showed decreased resistance against V. alginolyti- cus, but showed higher expressions of lipopolysaccharide and b-glucan- binding protein (LGBP), peroxinectin (PX), integrin b, prophenoloxidase (proPO), and a2-macroglobulin (a2-M), and other immune genes related to superoxide dismutase (SOD), and antimicrobial peptide. In another experiment, survival, immune parameters, resistance against V. alginoly- ticus, and gene expressions were examined in shrimp which had been Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752 1719
Transcript of Characterization of shrimp integrin β and its function in immunomodulation by dsRNA-mediated gene...
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752 1719
P-168.
Proinflammatory function of HMGB1 in the freshwater mussel Cris-taria plicata
Y. Li, C.G. Wen*, B.Q. Hu, D. Wu, Z.Y. Tao.
School of Life Sciences and Food Engineering, Institute of Life Science,Nanchang University, Nanchang 330031, China
AbstractThe freshwater mussel Cristaria plicata, which is of great economicalimportance and known as “pearl bivalves” in the aquaculture industry ofChina, widely respectively, have certain economic value.HMGB1 is a kind of nuclear proteinwhich has many functions in cells. Theexisting studies have shown that HMGB1 may be a late inflammatoryfactor in the extracellular. In the present study, the HMGB1 cDNA of thefreshwater mussel Cristaria plicata was cloned using the RT-PCR tech-nique. The comparison of the deduced amino acid sequences with HMGB1from other species showed that the CpHMGB1 had three functional areas,A box, B box and C box. While B box was considered to be the main area ofthe HMGB1 protein had a proinflammatory activity, the A box was a box Bantagonist. The CpHMGB1 was 621 nucleotides (nt) in length andencoding 207 amino acid residues, and the molecular weight of thepredicted HMGB1 was 24KDa with the calculated PI being 5.7. Further-more, the DNA products and vector pET-30a were digested by the Kpn I,EcoR I enzymes, and then ligated. The recombinant plasmids weretransformed into the prokaryotic cell Rosetta-gami (DE3) and inducedwith IPTG when the OD600 being 0.65 at 37�C for 12h, and then waspurified by using the native Ni2+ affinity chromatography. Finally, thepurpose of pure protein obtained. The levels of SDmice serum TNF-awereraised after be injected by rCpHMGB1, the maximum secretion wasobserved at 4 h later, and as rCpHMGB1 concentration rises, the mea-surement of TNF-a increased. It showed that the rCpHMGB1 had pro-inflammation capacity in SD mice.
* Corresponding author.E-mail address: [email protected] (C.G. Wen)
P-157.
Identification and expression analysis of two important molecules inTLR signal pathway: IRAK4 and TRAF6
Y. Li, X. Xiao, X. Li, A. Li*.
Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, The school of Life Sciences, Sun Yat-sen University, 135 Xingang West Street, Haizhu District, Guangzhou510275, Guangdong Province, People's Republic of China
AbstractTLR signal pathway plays a crucial role in host innate immune responses.Cryptocaryon irritans is a pathogenic ecto-parasite ciliate that infects salt-water fish causing heavy economic losses. However, the role of TLR signalpathway in host defense against C. irritans infection is poorly understood.In this paper, we firstly cloned two important signal transduction mole-cules of IRAK4 (EcIRAK4) and TRAF6 (EcTRAF6) from grouper (Epinepheluscoioides). Full-length of IRAK4 was 1859 bp containing an ORF of 1395 bpencoding a putative polypeptide of 464 amino acid residues. EcIRAK4 hasa conserved death domain (DD) and a serine/threonine/tyrosine proteinkinase domain (STYKc). TRAF6 has an ORF of 1713 bp encoding 570 aminoacids. The putative amino acids possess the characteristic motifs of otherTRAF6, including a ring domain, two zinc fingers, a coiled-coil region, anda MATH domain. Phylogenetic analysis and multiple alignments indicatedthat EcIRAK4 and EcTRAF6 was respectively clustered into fish group andshared a 62–83% and 65–86% sequence identity with that of other fishspecies. Both genes were constitutively expressed in all tested tissues,with the highest expression level in the head kidney and blood, respec-tively. Post infection with C. irritans, we detected both genes’ expressionin the local infection sites and system immune organs. Results demon-strated the both genes were significantly up-regulated in the skin, butslightly fluctuated in other three tissues at most time points, implying
that parasite infection could regulate TLR signal pathway responses inlocal attack site.
* Corresponding author.E-mail address: [email protected] (A. Li)
P-355.
Characterization of shrimp integrin b and its function in immunomo-dulation by dsRNA-mediated gene silencing
Y.C. Lin 1, J.C. Chen 1,*, W. Cheng 2, C.H. Liu 2, Y.H. Chang 1.
1Department of Aquaculture, National Taiwan Ocean University,Keelung 202, Taiwan;2Department of Aquaculture, National Pingtung University of Scienceand Technology, Pingtung 912, Taiwan
AbstractThe mature protein (764 aa) of white shrimp Litopenaeus vannameiintegrin b (LV-B) contains 1) an extracellular domain (ED) of 692 aa, 2) atransmembrane domain (TD) of 23 aa, and 3) a cytoplasmic domain (CD) of49 aa. The cloned LV-B grouped together with crayfish Pacifastacusleniusculus integrin b (PL-B1), but was far away from vertebrate integrin b1,b3, b5, b6, b7, and b8. A Southern blot analysis indicated that the cloned LV-B was a single copy of genomic DNA. LV-B mRNA was highly expressed inhaemocytes. LV-B was downregulated in shrimp 24 and 96 h after havingreceived white spot syndrome virus (WSSV). LV-B expression by haemo-cytes of shrimp was higher in the postmoult stage, and lower in the pre-moult (D2/D3) stage. LV-B expression was significantly higher by shrimpreared in 2.5& and 5& salinities. Shrimp injected with integrin b dsRNAshowed gene silencing of integrin b after 36 h. LV-B-silenced shrimpshowed decreased HC, GC (including semi-granular cell), total haemocytecount (THC), respiratory burst (RB), and lysozyme activity, but showedincreased RB/HC, superoxide dismutase (SOD) activity/HC, and the phe-noloxidase (PO) activity/GC. LV-B-silenced shrimp showed upregulatedexpressions of lipopolysaccharide- and b-glucan-binding protein (LGBP),peroxinectin (PX), prophenoloxidase I (proPO I), proPO II, proPO-activatingenzyme (ppA), a2-macroglobulin (a2-M), cytMnSOD, mtMnSOD, and heatshock protein 70 (HSP70). It was concluded that integrin b plays importantroles in proPO activation, phagocytosis, and the antioxidant system forimmunomodulation in shrimp.
* Corresponding author.E-mail address: [email protected] (J.C. Chen)
P-356.
Immunosuppression and immunomodulation of white shrimp at lowsalinity and under starvation
Y.C. Lin, J.C. Chen, Y.H. Kuo*, Y.H. Chang.
Department of Aquaculture, National Taiwan Ocean University,Keelung 202, Taiwan
AbstractSurvival, immune parameters, resistance against Vibrio alginolyticus, andgene expressions of white shrimp Litopenaeus vannamei juveniles whichhad been reared at different salinity levels were examined. The survivalrates of shrimp reared at 2.5& and 5&were significantly lower than thoseof shrimp reared at 15&, 25&, and 35&. Shrimp reared at lower salinitylevels showed lower hyaline cells (HCs), granular cells (GCs, includingsemi-granular cells), total haemocyte count (THC), phenoloxidase (PO)activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, andlysozyme activity, and showed decreased resistance against V. alginolyti-cus, but showed higher expressions of lipopolysaccharide and b-glucan-binding protein (LGBP), peroxinectin (PX), integrin b, prophenoloxidase(proPO), and a2-macroglobulin (a2-M), and other immune genes relatedto superoxide dismutase (SOD), and antimicrobial peptide. In anotherexperiment, survival, immune parameters, resistance against V. alginoly-ticus, and gene expressions were examined in shrimp which had been
