super-resolution-imaging-natural-killer-cell-immunological-synapse-on

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Texas Children's Hospital

Baylor College of Medicine

&#x524D;&#x915A;&#x6C27;&#x5316;&#x9176; (&#x6C27;&#x5316;&#x7C7B;&#x4EA7;&#x54C1;) &#x662F;&#x5B88;&#x6052;&#x7684;&#x542B;&#x94DC;&#x9176;&#xFF0C;&#x5728;&#x7532;&#x58F3;&#x7C7B;&#x52A8;&#x7269;&#x548C;&#x6606;&#x866B;&#x7684;&#x514D;&#x75AB;&#x53CD;&#x5E94;&#x4E2D;&#x626E;&#x6F14;&#x91CD;&#x8981;&#x89D2;&#x8272;&#x3002;&#x5728;&#x672C;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x524D;&#x915A;&#x6C27;&#x5316;&#x9176; (&#x6307;&#x5B9A; EsproPO) &#x5168;&#x957F; cDNA &#x514B;&#x9686;&#x4ECE;&#x6CB3;&#x87F9;&#x517B;&#x6B96;&#x4E2D;&#x534E;&#x7ED2;&#x87AF;&#x87F9;&#x8840;&#x7EC6;&#x80DE;&#x7684;&#x8868;&#x8FBE;&#x5E8F;&#x5217;&#x6807;&#x7B7E; (EST) &#x548C; PCR &#x6280;&#x672F;&#x3002;EsproPO &#x5168;&#x957F; cDNA &#x7684;&#x5B64;&#x7ACB;&#x7684; 3549bp &#x8F7D;&#x6709; 679 &#x6C28;&#x57FA;&#x9178;&#x3001; 5&#39; &#x975E;&#x7FFB;&#x8BD1;&#x533A; &#xFF08;&#x7F16;&#x7801;&#xFF09; &#x7684; 68bp &#x548C;&#x957F; 3&#39;-UTR &#x7684; 1441bp &#x5047;&#x5B9A;&#x6C27;&#x5316;&#x7C7B;&#x4EA7;&#x54C1;&#x86CB;&#x767D;&#x7F16;&#x7801; 2040bp &#x5F00;&#x653E;&#x9605;&#x8BFB;&#x6846;&#x67B6; (&#x53E3;&#x75AE;)&#x3002;EsproPO &#x63A8;&#x5BFC;&#x7684;&#x6C28;&#x57FA;&#x9178;&#x5E8F;&#x5217;&#x4E2D;&#x786E;&#x5B9A;&#x4E86;&#x4E24;&#x4E2A;&#x5047;&#x5B9A;&#x94DC;&#x7ED1;&#x5B9A;&#x7AD9;&#x70B9;&#x3001; &#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x7AD9;&#x70B9;&#x548C;&#x8865;&#x5145;&#x7C7B;&#x4F3C;&#x6BCD;&#x9898; &#xFF08;GCGWPQHM&#xFF09;&#x3002;&#x540C;&#x6E90;&#x6027;&#x5206;&#x6790;&#x8868;&#x660E; EsproPO &#x975E;&#x5E38;&#x7C7B;&#x4F3C;&#x4E8E;&#x5176;&#x4ED6;&#x547D;&#x9898;&#x4ECE;&#x7532;&#x58F3;&#x7C7B;&#x52A8;&#x7269;&#x4E0E;&#x6807;&#x8BC6;&#x4ECE; 52%&#x81F3; 68%&#x3002;&#x5B88;&#x6052;&#x7684;&#x57DF;&#x548C;&#x56FE;&#x6848;&#xFF0C;&#x4EE5;&#x53CA;&#x66F4;&#x9AD8;&#x76F8;&#x4F3C;&#x6027;&#x4E0E;&#x5176;&#x4ED6;&#x547D;&#x9898;&#x5EFA;&#x8BAE; EsproPO &#x662F;&#x6C27;&#x5316;&#x7C7B;&#x4EA7;&#x54C1;&#x5BB6;&#x65CF;&#x7684;&#x6210;&#x5458;&#x3002;EsproPO &#x548C;&#x5927;&#x57D4;&#x7684;&#x5177;&#x4F53;&#x6D3B;&#x52A8;&#x7684;&#x809D;&#x80F0;&#x810F;&#x3001; &#x9CC3;&#x3001; &#x6027;&#x817A;&#x3001; &#x808C;&#x8089;&#x3001; &#x5FC3;&#x810F;&#x3001; &#x773C;&#x775B;&#x548C;&#x8840;&#x7EC6;&#x80DE;&#x7EC4;&#x7EC7;&#x4E2D;&#x7684;&#x8868;&#x8FBE;&#x5206;&#x522B;&#x7531;&#x5B9E;&#x65F6;&#x8367;&#x5149;&#x5B9A;&#x91CF; PCR &#x548C;&#x5BF9;&#x6BD4;&#x8272;&#xFF0C;&#x6765;&#x8861;&#x91CF;&#x3002;EsproPO &#x548C;&#x5927;&#x57D4;&#x7684;&#x5177;&#x4F53;&#x6D3B;&#x52A8;&#x7684;&#x6210;&#x7EE9;&#x5355;&#x53EF;&#x4EE5;&#x62E5;&#x6709;&#x6700;&#x9AD8;&#x7684;&#x6240;&#x6709;&#x68C0;&#x67E5;&#x7EC4;&#x7EC7;&#x4E2D;&#x68C0;&#x6D4B;&#x5230;&#x7684; mRNA &#x6C34;&#x5E73;&#x5747;&#x5728;&#x809D;&#x80F0;&#x817A;&#x4E2D;&#x3002;&#x4E09;&#x5CF0; EsproPO mRNA &#x8868;&#x8FBE;&#x7684;&#x5F55;&#x5728; 2 h&#x3001; 12 h &#x548C; 48 h &#x7684;&#x6CB3;&#x87F9;&#x8840;&#x7EC6;&#x80DE;&#x4E2D;&#x5F20;&#x8D34;&#x5F27;&#x83CC;&#x9CD7;&#x6311;&#x6218;&#xFF0C;&#x4E0E;&#x5927;&#x57D4;&#x5177;&#x4F53;&#x6D3B;&#x52A8;&#x7684;&#x65F6;&#x7A7A;&#x914D;&#x7F6E;&#x6587;&#x4EF6;&#x662F;&#x4E00;&#x81F4;&#x7684;&#x3002;MRNA &#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x548C;&#x6D3B;&#x52A8;&#x6CE2;&#x52A8;&#x7684;&#x9CD7;&#x523A;&#x6FC0;&#x8BC9; EsproPO post &#x8868;&#x793A;&#x53EF;&#x80FD;&#x4F1A;&#x53C2;&#x4E0E;&#x6253;&#x51FB;&#x5165;&#x4FB5;&#x7684;&#x7EC6;&#x83CC;&#x5728;&#x6CB3;&#x87F9;&#x517B;&#x6B96;&#x4E2D;&#x7684;&#x6025;&#x6027;&#x53CD;&#x5E94;&#x3002;

&#x915A;&#x6C27;&#x5316;&#x9176;&#x539F;&#x4ECE;&#x6CB3;&#x87F9;&#x517B;&#x6B96;&#x4E2D;&#x534E;&#x7ED2;&#x87AF;&#x87F9;&#xFF1A; &#x57FA;&#x56E0;&#x7684;&#x514B;&#x9686;&#x3001; &#x8868;&#x8FBE;&#x53CA;&#x6D3B;&#x6027;&#x5206;&#x6790;&#x3002;

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549bp full-length cDNA of EsproPO contained a 2040bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68bp, and a long 3'-UTR of 1441bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2h, 12h and 48h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis.

Ph&eacute;noloxydase (proPO) est une enzyme contenant du cuivre conserv&eacute;e qui joue un r&ocirc;le important dans la r&eacute;ponse immunitaire des insectes et des crustac&eacute;s. Dans la pr&eacute;sente &eacute;tude, l&#39;ADNc pleine longueur d&#39;une ph&eacute;noloxydase (d&eacute;sign&eacute; EsproPO) a &eacute;t&eacute; clon&eacute; chez h&eacute;mocytes de crabe chinois Eriocheir sinensis par exprim&eacute; &eacute;tiquette de s&eacute;quence (EST) et des techniques de PCR. L&#39;ADNc pleine longueur 3549bp isol&eacute; de EsproPO contenait un cadre de lecture de 2040bp (ORF) codant pour une prot&eacute;ine putative proPO de 679 acides amin&eacute;s, une 5&#39;-r&eacute;gion non traduite (UTR) de 68bp et un long 3&#39;-UTR de 1441bp. Deux sites de liaison cuivre putatifs, un site d&#39;activation prot&eacute;olytique et un compl&eacute;ment comme motif (GCGWPQHM) ont &eacute;t&eacute; identifi&eacute;s dans la s&eacute;quence d&#39;acides amin&eacute;s d&eacute;duite de EsproPO. Homologie analyse a r&eacute;v&eacute;l&eacute; que le EsproPO &eacute;tait tr&egrave;s semblable aux autres proPOs de crustac&eacute;s avec les identit&eacute;s de 52 % &agrave; 68 %. Les domaines conserv&eacute;s et motifs et sup&eacute;rieur similitude avec d&#39;autres proPOs sugg&egrave;rent que EsproPO &eacute;tait un membre de la famille proPO. L&#39;expression de l&#39;ARNm de EsproPO et PO d&#39;activit&eacute;s ponctuelles dans les tissus de l&#39;h&eacute;patopancr&eacute;as, gill, gonades, muscle, coeur, yeux et h&eacute;mocytes ont &eacute;t&eacute; mesur&eacute;es par PCR en temps r&eacute;el quantitative et dosage colorim&eacute;trique, respectivement. Le mRNA transcriptions des activit&eacute;s sp&eacute;cifiques EsproPO et PO a peuvent &ecirc;tre d&eacute;tect&eacute;es dans tous les tissus examin&eacute;s avec le plus haut niveau aussi bien dans l&#39;h&eacute;patopancr&eacute;as. Trois pics d&#39;expression des ARNm de EsproPO ont &eacute;t&eacute; enregistr&eacute;s &agrave; 2 h, 12 h et 48 h dans les h&eacute;mocytes de crabe chinois apr&egrave;s d&eacute;fi de Vibrio anguillarum, qui concorde avec le profil temporel de l&#39;activit&eacute; sp&eacute;cifique de PO. Mod&egrave;le d&#39;expression de l&#39;ARNm et la fluctuation de l&#39;activit&eacute; du poste de EsproPO V. anguillarum stimulation a indiqu&eacute; que c&#39;&eacute;tait potentiellement impliqu&eacute; dans la r&eacute;ponse aigu&euml; contre l&#39;invasion des bact&eacute;ries en crabe chinois.

Une ph&eacute;noloxydase depuis le crabe chinois Eriocheir sinensis : clonage de g&egrave;nes, analyse de l&#39;expression et l&#39;activit&eacute;.

Prophenoloxidase (vorschlagen) ist eine konservierte Kupfer-haltigen Enzym, das die Immunantwort von Krebstieren und Insekten wichtige Rolle spielt. In der vorliegenden Studie, das in cDNA von einem Prophenoloxidase (bezeichneten EsproPO) aus Haemocytes der chinesischen Wollhandkrabbe Eriocheir Sinensis geklont wurde durch ausgedr&uuml;ckt, Sequenz-Tag (EST) und PCR-Techniken. Das isolierten 3549bp in cDNA von EsproPO enthalten eine 2040bp offene Leseraster (ORF) f&uuml;r eine vermeintliche vorschlagen-Protein 679 Aminos&auml;uren, nicht 5&#39;-&uuml;bersetzte Region (UTR) 68bp und eine lange 3&#39;-UTR des 1441bp. Zwei vermeintliche Kupfer-Bindungsstellen, eine proteolytische Aktivierung-Website und ein Komplement-&auml;hnliche Motiv (GCGWPQHM) wurden in der hergeleiteten Aminos&auml;ure-Sequenz des EsproPO identifiziert. Homologie-Analyse ergab, dass EsproPO sehr &auml;hnlich wie andere ProPOs von Krebstieren mit Identit&auml;ten von 52 % auf 68 %. Die konservierten Dom&auml;nen und Motive und h&ouml;her &Auml;hnlichkeit mit anderen ProPOs vorgeschlagen, EsproPO ein Mitglied der Familie vorschlagen war. Der mRNA-Ausdruck EsproPO und PO spezifische Aktivit&auml;ten in den Geweben der Hepatopankreas, Gill, Gonad, Muskel, Herz, Auge und Haemocytes wurden durch quantitative Echtzeit-PCR und kolorimetrische Assays, bzw. gemessen. Die mRNA, die Abschriften von EsproPO und PO spezifische T&auml;tigkeiten in allen untersuchten Geweben mit der h&ouml;chsten erkennbar waren Ebene sowohl in Hepatopankreas. Drei Gipfel der EsproPO mRNA Ausdruck wurden aufgezeichnet, um 2 h, 12 h und 48 h in Haemocytes der chinesischen Wollhandkrabbe post Vibrio Anguillarum Challenge, die konsistent mit dem zeitlichen Profil PO spezifischen T&auml;tigkeit war. Das mRNA Ausdrucksmuster und die Aktivit&auml;t-Schwankung der EsproPO Post V. Anguillarum Stimulierung angedeutet, dass es m&ouml;glicherweise die akute Reaktion gegen die eindringenden Bakterien im chinesischen Wollhandkrabbe beteiligt war.

Ein Prophenoloxidase aus der chinesischen Wollhandkrabbe Eriocheir Sinensis: Klonen, Ausdruck und Aktivit&auml;tsanalyse gen.

Prophenoloxidase (proPO) &egrave; un enzima contenente rame conservato che svolge ruoli importanti nella risposta immunitaria di crostacei e insetti. Nel presente studio, il full-length cDNA di un prophenoloxidase (designato EsproPO) &egrave; stato clonato da haemocytes di granchio cinese del mitten Eriocheir sinensis da espresso tag di sequenza (EST) e tecniche di PCR. Il cDNA full-length 3549bp isolato di EsproPO conteneva un 2040bp open reading frame (ORF) codifica per una proteina putativa proPO di 679 aminoacidi, un 5&#39;--regione non tradotta (UTR) del 68bp e una lunga 3&#39; UTR del 1441bp. Due siti di legame rame putativi, un sito di attivazione proteolitica e un motivo come complemento (GCGWPQHM) sono stati identificati nella sequenza dedotta dell&#39;amminoacido di EsproPO. Analisi di omologia ha rivelato che EsproPO era molto simile a quello di altre proPOs di crostacei con identit&agrave; dal 52% al 68%. I domini conservati e motivi e maggiore somiglianza con altre proPOs suggerito che EsproPO era un membro della famiglia proPO. L&#39;espressione del mRNA di EsproPO e PO attivit&agrave; specifiche nei tessuti dell&#39;epatopancreas, gill, gonadi, muscolo, cuore, occhi e haemocytes sono stati misurati da PCR quantitativa in tempo reale e analisi colorimetriche, rispettivamente. il mRNA le trascrizioni delle attivit&agrave; specifiche EsproPO e PO potrebbero essere rilevate in tutti i tessuti esaminati con il pi&ugrave; alto livello sia nell&#39;epatopancreas. Tre picchi di espressione del mRNA di EsproPO sono stati registrati a 2 h, 12 h e 48 h in haemocytes di granchio cinese del mitten posto Vibrio anguillarum sfida, che era coerenza con il profilo temporale dell&#39;attivit&agrave; specifica di PO. Il pattern di espressione di mRNA e la fluttuazione di attivit&agrave; di post EsproPO V. anguillarum stimolazione indicato che potenzialmente &egrave; stato coinvolto nella risposta acuta contro l&#39;invasione di batteri in granchio cinese del mitten.

Un prophenoloxidase dal granchio cinese del mitten Eriocheir sinensis: clonazione, espressione e attivit&agrave; di analisi genica.

Prophenoloxidase (&#x30D7;&#x30ED;&#x30DD;) &#x7532;&#x6BBB;&#x985E;&#x304A;&#x3088;&#x3073;&#x6606;&#x866B;&#x306E;&#x514D;&#x75AB;&#x5FDC;&#x7B54;&#x306B;&#x304A;&#x3044;&#x3066;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x3001;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x9285;&#x542B;&#x6709;&#x9175;&#x7D20;&#x3067;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;, prophenoloxidase (&#x6307;&#x5B9A; EsproPO) &#x306E; cDNA &#x30C1;&#x30E5;&#x30A6;&#x30B4;&#x30AF;&#x30E2;&#x30AF;&#x30BA;&#x30AC;&#x30CB; &#x30E2;&#x30AF;&#x30BA;&#x30AC;&#x30CB; sinensis &#x306E; haemocytes &#x304B;&#x3089;&#x8907;&#x88FD;&#x3055;&#x308C;&#x305F;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x672D; (EST) &#x304A;&#x3088;&#x3073; PCR &#x306E;&#x30C6;&#x30AF;&#x30CB;&#x30C3;&#x30AF;&#x306B;&#x3088;&#x3063;&#x3066;&#x8868;&#x73FE;&#x3002;&#x5B64;&#x7ACB;&#x3057;&#x305F; 3549bp &#x5B8C;&#x5168;&#x9577; cDNA EsproPO &#x306E;&#x63A8;&#x5B9A;&#x30D7;&#x30ED;&#x30DD;&#x86CB;&#x767D;&#x8CEA; 679 &#x30A2;&#x30DF;&#x30CE;&#x9178;&#x306E; 5&#39;-&#x975E;&#x7FFB;&#x8A33;&#x9818;&#x57DF; (UTR) 68bp&#x3001;&#x306A;&#x3089;&#x3073;&#x306B;&#x3001;&#x9577;&#x3044; 3&#39; &#x7B26;&#x53F7;&#x5316; 2040bp &#x30AA;&#x30FC;&#x30D7;&#x30F3;&#x30EA;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0; &#x30D5;&#x30EC;&#x30FC;&#x30E0; (ORF) &#x306B;&#x542B;&#x307E;&#x308C;&#x308B; 1441bp &#x306E; UTR&#x3002;2 &#x3064;&#x306E;&#x63A8;&#x5B9A;&#x9285;&#x7D50;&#x5408;&#x30B5;&#x30A4;&#x30C8;&#x3001;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x5316;&#x30B5;&#x30A4;&#x30C8;&#x3001;&#x304A;&#x3088;&#x3073;&#x88DC;&#x5B8C;&#x7684;&#x30E2;&#x30C1;&#x30FC;&#x30D5; (GCGWPQHM) EsproPO &#x306E;&#x63A8;&#x5B9A;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x914D;&#x5217;&#x3067;&#x540C;&#x5B9A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30DB;&#x30E2;&#x30ED;&#x30B8;&#x30FC;&#x89E3;&#x6790; EsproPO &#x4ED6; proPOs &#x306B; 68% &#x306B; 52% &#x304B;&#x3089;&#x306E; id &#x3092;&#x6301;&#x3064;&#x7532;&#x6BBB;&#x985E;&#x304B;&#x3089;&#x975E;&#x5E38;&#x306B;&#x985E;&#x4F3C;&#x3057;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x305F;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3068;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x3001;&#x304A;&#x3088;&#x3073;&#x3088;&#x308A;&#x9AD8;&#x3044;&#x985E;&#x4F3C;&#x6027;&#x304C;&#x4ED6; proPOs EsproPO &#x30D7;&#x30ED;&#x30DD;&#x306E;&#x5BB6;&#x65CF;&#x306E;&#x4E00;&#x54E1;&#x3060;&#x3063;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x809D;&#x81B5;&#x81D3;, &#x9C13;, &#x751F;&#x6B96;&#x817A;&#x3001;&#x7B4B;&#x8089;&#x3001;&#x5FC3;&#x81D3;&#x3001;&#x76EE;&#x3068; haemocytes &#x306E;&#x7D44;&#x7E54;&#x306E;&#x7279;&#x5B9A;&#x306E;&#x6D3B;&#x52D5;&#x3092; EsproPO PO &#x306E; mRNA &#x767A;&#x73FE;&#x6E2C;&#x5B9A;&#x3057;&#x305F;&#x5B9A;&#x91CF;&#x7684;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0; PCR &#x304A;&#x3088;&#x3073;&#x6BD4;&#x8272;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306B;&#x3088;&#x3063;&#x3066;&#x305D;&#x308C;&#x305E;&#x308C;&#x3002;&#x4E21;&#x65B9;&#x306E;&#x809D;&#x81B5;&#x81D3;&#x30EC;&#x30D9;&#x30EB;&#x306E; mRNA &#x306E;&#x8EE2;&#x5199;&#x7269; EsproPO &#x3068; PO &#x306E;&#x7279;&#x5B9A;&#x306E;&#x6D3B;&#x52D5;&#x306E;&#x6700;&#x9AD8;&#x306E;&#x3059;&#x3079;&#x3066;&#x306E;&#x691C;&#x67FB;&#x3055;&#x308C;&#x305F;&#x7D44;&#x7E54;&#x3067;&#x691C;&#x51FA;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;12 H 48 h &#x3067;&#x30C1;&#x30E5;&#x30A6;&#x30B4;&#x30AF;&#x30E2;&#x30AF;&#x30BA;&#x30AC;&#x30CB; haemocytes &#x6295;&#x7A3F; PO &#x7279;&#x5B9A;&#x6D3B;&#x52D5;&#x306E;&#x6642;&#x7A7A;&#x9593;&#x306E;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306B;&#x4E00;&#x8CAB;&#x3057;&#x3066;&#x3044;&#x305F;&#x8178;&#x708E;&#x30D3;&#x30D6;&#x30EA;&#x30AA;&#x75C5;&#x539F;&#x30C1;&#x30E3;&#x30EC;&#x30F3;&#x30B8;&#x3001;EsproPO mRNA &#x306E;&#x767A;&#x73FE;&#x306E;&#x30D4;&#x30FC;&#x30AF;&#x306F; 2 h &#x3067;&#x8A18;&#x9332;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;MRNA &#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3068; EsproPO &#x30DD;&#x30B9;&#x30C8;&#x9AD8;&#x3044;&#x523A;&#x6FC0;&#x5BFE;&#x306E;&#x6D3B;&#x6027;&#x5909;&#x52D5;&#x305D;&#x308C;&#x6F5C;&#x5728;&#x7684;&#x30C1;&#x30E5;&#x30A6;&#x30B4;&#x30AF;&#x30E2;&#x30AF;&#x30BA;&#x30AC;&#x30CB;&#x4E2D;&#x306E;&#x7D30;&#x83CC;&#x306E;&#x4FB5;&#x5165;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x6025;&#x6027;&#x306E;&#x5FDC;&#x7B54;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;

&#x30C1;&#x30E5;&#x30A6;&#x30B4;&#x30AF;&#x30E2;&#x30AF;&#x30BA;&#x30AC;&#x30CB; &#x30E2;&#x30AF;&#x30BA;&#x30AC;&#x30CB; sinensis &#x304B;&#x3089; prophenoloxidase&#xFF1A; &#x907A;&#x4F1D;&#x5B50;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3001;&#x6D3B;&#x6027;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306E;&#x5206;&#x6790;&#x3002;

Prophenoloxidase (proPO)&#xB294; &#xBCF4;&#xC874;&#xB41C; &#xAD6C;&#xB9AC; &#xD568;&#xC720; &#xD6A8;&#xC18C; &#xAC11;&#xAC01;&#xB958;&#xC640; &#xACE4;&#xCDA9;&#xC758; &#xBA74;&#xC5ED; &#xBC18;&#xC751;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; prophenoloxidase (&#xC9C0;&#xC815; &#xB41C; EsproPO)&#xC758; &#xC804;&#xCCB4; &#xAE38;&#xC774;&#xC758; Cdna&#xB294; &#xC911;&#xAD6D; &#xB531;&#xC9C0; &#xD06C;&#xB7A9; Eriocheir sinensis&#xC758; haemocytes&#xC5D0;&#xC11C; &#xBCF5;&#xC81C;&#xC5D0; &#xC758;&#xD574; &#xC2DC;&#xD000;&#xC2A4; &#xD0DC;&#xADF8; (EST) &#xBC0F; PCR &#xAE30;&#xBC95;&#xC744; &#xD45C;&#xD604;. Espropo&#xC758; &#xACA9;&#xB9AC; &#xB41C; 3549bp &#xC804;&#xCCB4; &#xAE38;&#xC774;&#xC758; cDNA 679 &#xC544;&#xBBF8;&#xB178;&#xC0B0;, 68bp&#xC758; 5&#39;-untranslated &#xC601;&#xC5ED; (UTR)&#xB294; &#xAE34; 3&#39;-UTR 1441bp&#xC758;&#xC758; &#xC0C1; proPO &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xCF54;&#xB529; 2040bp &#xC5F4;&#xB824;&#xC788;&#xB294; &#xB3C5;&#xC11C; &#xD504;&#xB808;&#xC784; (ORF) &#xD3EC;&#xD568;. &#xB450; &#xC0C1; &#xAD6C;&#xB9AC; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;, &#xB2E8;&#xBC31;&#xC9C8; &#xD65C;&#xC131;&#xD654; &#xC0AC;&#xC774;&#xD2B8;&#xC640; &#xBCF4;&#xC218; &#xAC19;&#xC740; &#xBAA8;&#xD2F0;&#xBE0C; (GCGWPQHM) Espropo&#xC758; &#xCD94;&#xB860;&#xB41C; &#xC544;&#xBBF8;&#xB178;&#xC0B0; &#xC2DC;&#xD000;&#xC2A4;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; &#xD588;&#xB2E4;. Homology &#xBD84;&#xC11D; &#xACF5;&#xAC1C; Espropo&#xB294; 68 %52 %&#xC5D0;&#xC11C; id&#xC640; &#xAC11;&#xAC01;&#xB958;&#xC5D0;&#xC11C; &#xB2E4;&#xB978; &#xC790; &#xACE0;&#xC640; &#xB9E4;&#xC6B0; &#xBE44;&#xC2B7;&#xD569;&#xB2C8;&#xB2E4;. &#xBCF4;&#xC874;&#xB41C; &#xB3C4;&#xBA54;&#xC778; &#xBC0F; &#xBAA8;&#xD2F0;&#xD504;, &#xADF8;&#xB9AC;&#xACE0; &#xB2E4;&#xB978; &#xC790; &#xACE0;&#xC640; &#xB192;&#xC740; &#xC720;&#xC0AC;&#xC131; &#xC81C;&#xC548; EsproPO proPO &#xAC00;&#xC871;&#xC758; &#xC77C;&#xC6D0;&#xC774; &#xB418;&#xC5C8;&#xB2E4;. Espropo&#xC640; &#xD3EC; hepatopancreas, &#xAE38;, gonad, &#xADFC;&#xC721;, &#xC2EC;&#xC7A5;, &#xB208; &#xBC0F; haemocytes&#xC758; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xD2B9;&#xC815; &#xD65C;&#xB3D9;&#xC758; mRNA &#xC2DD; &#xC815;&#xB7C9; &#xC2E4;&#xC2DC;&#xAC04; PCR &#xBC0F; &#xC0C9;&#xCC44; &#xBD84;&#xC11D; &#xACB0;&#xACFC;, &#xAC01;&#xAC01; &#xCE21;&#xC815; &#xD588;&#xB2E4;. EsproPO &#xD3EC; &#xD2B9;&#xC815; &#xD65C;&#xB3D9;&#xC758; &#xC131;&#xC801; &#xCD5C;&#xACE0; &#xBAA8;&#xB4E0; &#xAC80;&#xC0AC; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xAC10;&#xC9C0; &#xC218; mRNA &#xC218;&#xC900; &#xBAA8;&#xB450; hepatopancreas&#xC5D0;&#xC11C;. EsproPO mRNA &#xC2DD;&#xC758; 3 &#xAC1C;&#xC758; &#xBD09;&#xC6B0;&#xB9AC;&#xC5D0; 2 h &#xAE30;&#xB85D; &#xD588;&#xB2E4;, 12h&#xC640; &#xC911;&#xAD6D; &#xB531;&#xC9C0; &#xD06C;&#xB7A9;&#xC758; haemocytes&#xC5D0;&#xC11C; 48 h &#xAC8C;&#xC2DC; &#xBE0C;&#xB9AC; anguillarum &#xB3C4;&#xC804;, PO &#xD2B9;&#xC815; &#xD65C;&#xB3D9;&#xC758; &#xC784;&#xC2DC; &#xD504;&#xB85C;&#xD544;&#xACFC; &#xC77C;&#xCE58; &#xD588;&#xB2E4;. MRNA &#xC2DD; &#xD328;&#xD134; &#xBC0F; anguillarum &#xC790;&#xADF9; V. EsproPO &#xAC8C;&#xC2DC;&#xBB3C; &#xD65C;&#xB3D9; &#xBCC0;&#xB3D9; &#xD45C;&#xC2DC; &#xC911;&#xAD6D; &#xB531;&#xC9C0; &#xD06C;&#xB7A9;&#xC758; &#xBC15;&#xD14C;&#xB9AC;&#xC544; &#xCE68;&#xC785;&#xC5D0; &#xB300; &#xD55C; &#xAE09;&#xC131; &#xBC18;&#xC751;&#xC5D0; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; &#xCC38;&#xC5EC; &#xD588;&#xB2E4;.

&#xC911;&#xAD6D; &#xB531;&#xC9C0; &#xD06C;&#xB7A9; Eriocheir sinensis&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; prophenoloxidase: &#xC720;&#xC804;&#xC790; &#xD074;&#xB85C;&#xB2DD;, &#xD45C;&#xD604;&#xACFC; &#xD65C;&#xB3D9; &#xBD84;&#xC11D;.

Prophenoloxidase (proPO) es una enzima que contiene cobre conservada que juega un papel importante en la respuesta inmune de crust&aacute;ceos e insectos. En el presente estudio, fue clonado el cDNA de cuerpo entero de un prophenoloxidase (designado EsproPO) de haemocytes de cangrejo chino manopla Eriocheir sinensis por expres&oacute; etiqueta de secuencia (EST) y t&eacute;cnicas de PCR. El cDNA de cuerpo entero de 3549bp aislados de EsproPO contiene un marco de lectura abierto de 2040bp (ORF) codificar una prote&iacute;na proPO putativos de 679 amino&aacute;cidos, una 5&#39;-regi&oacute;n sin traducir (UTR) de 68bp y un largo 3&#39;-UTR de 1441bp. Se identificaron dos posibles sitios de Uni&oacute;n cobre, un sitio de activaci&oacute;n proteol&iacute;tica y un motivo de complemento como (GCGWPQHM) en la secuencia de amino&aacute;cidos deducida de EsproPO. An&aacute;lisis de homolog&iacute;a revel&oacute; que EsproPO era muy similar a otro acerca de crust&aacute;ceos con identidades del 52% al 68%. La mayor similitud con otros proPOs, dominios conservados y motivos sugirieron que EsproPO era un miembro de la familia proPO. La expresi&oacute;n del mRNA de EsproPO y PO actividades espec&iacute;ficas en los tejidos del hepatop&aacute;ncreas, gill, g&oacute;nada, m&uacute;sculo, coraz&oacute;n, ojos y haemocytes fueron medidos por PCR cuantitativa en tiempo real y an&aacute;lisis colorim&eacute;trico, respectivamente. El mRNA transcritos de actividades espec&iacute;ficas EsproPO y PO podr&iacute;an detectarse en todos los tejidos examinados con el m&aacute;s alto nivel tanto en hepatop&aacute;ncreas. Tres picos de la expresi&oacute;n del mRNA de la EsproPO se registraron a las 2 h, 12 h y 48 h en haemocytes de cangrejo chino manopla post desaf&iacute;o de Vibrio anguillarum, que era constante con el perfil temporal de la actividad espec&iacute;fica de PO. El patr&oacute;n de expresi&oacute;n de mRNA y la fluctuaci&oacute;n de la actividad de EsproPO post V. anguillarum estimulaci&oacute;n indican que potencialmente estuvo implicado en la respuesta aguda contra la invasi&oacute;n de bacterias en el cangrejo chino manopla.

Un prophenoloxidase del cangrejo chino manopla Eriocheir sinensis: gene clonaci&oacute;n, expresi&oacute;n y actividad de an&aacute;lisis.

Lectins are a family of carbohydrate-recognition proteins which play crucial roles in innate immunity. In this study, a new lectin (CfLec-2) gene was cloned from Chlamys farreri by EST and RACE approaches. The full-length cDNA of CfLec-2 was composed of 708bp, encoding a typical long form carbohydrate-recognition domain of 130 residues. The deduced amino acid sequence showed high similarity to Brevican in Homo sapiens, C-type lectin-1 and lectin-2 in Anguilla japonica. The cDNA fragment encoding the mature peptide of CfLec-2 was recombined into plasmid pET-32a (+) and expressed in Escherichia coli Rosseta-Gami (DE3). The recombinant CfLec-2 (rCfLec-2) protein exhibited aggregative activity toward Staphylococcus haemolyticus, and the agglutination could be inhibited by d-mannose but not EDTA or d-galactose, indicating that CfLec-2 was a Ca2+ independent lectin. Moreover, rCfLec-2 could suppress the growth of E. coli TOP10F'. These results suggested that CfLec-2 was perhaps involved in the recognition and clearance of bacterial pathogens in scallop.

A lectin (CfLec-2) aggregating Staphylococcus haemolyticus from scallop Chlamys farreri.

&#x51DD;&#x96C6;&#x7D20;&#x662F;&#x4E00;&#x5BB6;&#x4EBA;&#xFF0C;&#x78B3;&#x6C34;&#x5316;&#x5408;&#x7269;&#x8BC6;&#x522B;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x5929;&#x7136;&#x514D;&#x75AB;&#x4E2D;&#x53D1;&#x6325;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x5728;&#x6B64;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x65B0;&#x7684;&#x51DD;&#x96C6;&#x7D20; (CfLec 2) &#x57FA;&#x56E0;&#x88AB;&#x514B;&#x9686;&#x6809;&#x5B54;&#x6247;&#x8D1D;&#x7684; EST &#x548C;&#x7ADE;&#x8D5B;&#x529E;&#x6CD5;&#x4E86;&#x3002;CfLec-2 &#x7684;&#x5168;&#x957F; cDNA &#x7684; 708bp&#xFF0C;&#x7F16;&#x7801; 130 &#x6B8B;&#x7559;&#x7684;&#x5178;&#x578B;&#x7684;&#x957F;&#x7A97;&#x4F53;&#x78B3;&#x6C34;&#x5316;&#x5408;&#x7269;&#x8BC6;&#x522B;&#x57DF;&#x7EC4;&#x6210;&#x3002;&#x63A8;&#x5BFC;&#x7684;&#x6C28;&#x57FA;&#x9178;&#x5E8F;&#x5217;&#x663E;&#x793A;&#x9AD8;&#x76F8;&#x4F3C;&#x5EA6;&#x4E0A;&#x8C03;&#x5230;&#x667A;&#x4EBA;&#xFF0C;C &#x578B;&#x51DD;&#x96C6;&#x7D20;-1 &#x548C;&#x51DD;&#x96C6;&#x7D20;-2 &#x5728;&#x65E5;&#x672C;&#x9CD7;&#x9CA1;&#x3002;&#x7F16;&#x7801;&#x7684; CfLec 2 &#x7684;&#x6210;&#x719F;&#x80BD; cDNA &#x7247;&#x6BB5;&#x662F;&#x91CD;&#x7EC4;&#x5230;&#x8D28;&#x7C92;&#x5BA0;&#x7269;-32a &#x53F7; (+)&#xFF0C;&#x5E76;&#x5728;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC; Rosseta-Gami &#xFF08;DE3&#xFF09; &#x8868;&#x793A;&#x3002;&#x91CD;&#x7EC4; CfLec-2 (rCfLec 2) &#x86CB;&#x767D;&#x5C55;&#x51FA;&#x805A;&#x96C6;&#x6D3B;&#x52A8;&#x5BF9;&#x8461;&#x8404;&#x7403;&#x83CC;&#xFF0C;&#x5E76;&#x53EF;&#x4EE5;&#x7531; d-&#x7518;&#x9732;&#x7CD6;&#x4F46;&#x4E0D;&#x662F; EDTA &#x6216; d-&#x534A;&#x4E73;&#x7CD6;&#xFF0C;&#x8868;&#x660E; CfLec 2 Ca2 + &#x72EC;&#x7ACB;&#x51DD;&#x96C6;&#x6291;&#x5236;&#x51DD;&#x96C6;&#x3002;&#x6B64;&#x5916;&#xFF0C;rCfLec-2 &#x53EF;&#x6291;&#x5236;&#x751F;&#x957F;&#x7684;&#x5927;&#x80A0;&#x6746;&#x83CC; TOP10F&#39;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E; CfLec 2 &#x53EF;&#x80FD;&#x53C2;&#x4E0E;&#x7684;&#x8BC6;&#x522B;&#x548C;&#x6E05;&#x9664;&#x7EC6;&#x83CC;&#x75C5;&#x539F;&#x4F53;&#x5728;&#x6247;&#x8D1D;&#x3002;

&#x51DD;&#x96C6;&#x7D20; (CfLec 2) &#x805A;&#x5408;&#x8461;&#x8404;&#x7403;&#x83CC;&#x4ECE;&#x6809;&#x5B54;&#x6247;&#x8D1D;&#x3002;

Lectines sont une famille de prot&eacute;ines glucides-reconnaissance qui jouent un r&ocirc;le crucial dans l&#39;immunit&eacute; inn&eacute;e. Dans cette &eacute;tude, un nouveau g&egrave;ne de la lectine (CfLec-2) a &eacute;t&eacute; clon&eacute; de Chlamys farreri par des approches EST et de la RACE. L&#39;ADNc pleine longueur de CfLec-2 &eacute;tait compos&eacute; de 708bp, un domaine de glucides-reconnaissance typique forme longue de 130 r&eacute;sidus d&#39;encodage. La s&eacute;quence d&#39;acides amin&eacute;s a montr&eacute; forte similarit&eacute; &agrave; Brevican chez Homo sapiens, lectine de type C-1 et 2-lectine &agrave; Anguilla japonica. Les fragments d&#39;ADNc codant le peptide mature du CfLec-2 a &eacute;t&eacute; recombin&eacute;s dans le plasmide pET-32 a (+) et exprim&eacute; chez Escherichia coli Rosseta-Gami (DE3). La prot&eacute;ine recombinante de (rCfLec-2) CfLec-2 a pr&eacute;sent&eacute; une activit&eacute; agr&eacute;gatives vers Staphylococcus haemolyticus et l&#39;agglutination peut &ecirc;tre inhib&eacute;e par le d-mannose mais pas d&#39;EDTA ou le d-galactose, indiquant que CfLec-2 &eacute;tait une lectine ind&eacute;pendante de Ca2 +. En outre, rCfLec-2 pourrait emp&ecirc;cher la croissance d&#39;Escherichia coli TOP10F&#39;. Ces r&eacute;sultats sugg&egrave;rent que CfLec-2 a peut-&ecirc;tre particip&eacute; &agrave; la reconnaissance et l&#39;apurement des bact&eacute;ries pathog&egrave;nes dans les p&eacute;toncles.

Une lectine (CfLec-2) agr&eacute;gation Staphylococcus haemolyticus de p&eacute;toncle Chlamys farreri.

Lektine sind eine Familie der Kohlenhydrat-Anerkennung-Proteine die entscheidende Rolle in der angeborenen Immunit&auml;t zu spielen. In dieser Studie wurde ein neues Lektin (CfLec-2)-gen durch EST und Rennen Ans&auml;tze von Chlamys Farreri geklont. Das in cDNA CfLec-2 wurde von 708bp, Codierung einer typischen Langform-Kohlenhydrat-Anerkennung-Dom&auml;ne 130 R&uuml;ckst&auml;nde komponiert. Die hergeleiteten Aminos&auml;ure-Sequenz zeigte hohe &Auml;hnlichkeit Brevican in der Homo Sapiens, das C-f&ouml;rmige Lektin-1 und Lektin-2 in Anguilla Japonica. Das cDNA-Fragment, das ausgereifte Peptid des CfLec-2-Codierung war rekombiniert in Plasmid Haustier-32a (+) und in Escherichia coli Rosseta-Gami (DE3) ausgedr&uuml;ckt. Das rekombinante Protein CfLec-2 (rCfLec-2) stellte aggregative Aktivit&auml;t gegen&uuml;ber Staphylococcus Haemolyticus und die Agglutination von d-Mannose aber nicht EDTA oder d-Galaktose, darauf hinweist, dass CfLec-2 eine unabh&auml;ngige Ca2 +-Lektin war gehemmt werden k&ouml;nnte. RCfLec-2 konnte dar&uuml;ber hinaus das Wachstum von E. Coli TOP10F unterdr&uuml;cken &#39;. Diese Ergebnisse vorgeschlagen, dass die Anerkennung und die Abfertigung von bakteriellen Krankheitserregern bei Jakobsmuschel CfLec-2 vielleicht beteiligt war.

Ein Lektin (CfLec-2) aggregieren Staphylococcus Haemolyticus aus Muschel Chlamys Farreri.

Le lectine sono una famiglia di proteine carboidrati-riconoscimento che giocano un ruolo cruciale nell&#39;immunit&agrave; innata. In questo studio, un nuovo gene lectin (CfLec-2) &egrave; stato clonato da Chlamys farreri da EST e gara di approcci. Il full-length cDNA di CfLec-2 era composto da 708bp, un dominio di carboidrati-riconoscimento tipica forma lunga di 130 residui di codifica. La sequenza aminoacidica dedotta ha mostrato elevata somiglianza a Brevican in Homo sapiens, C-type lectin-1 e lectina-2 in Anguilla japonica. Il frammento di cDNA che codifica il peptide maturo di CfLec-2 era ricombinato in plasmide pET-32a (+) ed espresso in Escherichia coli Rosetta-Gami (DE3). CfLec-2 (rCfLec-2) proteina ricombinante esposte attivit&agrave; aggregativa verso Staphylococcus haemolyticus, e l&#39;agglutinazione potrebbe essere inibito dal d-mannosio ma non EDTA o d-galattosio, che indica che CfLec-2 era un lectina Ca2 + indipendente. Inoltre, rCfLec-2 potrebbe sopprimere la crescita di e. coli TOP10F&#39;. Questi risultati ha suggerito che CfLec-2 &egrave; stato forse coinvolti nel riconoscimento e liquidazione degli agenti patogeni batterici nel pettine.

Una lectina (CfLec-2) aggregando Staphylococcus haemolyticus dal pettine farreri Chlamys.

&#x30EC;&#x30AF;&#x30C1;&#x30F3;&#x306F;&#x3001;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x306B;&#x304A;&#x3051;&#x308B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x70AD;&#x6C34;&#x5316;&#x7269;&#x8A8D;&#x8B58;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x5BB6;&#x65CF;&#x3067;&#x3059;&#x3002;&#x3053;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;, &#x65B0;&#x898F;&#x30EC;&#x30AF;&#x30C1;&#x30F3; (CfLec 2) &#x907A;&#x4F1D;&#x5B50;&#x5C64;&#x7523; chlamys &#x5C5E; farreri &#x304B;&#x3089;&#x7C73;&#x56FD;&#x6771;&#x90E8;&#x6A19;&#x6E96;&#x6642;&#x523B;&#x3068;&#x30EC;&#x30FC;&#x30B9;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306B;&#x3088;&#x3063;&#x3066;&#x8907;&#x88FD;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x5B8C;&#x5168;&#x9577; cDNA CfLec 2 &#x306E;&#x5178;&#x578B;&#x7684;&#x306A;&#x9577;&#x3044;&#x5F62;&#x5F0F;&#x306E;&#x70AD;&#x6C34;&#x5316;&#x7269;&#x8A8D;&#x8B58;&#x30C9;&#x30E1;&#x30A4;&#x30F3; 130 &#x6B8B;&#x57FA;&#x306E;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C9;&#x3001;708bp &#x306E;&#x69CB;&#x6210;&#x3055;&#x308C;&#x3066;&#x3044;&#x305F;&#x3002;&#x63A8;&#x5B9A;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x914D;&#x5217;&#x985E;&#x4F3C;&#x6027;&#x9AD8;&#x3044; Brevican &#x306B;&#x30DB;&#x30E2;&#x30B5;&#x30D4;&#x30A8;&#x30F3;&#x30B9;&#x3001;C &#x578B;&#x30EC;&#x30AF;&#x30C1;&#x30F3;-1 &#x304A;&#x3088;&#x3073;&#x30EC;&#x30AF;&#x30C1;&#x30F3;-2 &#x30A2;&#x30F3;&#x30AE;&#x30E9; &#x30B8;&#x30E3;&#x30DD;&#x30CB;&#x30AB;&#x3067;&#x793A;&#x3057;&#x305F;&#x3002;&#x6210;&#x719F;&#x30DA;&#x30D7;&#x30C1;&#x30C9; CfLec 2 &#x306E;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0; cDNA &#x65AD;&#x7247;&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30C9; &#x30DA;&#x30C3;&#x30C8; 32a (+) &#x306B;&#x7D44;&#x63DB;&#x3048;&#x305F;&#x3057;&#x3001;&#x5927;&#x8178;&#x83CC;&#x30ED;&#x30BC;&#x30C3;&#x30BF;&#x307F;&#x3060;&#x308C; (DE3) &#x8868;&#x73FE;&#x3002;&#x7D44;&#x63DB;&#x3048; CfLec 2 &#xFF08;rCfLec 2&#xFF09; &#x86CB;&#x767D;&#x8CEA;&#x8695;&#x306E;&#x6D3B;&#x52D5;&#x3092;&#x57FA;&#x3001;&#x51FA;&#x5C55;&#x3057;&#x3001;&#x51DD;&#x96C6; d &#x2010; &#x30DE;&#x30F3;&#x30CE;&#x30FC;&#x30B9;&#x304C;&#x306A;&#x3044; EDTA &#x307E;&#x305F;&#x306F; d-&#x30AC;&#x30E9;&#x30AF;&#x30C8;&#x30FC;&#x30B9;, CfLec 2 ca 2 + &#x306E;&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x30EC;&#x30AF;&#x30C1;&#x30F3;&#x3067;&#x3042;&#x3063;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x306B;&#x3088;&#x3063;&#x3066;&#x6291;&#x5236;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;rCfLec 2 &#x5927;&#x8178;&#x83CC; TOP10F &#x306E;&#x6210;&#x9577;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B; &#39;&#x3002;CfLec 2 &#x304C;&#x8A8D;&#x8B58;&#x3068;&#x30DB;&#x30BF;&#x30C6;&#x306E;&#x7D30;&#x83CC;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x306E;&#x30AF;&#x30EA;&#x30A2;&#x30E9;&#x30F3;&#x30B9;&#x3067;&#x304A;&#x305D;&#x3089;&#x304F;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x305F;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#x30EC;&#x30AF;&#x30C1;&#x30F3; (CfLec 2) &#x96C6;&#x7D04;&#x9EC4;&#x8272;&#x30D6;&#x30C9;&#x30A6;&#x7403;&#x83CC;&#x57FA;&#x30DB;&#x30BF;&#x30C6;&#x5C64;&#x7523; chlamys &#x5C5E; farreri &#x304B;&#x3089;&#x3002;

Lectins&#xC5D0;&#xB294; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC7AC;&#xC0DD;&#xD560; &#xC218; &#xC788;&#xB294; &#xD0C4;&#xC218;&#xD654;&#xBB3C; &#xC778;&#xC2DD; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xAC00;&#xC871;&#xC785;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C;&#xB294; &#xC0C8;&#xB85C;&#xC6B4; lectin (CfLec-2) &#xC720;&#xC804;&#xC790;&#xB294; Chlamys farreri&#xC5D0;&#xC11C; EST&#xC640; &#xB808;&#xC774;&#xC2A4; &#xC811;&#xADFC; &#xD558; &#xC5EC; &#xBCF5;&#xC81C; &#xB429;&#xB2C8;&#xB2E4;. CfLec-2&#xC758; &#xC804;&#xCCB4; &#xAE38;&#xC774;&#xC758; cDNA 708bp, 130 &#xC794;&#xB958;&#xBB3C;&#xC758; &#xC804;&#xD615;&#xC801;&#xC778; &#xAE34; &#xD615;&#xD0DC;&#xB85C; &#xD0C4;&#xC218;&#xD654;&#xBB3C; &#xC778;&#xC2DD; &#xB3C4;&#xBA54;&#xC778; &#xC778;&#xCF54;&#xB529; &#xAD6C;&#xC131; &#xB418;&#xC5C8;&#xB2E4;. &#xCD94;&#xB860;&#xB41C; &#xC544;&#xBBF8;&#xB178;&#xC0B0; &#xC2DC;&#xD000;&#xC2A4; &#xD638;&#xBAA8; &#xC0AC;&#xD53C;&#xC5D4;&#xC2A4;, C &#xD0C0;&#xC785; lectin-1 &#xBC0F; &#xC575;&#xADC8;&#xB77C; japonica&#xC5D0; lectin-2&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; Brevican&#xC5D0; &#xB192;&#xC740; &#xC720;&#xC0AC;&#xC131;&#xC744; &#xBCF4;&#xC600;&#xB2E4;. CfLec-2&#xC758; &#xC131;&#xC219;&#xD55C; &#xD3A9;&#xD0C0;&#xC774;&#xB4DC; &#xC778;&#xCF54;&#xB529; cDNA &#xC870;&#xAC01; &#xD50C;&#xB77C;&#xC2A4; &#xBBF8;&#xB4DC; &#xC560;&#xC644; &#xB3D9;&#xBB3C;-32a (+)&#xC73C;&#xB85C; &#xC62E;&#xAE30;&#xB294; &#xACE0; &#xB300;&#xC7A5;&#xADE0; Rosseta &#xB3C4;&#xC640;&#xC8FC; (DE3)&#xB85C; &#xD45C;&#xD604;. &#xC7AC;&#xC870;&#xD569; CfLec-2 (rCfLec 2) &#xB2E8;&#xBC31;&#xC9C8; &#xD669;&#xC0C9; &#xD3EC;&#xB3C4; &#xC0C1; &#xAD6C; haemolyticus&#xB85C; aggregative &#xD65C;&#xB3D9;&#xC744; &#xC804;&#xC2DC; &#xD558; &#xACE0;&#xB294; &#xC735;&#xD569; &#xB4E4; &#xD558;&#xC9C0;&#xB9CC; &#xD558;&#xC9C0; EDTA &#xB610;&#xB294; d-galactose, CfLec 2&#xB294; Ca2 + &#xB3C5;&#xB9BD; lectin&#xB97C; &#xB098;&#xD0C0;&#xB0B4;&#xB294; d-&#xC2A4;&#xC5D0; &#xC758;&#xD574; &#xC800;&#xD574; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, rCfLec-2 &#xB300;&#xC7A5;&#xADE0; top10f&#xC758; &#xC131;&#xC7A5;&#xC744; &#xC5B5;&#xC81C;&#xD560; &#xC218; &#39;. &#xC774; &#xACB0;&#xACFC; CfLec 2 &#xC544;&#xB9C8;&#xB3C4; &#xC778;&#xC2DD;&#xACFC; &#xAC00;&#xB9AC;&#xBE44;&#xC5D0; &#xC138;&#xADE0;&#xC131; &#xBCD1;&#xC6D0; &#xCCB4;&#xC758; &#xD074;&#xB9AC;&#xC5B4;&#xB7F0;&#xC2A4;&#xC5D0; &#xCC38;&#xC5EC; &#xD588;&#xB2E4; &#xC81C;&#xC548; &#xD588;&#xB2E4;.

Lectin (CfLec-2) &#xC9D1;&#xACC4; &#xD669;&#xC0C9; &#xD3EC;&#xB3C4; &#xC0C1; &#xAD6C; haemolyticus &#xAC00;&#xB9AC;&#xBE44; Chlamys farreri&#xC5D0;&#xC11C;.

Lectinas son una familia de prote&iacute;nas de reconocimiento de carbohidratos que juegan papeles cruciales en la inmunidad innata. En este estudio, un nuevo gen de la lectina (CfLec-2) fue reproducido de Chlamys farreri por acercamientos EST y raza. El cDNA de longitud completo de CfLec-2 estaba compuesto por 708bp, un dominio de reconocimiento de carbohidratos t&iacute;picos forma larga de 130 residuos de codificaci&oacute;n. La secuencia de amino&aacute;cidos deducida demostr&oacute; alta similitud a Brevican en el Homo sapiens, lectinas tipo C-1 y 2-lectina en Anguilla japonica. El fragmento de cDNA codificaci&oacute;n el p&eacute;ptido maduro de CfLec-2 fue recombinado en pl&aacute;smido pET-32a (+) y expresado en Escherichia coli Rosseta-Gami (DE3). La prote&iacute;na recombinante de (rCfLec-2) CfLec-2 mostraron actividad agregativo hacia Staphylococcus haemolyticus y la aglutinaci&oacute;n puede ser inhibida por d-Mannosio pero no EDTA o d-galactosa, indicando que CfLec-2 era una lectina independiente de Ca2 +. Por otra parte, rCfLec-2 podr&iacute;a suprimir el crecimiento de e. coli TOP10F&#39;. Estos resultados sugirieron que CfLec-2 tal vez estuvo implicado en el reconocimiento y la remoci&oacute;n de los pat&oacute;genos bacterianos en Vieira.

Una adici&oacute;n lectinas (CfLec-2) Staphylococcus haemolyticus de Vieira Chlamys farreri.

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3h post-challenge. After a decrease at 6h, the expression level increased again and reached 207.8-fold at 12h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was 173 nmol L(-1). These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response.

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain.

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036bp ESPLC cDNA contained an 831bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67bp, and a 3'-UTR of 138bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2h and 12h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E. sinensis.

cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis.

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds, interacts with a variety of proteins and therefore regulates their activities. One CypA (designated CfCypA) cDNA was cloned from Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CfCypA consisted of 1,248 nucleotides with a canonical polyadenylation signal sequence AATAAA, a poly (A) tail, and an open reading frame (ORF) of 495 nucleotides encoding a polypeptide of 164 amino acids. The deduced amino acid sequence shared high similarity with CypA from the other species, indicating that CfCypA should be a new member of the CypA family. Quantitative real-time (RT) PCR was employed to assess the mRNA expression of CfCypA in various tissues and its temporal expression in haemocytes and gonad of scallops challenged with Vibrio anguillarum. The mRNA transcripts of CfCypA could be detected in all the examined tissues with highest expression level in gonad. After bacterial challenge, the expression level of CfCypA was almost unchanged in haemocytes, but up-regulated in gonad and increased to the peak (22.59-fold; P < 0.05) at 4 h post-injection, and then dropped to the original level at 8 h post-injection. These results indicated that CfCypA was constitutive expressed in haemocytes, but could be induced in gonad, and perhaps played a critical role in response to the bacterial challenge in gonad.

A cyclophilin A inducible expressed in gonad of zhikong scallop Chlamys farreri.

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA.

Crustin is a cysteine-rich antibacterial peptide which widely distributes within decapod crustaceans. In the present study, the cDNA encoding crustin-like peptide (designated CrusEs) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag analysis. The full-length cDNA of CrusEs was of 796bp, containing a 5' untranslated region (UTR) of 214bp, a 3' UTR of 267bp with a poly(A) tail, and an open reading frame (ORF) of 315bp encoding a polypeptide of 104 amino acids including a putative signal peptide of 21 amino acids. A WAP domain and the consensus framework existing in class I crustins were both identified in CrusEs, suggesting that CrusEs is a new member of type I crustins. Quantitative real-time RT-PCR was employed to examine the expression of CrusEs, and its mRNA transcript was mainly detected in haemocytes and gill. The CrusEs mRNA transcript in haemocytes was down-regulated after the challenge with Gram-positive bacteria Micrococcus luteus, while Gram-negative bacteria Listonella anguillarum did not induce significant variation of CrusEs mRNA. The mature peptide of CrusEs was cloned into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli, and the recombinant CrusEs inhibited the growth of Gram-positive bacteria with low MIC. The results indicate that CrusEs is a potent antibacterial protein against Gram-positive bacteria infection, and it may play an important role in innate immune response of E. sinensis.

Molecular characterization and expression of a crustin-like gene from Chinese mitten crab, Eriocheir sinensis.

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated CrusEs2) was cloned from Chinese mitten crab Eriocheir sinensis by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CrusEs2 was of 1237 bp, containing a 5' untranslated region (UTR) of 12 bp, a 3' UTR of 886 bp with a poly (A) tail, and an open reading frame (ORF) of 339 bp encoding a polypeptide of 112 amino acids with a signal peptide of 19 amino acids. The CrusEs2 contained a typical WAP domain, but lacked the Gly-rich domain of the type II crustin and the Cys-rich region present in both type I and type II crustin, suggesting that CrusEs2 should be classified as a type III crustin. The mRNA transcripts of CrusEs2 could be detected in haemocytes and gill, and its expression level in haemocytes was up-regulated after Listonella anguillarum challenge, while decreased after Micrococcus luteus challenge. The mature peptide coding region of CrusEs2 was cloned into pET-21a+ and expressed in Escherichia coli. The purified recombinant CrusEs2 inhibited the growth of Gram-positive bacteria at MIC of 0.093-0.37 μM. The results indicated that CrusEs2 was involved in immune response of E. sinensis against bacterial challenge.

A novel type III crustin (CrusEs2) identified from Chinese mitten crab Eriocheir sinensis.

CTLA4 gene variation associates with multiple autoimmune disorders, including type 1 diabetes. The CTLA4 susceptibility allele was found to generate decreased levels of mRNA encoding soluble CTLA-4 (sCTLA-4) relative to the full-length isoform, the functional consequence of which is as yet unknown. In this study, we investigated the contribution of sCTLA-4 to immune regulation with the aim to elucidate the functional basis of the disease association of CTLA4.

The soluble CTLA-4 splice variant protects from type 1 diabetes and potentiates regulatory T-cell function.

PTPN22 encodes the lymphoid tyrosine phosphatase (LYP) and is the second strongest non-HLA genetic risk factor for type 1 diabetes. The PTPN22 susceptibility allele generates an LYP variant with an arginine-to-tryptophan substitution at position 620 (R620W) that has been reported by several studies to impart a gain of function. However, a recent report investigating both human cells and a knockin mouse model containing the R620W homolog suggested that this variation causes faster protein degradation. Whether LYP R620W is a gain- or loss-of-function variant, therefore, remains controversial. To address this issue, we generated transgenic NOD mice (nonobese diabetic) in which Ptpn22 can be inducibly silenced by RNA interference. We found that Ptpn22 silencing in the NOD model replicated many of the phenotypes observed in C57BL/6 Ptpn22 knockout mice, including an increase in regulatory T cells. Notably, loss of Ptpn22 led to phenotypic changes in B cells opposite to those reported for the human susceptibility allele. Furthermore, Ptpn22 knockdown did not increase the risk of autoimmune diabetes but, rather, conferred protection from disease. Overall, to our knowledge, this is the first functional study of Ptpn22 within a model of type 1 diabetes, and the data do not support a loss of function for the PTPN22 disease variant.

Peilin Zheng

Center for Human Immunobiology

Texas Children's Hospital

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer