hela-based-cell-free-expression-systems-for-expression-plasmodium

HeLa Based Cell Free Expression Systems for Expression of <em>Plasmodium</em> Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

Cleveland State University

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.

A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling.

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosine-sorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria.

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites.

In the present study, Rhop-3 polymorphism among Plasmodium falciparum field and laboratory isolates and among rodent Plasmodium species was investigated and identified. The Rhop-3 gene was found in all Plasmodium species so far tested. The overall structure of the Rhop-3 protein was found conserved among P. falciparum, Plasmodium yoelii, and Plasmodium berghei. However, it was more conserved among rodent Plasmodium species than between P. falciparum and Plasmodium vivax. The most conserved regions of Rhop-3 are the second half of exon 6 (amino acid #548 to #665) and the beginning of exon 3 (amino acid #59 to #210). Recombinant C-terminal partial and full-length Rhop-3 proteins of P. yoelii and P. berghei were expressed in Escherichia coli and purified. Immunization-challenge experiments in mice using recombinant Rhop-3 proteins led to a delay in parasite development and protected mice from a homologous lethal challenge infection. In a group of eight outbred Carworth Farm White (CFW) mice immunized with P. yoelii C-terminal recombinant His-Y1412 protein, three mice (37.5%) were protected from a lethal P. yoelii challenge. In BALB/cJ mice one mouse (20%) survived the infection. Immunization of mice with P. berghei recombinant full-length Rhop-3 protein in BALB/cJ mice led to a 40% survival from lethal P. berghei challenge. CFW mice immunized with P. berghei recombinant full-length Rhop-3 protein showed a significant delay in parasite development with a heterologous P. yoelii challenge. The Rhop-3 protein is a promising candidate for an asexual stage malaria vaccine.

Rhop-3 protein conservation among Plasmodium species and induced protection against lethal P. yoelii and P. berghei challenge.

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria.

Plasmodium falciparum Maurer's clefts participate in the transport of macromolecules within the cytoplasm, including the transport of virulence proteins to the erythrocyte membrane surface. We identified a family of genes PfMC-2TM encoding transmembrane proteins located within the intramembranous network of the infected erythrocyte using monoclonal antibody SP1C1. The distribution of the PfMC-2TM protein family within domains of the network was investigated by colocalization and confocal microscopy studies using monoclonal antibody SP1C1 specific for PFMC-2TM and monoclonal antibody SP1A6 specific for the130 kDa Maurer's cleft protein. Peptide-specific antibodies were prepared against six peptides from different domains of PfMC-2TM and used with the Mabs, as well as known antibodies specific to Maurer's clefts proteins (ring-expressed protein and membrane-associated histidine-rich protein 1), the erythrocyte membrane protein 1 (PfEMP-1), and serine-rich antigen in colocalization studies. We show that PfMC-2TM is located in the Maurer's clefts throughout the intracellular blood stage, and immunoelectron microscopy shows domains of PfMC-2TM localized in the parasitophorous vacuole and parasitophorous vacuole membrane. The distribution of the 130 kDa Maurer's cleft protein changes from within the parasite to the clefts during intracellular development as the parasite matures from young trophozoite to segmented schizont.

Proteins of the Plasmodium falciparum two transmembrane Maurer's cleft protein family, PfMC-2TM, and the 130 kDa Maurer's cleft protein define different domains of the infected erythrocyte intramembranous network.

Maurer's clefts (MCs) are membranous structures that are formed by Plasmodium falciparum and used by the parasite for protein sorting and protein export. Virulence proteins, as well as other proteins used to remodel the erythrocyte, are exported. Discontinuity between major membrane compartments within the infected erythrocyte cytoplasm suggests multiple traffic routes for exported proteins. The sequences of the conserved Plasmodium export element seem insufficient for export of all parasite proteins. The parasite displays remarkable versatility in the types of proteins exported to the MCs and in the functions of the proteins within the MCs. In this Review, protein export to the MCs and the role of the MCs in the transport of proteins to the erythrocyte membrane are summarized.

The role of the Maurer's clefts in protein transport in Plasmodium falciparum.

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The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 Â°C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 Â°C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker.

Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria.

Les anticorps sp&eacute;cifiques de la prot&eacute;ine de paludisme, Rhop-3 et FL-Rhop-3, ont &eacute;t&eacute; immobilis&eacute;s sur la surface d&#39;une &eacute;lectrode d&#39;or modifi&eacute;e par la cyst&eacute;amine. L&#39;or collo&iuml;dal a &eacute;t&eacute; utilis&eacute; pour am&eacute;liorer le signal de d&eacute;tection d&#39;antig&egrave;nes Rhop-3. L&#39;anticorps Rhop-3 a &eacute;t&eacute; &eacute;galement immobilis&eacute; sur des &eacute;lectrodes en or pr&eacute;activ&eacute;es avec dithiobis(succinimidyl proprionate) (DSP). Immobilisation a &eacute;t&eacute; r&eacute;alis&eacute;e &agrave; temp&eacute;rature ambiante et &agrave; 37 &deg; C. Voltam&eacute;trie cyclique (CV) a &eacute;t&eacute; utilis&eacute;e pour surveiller l&#39;interaction entre l&#39;anticorps immobilis&eacute; et son antig&egrave;ne apparent&eacute; en solution, &agrave; l&#39;aide de ferricyanure, K3Fe (CN) 6, comme d&eacute;claration &eacute;lectroactifs sonde. Tests indiquent la reconnaissance de la prot&eacute;ine Rhop-3 par l&#39;anticorps immobilis&eacute;. Reconnaissance de l&#39;antig&egrave;ne est stimul&eacute;e par une incubation &agrave; 37 &deg; C par rapport &agrave; l&#39;incubation &agrave; temp&eacute;rature ambiante. Nos r&eacute;sultats sugg&egrave;rent qu&#39;une immunosensor peut &ecirc;tre d&eacute;velopp&eacute; et optimis&eacute; pour faciliter la d&eacute;tection des antig&egrave;nes Rhop-3 &eacute;chantillons de patients atteints de paludisme. Que l&#39;on sait, c&#39;est la premi&egrave;re immunosensor amp&eacute;rom&eacute;trique ciblant l&#39;antig&egrave;ne Rhop-3 comme un biomarqueur de paludisme.

Sonde fonctionnalisation avec un anticorps Rhop-3: vers une immunosensor d&#39;antig&egrave;ne Rhop-3 pour la d&eacute;tection du paludisme.

Der Antik&ouml;rper, die spezifisch f&uuml;r die Malaria-Protein, Rhop-3 und FL-Rhop-3, wurden auf der Oberfl&auml;che einer gold-Elektrode mit Cysteamin modifizierte immobilisiert. Kolloidales Gold wurde verwendet, um das Erkennungssignal f&uuml;r Rhop-3-Antigene zu verbessern. Der Rhop-3-Antik&ouml;rper wurde auch auf gold Elektroden vorhanden mit Dithiobis(succinimidyl proprionate) (DSP) immobilisiert. Immobilisierung erfolgte bei Raumtemperatur und bei 37 &deg; C. Cyclovoltammetrie (CV) wurde verwendet, um die Interaktion zwischen der immobilisierte Antik&ouml;rper und seine cognate Antigen in-L&ouml;sung, mit Ferricyanide, K3Fe (CN) 6, als Berichterstattung Elektroaktive Sonde &uuml;berwachen. Tests zeigen Anerkennung von Rhop-3-Protein von der immobilisierte Antik&ouml;rper. Antigen-Anerkennung wurde durch Inkubation bei 37 &deg; C Raumtemperatur Inkubation gegen&uuml;ber gef&ouml;rdert. Unsere Ergebnisse legen nahe, dass ein Immunosensor entwickelt und optimiert, um die Erkennung von Rhop-3-Antigenen in Malaria-Patienten zu helfen. Soweit uns bekannt ist, ist dies die erste Amperometrisch Immunosensor Zielgruppenadressierung Rhop-3-Antigen als Malaria Biomarker.

Pr&uuml;fpunkt Funktionalisierung mit einem Rhop-3-Antik&ouml;rper: in Richtung einer Rhop-3-Antigen-Immunosensor zum Nachweis von Malaria.

Gli anticorpi specifici per la proteina di malaria, Rhop-3 e FL-Rhop-3, sono stati immobilizzati sulla superficie di un elettrodo d&#39;oro modificato con cisteammina. L&#39;oro colloidale &egrave; stato usato per migliorare il segnale di rilevazione per gli antigeni Rhop-3. L&#39;anticorpo Rhop-3 &egrave; stato immobilizzato anche su elettrodi in oro preattivati con dithiobis(succinimidyl proprionate) (DSP). Immobilizzazione &egrave; stata eseguita a temperatura ambiente e a 37 &deg; C. Ciclovoltammetria (CV) &egrave; stato utilizzato per monitorare l&#39;interazione tra l&#39;anticorpo immobilizzato e relativo antigene cognata in soluzione, utilizzando ferricianuro, K3Fe (CN) 6, come segnalazione elettroattivi sonda. Prove indicano riconoscimento della proteina Rhop-3 con l&#39;anticorpo immobilizzato. Riconoscimento dell&#39;antigene &egrave; stata migliorata di incubazione a 37 &deg; C, confrontato con incubazione a temperatura ambiente. I nostri risultati suggeriscono che un immunosensor pu&ograve; essere sviluppato e ottimizzato per facilitare la rilevazione degli antigeni Rhop-3 in campioni di pazienti la malaria. Per quanto siamo consapevoli, questo &egrave; il primo immunosensor amperometrico targeting antigene Rhop-3 come un biomarker di malaria.

Sonda funzionalizzazione con un anticorpo Rhop-3: verso un immunosensor di antigene Rhop-3 per il rilevamento della malaria.

&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092; Rhop 3&#x3001;&#x30D5;&#x30ED;&#x30EA;&#x30C0;-Rhop-3 &#x7279;&#x5B9A;&#x306E;&#x6297;&#x4F53;&#x3068;&#x30B7;&#x30B9;&#x30C6;&#x30A2;&#x30DF;&#x30F3;&#x3092;&#x4FEE;&#x98FE;&#x91D1;&#x96FB;&#x6975;&#x306E;&#x8868;&#x9762;&#x306B;&#x56FA;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x91D1;&#x30B3;&#x30ED;&#x30A4;&#x30C9; Rhop 3 &#x6297;&#x539F;&#x306E;&#x691C;&#x51FA;&#x4FE1;&#x53F7;&#x3092;&#x5F37;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;Rhop 3 &#x6297;&#x4F53;&#x306F;&#x307E;&#x305F; dithiobis(succinimidyl proprionate) (DSP) &#x30E9;&#x30A4;&#x30BB;&#x30F3;&#x30B9;&#x8A8D;&#x8A3C;&#x6E08;&#x307F;&#x91D1;&#x96FB;&#x6975;&#x4E0A;&#x56FA;&#x5B9A;&#x5316;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x56FA;&#x5B9A;&#x5316;&#x306F;, &#x5BA4;&#x6E29;&#x3068; 37 &#x2103; &#x3067;&#x884C;&#x3063;&#x305F;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#x30DC;&#x30EB;&#x30BF;&#x30F3;&#x30E1;&#x30C8;&#x30EA;&#x30FC; (CV) &#x56FA;&#x5B9A;&#x5316;&#x6297;&#x4F53;&#x3068;&#x305D;&#x306E;&#x540C;&#x7A2E;&#x6297;&#x539F;&#x30D5;&#x30A7;&#x30EA;&#x30B7;&#x30A2;&#x30F3;&#x3001;K3Fe (&#x30B2;&#x30EB;&#x30ED;&#x30DC;&#x30C3;&#x30C8; &#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x3092;&#x5831;&#x544A;&#x3068;&#x3057;&#x3066; CN) 6 &#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30C6;&#x30B9;&#x30C8;&#x306B;&#x3088;&#x308B;&#x56FA;&#x5B9A;&#x5316;&#x6297;&#x4F53; Rhop 3 &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x8A8D;&#x8B58;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6297;&#x539F;&#x8A8D;&#x8B58;&#x3068;&#x90E8;&#x5C4B;&#x306E;&#x6E29;&#x5EA6;&#x306E;&#x5B75;&#x5316;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066; 37 &#x2103; &#x3067;&#x5B75;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F37;&#x5316;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x514D;&#x75AB;&#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x3092;&#x958B;&#x767A;&#x3057;&#x3001;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x60A3;&#x8005;&#x304B;&#x3089;&#x306E;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x3067; Rhop 3 &#x6297;&#x539F;&#x306E;&#x691C;&#x51FA;&#x3092;&#x652F;&#x63F4;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x6700;&#x9069;&#x5316;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x77E5;&#x3063;&#x3066;&#x3044;&#x308B;&#x9650;&#x308A;&#x3067;&#x306F;&#x3001;&#x3053;&#x308C;&#x306F;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x306E;&#x30D0;&#x30A4;&#x30AA; &#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3068;&#x3057;&#x3066;&#x306E; Rhop 3 &#x6297;&#x539F;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x6700;&#x521D;&#x306E;&#x30A2;&#x30F3;&#x30DA;&#x30ED; &#x30E1;&#x30C8;&#x30EA;&#x30C3;&#x30AF; &#x30A4;&#x30E0;&#x30CE; &#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x3067;&#x3059;&#x3002;

Rhop 3 &#x6297;&#x4F53;&#x591A;&#x6A5F;&#x80FD;&#x5316;&#x306B;&#x4F34;&#x3044;&#x30D7;&#x30ED;&#x30FC;&#x30D6;: Rhop 3 &#x6297;&#x539F;&#x514D;&#x75AB;&#x691C;&#x51FA;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x306E;&#x305F;&#x3081;&#x306B;&#x5411;&#x304B;&#x3063;&#x3066;&#x3002;

Rhop-3, &#xADF8;&#xB9AC;&#xACE0; &#xD50C;&#xB85C;&#xB9AC;&#xB2E4;-Rhop-3, &#xB9D0;&#xB77C;&#xB9AC;&#xC544; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xC704;&#xD574; &#xD2B9;&#xC815; &#xD56D; &#xCCB4; cysteamine &#xC218;&#xC815; &#xACE8;&#xB4DC; &#xC804;&#xADF9;&#xC758; &#xD45C;&#xBA74;&#xC5D0; &#xC6C0;&#xC9C1;&#xC77C; &#xC218; &#xC788;&#xC5C8;&#xB2E4;. &#xCF5C; &#xB85C;&#xC774;&#xB4DC; &#xACE8;&#xB4DC; Rhop-3 &#xD56D; &#xC6D0;&#xC5D0; &#xB300; &#xD55C; &#xD0D0;&#xC9C0; &#xC2E0;&#xD638;&#xB97C; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0A4;&#xAE30; &#xC704;&#xD574; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xB2E4;. Rhop-3 &#xD56D; &#xCCB4;&#xB294; &#xB610;&#xD55C; dithiobis(succinimidyl proprionate) (DSP)&#xC640; preactivated &#xACE8;&#xB4DC; &#xC804;&#xADF9;&#xC5D0; &#xC6C0;&#xC9C1;&#xC77C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC2E4; &#xC628;&#xC5D0;&#xC11C; &#xD558; &#xACE0; 37 &deg; C&#xC5D0;&#xC11C; immobilization &#xC218;&#xD589; &#xC8FC;&#xAE30;&#xC801; voltammetry (CV) &#xC6C0;&#xC9C1;&#xC774;&#xC9C0; &#xD56D; &#xCCB4;&#xC640; ferricyanide, K3Fe (CN) 6, electroactive &#xAC80;&#xC0AC; &#xBCF4;&#xACE0;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC194;&#xB8E8;&#xC158;&#xC5D0;&#xC11C; &#xB3D9;&#xC871;&#xC758; &#xD56D; &#xC6D0; &#xAC04;&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9;&#xD55C;. &#xD14C;&#xC2A4;&#xD2B8; &#xC6C0;&#xC9C1;&#xC774;&#xC9C0; &#xD56D; &#xCCB4;&#xC5D0; &#xC758;&#xD574; Rhop 3 &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC778;&#xC2DD;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xD56D; &#xC6D0; &#xC778;&#xC2DD; &#xC778;&#xD050;&#xBCA0;&#xC774;&#xC158; &#xB8F8; &#xC628;&#xB3C4; &#xBD80; &#xD654;&#xC5D0; &#xBE44;&#xD574; 37 &deg; C&#xC5D0; &#xC758;&#xD574; &#xD5A5;&#xC0C1; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; immunosensor &#xAC1C;&#xBC1C; &#xBC0F; &#xB9D0;&#xB77C;&#xB9AC;&#xC544; &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xC0D8;&#xD50C; Rhop 3 &#xD56D; &#xC6D0;&#xC758; &#xAC80;&#xCD9C;&#xC744; &#xB3D5;&#xAE30; &#xC704;&#xD574; &#xCD5C;&#xC801;&#xD654; &#xB41C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAD8C;&#xD574; &#xB4DC;&#xB9BD;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC54C;&#xACE0; &#xC788;&#xB2E4;, &#xC774;&#xAC83;&#xC740; &#xCCAB; &#xBC88;&#xC9F8; amperometric immunosensor &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xB9D0;&#xB77C;&#xB9AC;&#xC544; biomarker&#xB85C; Rhop-3 &#xD56D; &#xC6D0; &#xC774;&#xB2E4;.

Rhop-3 &#xD56D; &#xCCB4;&#xB85C; &#xAE30;&#xB2A5;&#xD654; &#xD504;&#xB85C;&#xBE0C;: &#xB9D0;&#xB77C;&#xB9AC;&#xC544;&#xC758; &#xAC80;&#xCD9C;&#xC744; &#xC704;&#xD55C; Rhop 3 &#xD56D; &#xC6D0; immunosensor&#xC744; &#xD5A5;&#xD574;.

El anticuerpo espec&iacute;fico para la prote&iacute;na de la malaria, Rhop-3 y FL-Rhop-3, fueron inmovilizados en la superficie de un electrodo de oro modificado con cisteamina. Oro coloidal fue utilizado para mejorar la se&ntilde;al de detecci&oacute;n de ant&iacute;genos de Rhop-3. El anticuerpo de Rhop-3 tambi&eacute;n fue inmovilizado en electrodos de oro previamente activados con dithiobis(succinimidyl proprionate) (DSP). Inmovilizaci&oacute;n se realiz&oacute; a temperatura ambiente y a 37 &deg; C. Voltametr&iacute;a c&iacute;clica (CV) fue utilizada para supervisar la interacci&oacute;n entre el anticuerpo inmovilizado y su cognado ant&iacute;geno en soluci&oacute;n, utilizando ferricianuro, K3Fe (CN) 6, como informes electroactivos sonda. Pruebas indican el reconocimiento de la prote&iacute;na Rhop-3 por el anticuerpo inmovilizado. Reconocimiento del ant&iacute;geno fue realzada por la incubaci&oacute;n a 37 &deg; C en comparaci&oacute;n con la incubaci&oacute;n de la temperatura. Nuestros resultados sugieren que un inmunosensor puede ser desarrollado y optimizado para facilitar la detecci&oacute;n de ant&iacute;genos de Rhop-3 muestras de pacientes con malaria. Como lo que somos conscientes, se trata de la primera inmunosensor amperom&eacute;trico dirigidos a ant&iacute;geno Rhop-3 como un biomarcador de la malaria.

Article	Year
A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling. Genome research\| PubMed ID: 15140830	2004
Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites. Journal of proteome research\| PubMed ID: 15473688	2004
Rhop-3 protein conservation among Plasmodium species and induced protection against lethal P. yoelii and P. berghei challenge. Parasitology research\| PubMed ID: 16541261	2006
Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria. Experimental parasitology\| PubMed ID: 18606406	2008
Proteins of the Plasmodium falciparum two transmembrane Maurer's cleft protein family, PfMC-2TM, and the 130 kDa Maurer's cleft protein define different domains of the infected erythrocyte i... Parasitology research\| PubMed ID: 19130087	2009
The role of the Maurer's clefts in protein transport in Plasmodium falciparum. Trends in parasitology\| PubMed ID: 19442584	2009
Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria. Analytical and bioanalytical chemistry\| PubMed ID: 22297859	2012

Tobili Sam-Yellowe

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