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Partager le résultat de cette recherche Interroger des sources externesPurification et caractérisation d’un antigène somatique et d’une protéase à cystéine excrétée-sécrétée de Fasciola hepatica à intérêt immunodiagnostic. / Ahmed Hemici
Titre : Purification et caractérisation d’un antigène somatique et d’une protéase à cystéine excrétée-sécrétée de Fasciola hepatica à intérêt immunodiagnostic. Type de document : texte imprimé Auteurs : Ahmed Hemici, Auteur ; Dalila Bendjeddou, Directeur de thèse Editeur : جامعة الإخوة منتوري قسنطينة Année de publication : 2019 Importance : 206 f. Format : 30 cm. Note générale : 2 copies imprimées disponibles
Langues : Français (fre) Catégories : Français - Anglais
BiologieTags : Biologie Animale: Immunologie Fasciola hepatica antigènes somatiques antigènes excrétés-sécrétés protéase typecystéine purification caractérisation ELISA Western blot somatic antigens excreted-secreted antigens cystéine like- protease characterization المستضدات الجسدية المستضدات الافرازية بروتيياز السستيين تنقية توصيف Index. décimale : 570 Sciences de la vie. Biologie Résumé :
The present study describes in its first experimental part, the isolation of an antigenic molecule from the total homogenate of Fasciola hepatica adult flukes, then purified to homogeneity using gel filtration chromatography followed by a specific affinity chromatography. Among the five antigenic fractions obtained by fractionation of the crude somatic extract on Sephadex G-200 gel, only the first fraction (F.1) gave a highly significant recipitation reaction with the Concanavalin A lectin. It was then purified to homogeneity through a ConA-Sepharose 4B affinity column and characterized by SDS-PAGE electrophoresis as a single-band with apparent MW of about 40 kDa. This pure molecule, whose immunoreactivity was very positive in the presence of rabbit specific antiserum, is therefore endowed with a significant antigenic activity. In addition, the immunodiagnostic reliability of this molecule was verified by Elisa; the assay using the purified fraction as antigen (ELISAPf) proved more sensitive than the Elisa using the excreted-secreted product (ELISAESP) (92.3% of sensitivity for Elisa(Pf) against 72% for Elisa(ESP)). However, the ELISA specificity using the purified fraction was slightly lower compared to that corresponding to excreted-secreted product (93.3% of specificity for ELISA(Pf) against 95.5% for ELISA(ESP)). In the second practical part, a proteolytic activity cysteine-like produced in the supernatant of adult fluke culture medium was purified and characterized by combination of acetone precipitation and two chromatographic methods. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9 U/mg and 31.5% recovery. Ultrafiltration was required to increase the purification fold to 13.1 with an overall specific activity of 2268.8 U/mg. The MW of the purified protease was estimated by SDSŔPAGE to be 22 kDa, while the proteolytic activity detection was carried out by zymography on non-denaturing SDSŔPAGE containing the casein as substrate. The enzyme has relative stability over a wide range of pH and temperature with optimal activity at pH 5.5 and 40 °C. Its activity was completely inhibited by specific inhibitors of cysteine proteases, namely 5 mM E-64 and 10 mM iodoacetamide. The practical interest of this purified molecule as an antigen was evaluated by Western blot using sera from sheep experimentally infected with metacercariae of F. hepatica. The pool of sera recognized a single band corresponding to this protease just after the 2nd week of infestation. The early detection of specific antibodies in the sera of infested animals suggests the application of this molecule to the immunological screening of fasciolosis by this technique.
Note de contenu :
Annexes.
Diplôme : Doctorat en sciences En ligne : ../theses/biologie/HEM7449.pdf Format de la ressource électronique : Permalink : index.php?lvl=notice_display&id=11235 Purification et caractérisation d’un antigène somatique et d’une protéase à cystéine excrétée-sécrétée de Fasciola hepatica à intérêt immunodiagnostic. [texte imprimé] / Ahmed Hemici, Auteur ; Dalila Bendjeddou, Directeur de thèse . - جامعة الإخوة منتوري قسنطينة, 2019 . - 206 f. ; 30 cm.
2 copies imprimées disponibles
Langues : Français (fre)
Catégories : Français - Anglais
BiologieTags : Biologie Animale: Immunologie Fasciola hepatica antigènes somatiques antigènes excrétés-sécrétés protéase typecystéine purification caractérisation ELISA Western blot somatic antigens excreted-secreted antigens cystéine like- protease characterization المستضدات الجسدية المستضدات الافرازية بروتيياز السستيين تنقية توصيف Index. décimale : 570 Sciences de la vie. Biologie Résumé :
The present study describes in its first experimental part, the isolation of an antigenic molecule from the total homogenate of Fasciola hepatica adult flukes, then purified to homogeneity using gel filtration chromatography followed by a specific affinity chromatography. Among the five antigenic fractions obtained by fractionation of the crude somatic extract on Sephadex G-200 gel, only the first fraction (F.1) gave a highly significant recipitation reaction with the Concanavalin A lectin. It was then purified to homogeneity through a ConA-Sepharose 4B affinity column and characterized by SDS-PAGE electrophoresis as a single-band with apparent MW of about 40 kDa. This pure molecule, whose immunoreactivity was very positive in the presence of rabbit specific antiserum, is therefore endowed with a significant antigenic activity. In addition, the immunodiagnostic reliability of this molecule was verified by Elisa; the assay using the purified fraction as antigen (ELISAPf) proved more sensitive than the Elisa using the excreted-secreted product (ELISAESP) (92.3% of sensitivity for Elisa(Pf) against 72% for Elisa(ESP)). However, the ELISA specificity using the purified fraction was slightly lower compared to that corresponding to excreted-secreted product (93.3% of specificity for ELISA(Pf) against 95.5% for ELISA(ESP)). In the second practical part, a proteolytic activity cysteine-like produced in the supernatant of adult fluke culture medium was purified and characterized by combination of acetone precipitation and two chromatographic methods. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9 U/mg and 31.5% recovery. Ultrafiltration was required to increase the purification fold to 13.1 with an overall specific activity of 2268.8 U/mg. The MW of the purified protease was estimated by SDSŔPAGE to be 22 kDa, while the proteolytic activity detection was carried out by zymography on non-denaturing SDSŔPAGE containing the casein as substrate. The enzyme has relative stability over a wide range of pH and temperature with optimal activity at pH 5.5 and 40 °C. Its activity was completely inhibited by specific inhibitors of cysteine proteases, namely 5 mM E-64 and 10 mM iodoacetamide. The practical interest of this purified molecule as an antigen was evaluated by Western blot using sera from sheep experimentally infected with metacercariae of F. hepatica. The pool of sera recognized a single band corresponding to this protease just after the 2nd week of infestation. The early detection of specific antibodies in the sera of infested animals suggests the application of this molecule to the immunological screening of fasciolosis by this technique.
Note de contenu :
Annexes.
Diplôme : Doctorat en sciences En ligne : ../theses/biologie/HEM7449.pdf Format de la ressource électronique : Permalink : index.php?lvl=notice_display&id=11235 Exemplaires (1)
Code-barres Cote Support Localisation Section Disponibilité HEM/7449 HEM/7449 Thèse Bibliothèque principale Thèses Disponible
Titre : Sélection in vitro pour la tolérance au stress hydrique chez le blé dur (Triticum durum Desf) : approche protéomique, transcriptomique et génétique. Type de document : texte imprimé Auteurs : Nadia Sandra Kacem, Auteur ; Abdelhamid Djekoun, Directeur de thèse ; Yordan Muhovski, Directeur de thèse Editeur : جامعة الإخوة منتوري قسنطينة Année de publication : 2017 Note générale : 2 copies imprimées disponibles
Langues : Français (fre) Catégories : Français - Anglais
BiologieTags : Durum wheat water stress PEG 6000 in vitro selection somaclonal
variation molecular marker diagonal two-dimensional electrophoresis , qRT-PCR Western blot Blé dur électrophorèse diagonale bidimensionnelle marqueur moléculaire sélection in vitro stress hydrique variation somaclonaleIndex. décimale : 570 Sciences de la vie. Biologie Résumé : The in vitro evaluation of three durum wheat genotypes (Djenah Khetifa, Oued Zenati
and Waha( to water stress tolerance simulated in vitro by different concentrations of
PEG 6000 indicated significant differences among genotypes, PEG concentrations and
their interactions for all the studied characters, indicating genetic variability in PEGsimulated drought stress. The osmotic pressure has negatively affected all parameters
studied. However, cells that continue to grow under severe osmotic stress are
considered as tolerant to water stress. In vitro screening for stress tolerance reveals the
variety Djenah Khetifa as the most tolerant and Waha as sensitive. The studied traits can
be recommended as suitable selection criteria for screening drought-tolerant genotypes.
Three amplified and reproducible locus (gwm331, gwm335 and gwm427) were retained.
These loci provided in total 78 monomorphic loci of which 69 were detected in somaclones.
Five somaclonal variants (DKR1-S1, DKR1-S2, OZR1-S2, WR1-C3 and WR1-S1) were
identified by only two SSR markers. Genetic variation rate was 21.74%. The presence of
selective agent in the medium could explain the observed genetic variations. Somaclonal
variation cannot always be detected at the gross morphological level. The selected SSR
markers could be used to study the uniformity of plants obtained from tissue culture and
varietal identification. Analysis of the proteins by a new two-dimensional diagonal
electrophoresis approach named (D-2DE) followed by MALDI-TOF mass spectrometry
has identified six differentially expressed proteins. For each variety, the level of mRNA
expression of three candidate genes (globulin, GAPDH and peroxidase) and a
ubiquitous (Actine) gene was determined by qRT-PCR. The amounts of mRNA of the
three genes are partially correlated with the amounts of proteins. These genes were
slightly up-regulated over the control. The D-2DE results were validated using Western
blot analysis with specific anti-globulin and anti-glyceraldehyde in the embryogenic
calli of wheat, in response to water stress. The proteins identified through this technique
provide new insights as to how calli respond to osmotic stress. Our method of study
provides an original and relevant approach that could contribute significantly to a better
understanding of possible linkages between the abundance of specific proteins and the
acclimation of durum wheat to water stress at the cellular level.Diplôme : Doctorat en sciences En ligne : ../theses/biologie/KAC7070.pdf Format de la ressource électronique : Permalink : index.php?lvl=notice_display&id=10531 Sélection in vitro pour la tolérance au stress hydrique chez le blé dur (Triticum durum Desf) : approche protéomique, transcriptomique et génétique. [texte imprimé] / Nadia Sandra Kacem, Auteur ; Abdelhamid Djekoun, Directeur de thèse ; Yordan Muhovski, Directeur de thèse . - جامعة الإخوة منتوري قسنطينة, 2017.
2 copies imprimées disponibles
Langues : Français (fre)
Catégories : Français - Anglais
BiologieTags : Durum wheat water stress PEG 6000 in vitro selection somaclonal
variation molecular marker diagonal two-dimensional electrophoresis , qRT-PCR Western blot Blé dur électrophorèse diagonale bidimensionnelle marqueur moléculaire sélection in vitro stress hydrique variation somaclonaleIndex. décimale : 570 Sciences de la vie. Biologie Résumé : The in vitro evaluation of three durum wheat genotypes (Djenah Khetifa, Oued Zenati
and Waha( to water stress tolerance simulated in vitro by different concentrations of
PEG 6000 indicated significant differences among genotypes, PEG concentrations and
their interactions for all the studied characters, indicating genetic variability in PEGsimulated drought stress. The osmotic pressure has negatively affected all parameters
studied. However, cells that continue to grow under severe osmotic stress are
considered as tolerant to water stress. In vitro screening for stress tolerance reveals the
variety Djenah Khetifa as the most tolerant and Waha as sensitive. The studied traits can
be recommended as suitable selection criteria for screening drought-tolerant genotypes.
Three amplified and reproducible locus (gwm331, gwm335 and gwm427) were retained.
These loci provided in total 78 monomorphic loci of which 69 were detected in somaclones.
Five somaclonal variants (DKR1-S1, DKR1-S2, OZR1-S2, WR1-C3 and WR1-S1) were
identified by only two SSR markers. Genetic variation rate was 21.74%. The presence of
selective agent in the medium could explain the observed genetic variations. Somaclonal
variation cannot always be detected at the gross morphological level. The selected SSR
markers could be used to study the uniformity of plants obtained from tissue culture and
varietal identification. Analysis of the proteins by a new two-dimensional diagonal
electrophoresis approach named (D-2DE) followed by MALDI-TOF mass spectrometry
has identified six differentially expressed proteins. For each variety, the level of mRNA
expression of three candidate genes (globulin, GAPDH and peroxidase) and a
ubiquitous (Actine) gene was determined by qRT-PCR. The amounts of mRNA of the
three genes are partially correlated with the amounts of proteins. These genes were
slightly up-regulated over the control. The D-2DE results were validated using Western
blot analysis with specific anti-globulin and anti-glyceraldehyde in the embryogenic
calli of wheat, in response to water stress. The proteins identified through this technique
provide new insights as to how calli respond to osmotic stress. Our method of study
provides an original and relevant approach that could contribute significantly to a better
understanding of possible linkages between the abundance of specific proteins and the
acclimation of durum wheat to water stress at the cellular level.Diplôme : Doctorat en sciences En ligne : ../theses/biologie/KAC7070.pdf Format de la ressource électronique : Permalink : index.php?lvl=notice_display&id=10531 Exemplaires (1)
Code-barres Cote Support Localisation Section Disponibilité KAC/7070 KAC/7070 Thèse Bibliothèque principale Thèses Disponible
