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Grácia Maria Soares Rosinha - One of the best experts on this subject based on the ideXlab platform.
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Identification of new Corynebacterium pseudotuberculosis antigens by Immunoscreening of gene expression library
BMC Microbiology, 2017Co-Authors: Cleber Eduardo Galvão, Stenio Perdigão Fragoso, Carina Elisei De Oliveira, Odinéia Forner, Renata Ribeiro Bastos Pereira, Cleber Oliveira Soares, Grácia Maria Soares RosinhaAbstract:Background Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use Immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. Results A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli . For Immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2 , fagA , fagB , NlpC/P60 protein family and LPxTG putative protein family. Conclusion Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.
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Identification of new Corynebacterium pseudotuberculosis antigens by Immunoscreening of gene expression library.
BMC Microbiology, 2017Co-Authors: Cleber Eduardo Galvão, Stenio Perdigão Fragoso, Carina Elisei De Oliveira, Odinéia Forner, Renata Ribeiro Bastos Pereira, Cleber Oliveira Soares, Grácia Maria Soares RosinhaAbstract:Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use Immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For Immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.
Thasaneeya Harnnoi - One of the best experts on this subject based on the ideXlab platform.
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identification of genes encoding cement like antigens expressed in the salivary glands of haemaphysalis longicornis
Journal of Veterinary Medical Science, 2006Co-Authors: Thasaneeya Harnnoi, Takeshi Sakaguchi, Xuenan Xuan, Kozo FujisakiAbstract:A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine.
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Molecular cloning of a gene encoding matrix metalloproteinase-like protein from Gnathostoma spinigerum
Parasitology Research, 2001Co-Authors: Pichart Uparanukraw, Thasaneeya Harnnoi, Nimit Morakote, Anchalee DantrakoolAbstract:The advanced third-stage larvae (aL3) of Gnathostoma spinigerum contain a 24 kDa glycoprotein with diagnostic potential. Immunoscreening with the monoclonal antibody to the 24-kDa protein (mAb GN6/24) has identified a cDNA clone with an insert of 932 base pairs (bp). The insert contains a full-length gene of 732 bp encoding a protein that is 33–39% similar to matrix metalloproteinases (MMPs) of Caenorhabditis elegans and several lower and higher vertebrates. The MMP-like protein of G. spinigerum possesses the catalytic domain, but lacks the propeptide and hemopexin-like domains found in other MMPs. A signal peptide of 23 amino acids at its amino terminus indicates that it is a secretory protein, which is confirmed by Western blot analysis showing the presence of the 24 kDa protein in the excretory–secretory products of aL3.
Markus Von Nickisch-rosenegk - One of the best experts on this subject based on the ideXlab platform.
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Rapid identification of novel antigens of Salmonella Enteritidis by microarray-based Immunoscreening.
Microchimica Acta, 2014Co-Authors: Lena Danckert, Sebastian Hoppe, Frank F Bier, Markus Von Nickisch-rosenegkAbstract:We report on an approach to rapidly screen thousands of Salmonella Enteritidis proteins with the goal of identifying novel immunodominant proteins. We used a microarray-based system that warrants high throughput and easy handling. Seven immunogenic candidates were selected after screening. Comparative analyses by ELISA and microarrays manifested their immunodominant character. The large repetitive protein (SEN4030) that plays a role as a putative adhesin in initial cell surface interaction and is highly specific to Salmonella is considered to be the most suitable protein for a diagnostic approach. The results further demonstrate that the strategy applied herein is convenient for specifically identifying immunogenic proteins of pathogenic microorganisms. Consequently, it enables a sound assessment of promising candidates for diagnostic applications and vaccine development. Moreover, the elucidation of immunogenic proteins may assist in unveiling unknown virulence-associated factors, thus furthering the understanding of the underlying pathogenicity of Salmonella in general, and of S. Enteritidis, one of the most frequently detected serovars of this pathogen, in particular. FigureThe microarray-based approach was aimed at identifying novel immunodominant proteins of S. Enteritidis. Seven antigens were revealed by screening a cDNA expression library. SEN4030, a large repetitive protein specific for salmonella, is considered an optimal candidate for future applications.
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Rapid identification of novel antigens of Salmonella Enteritidis by microarray-based Immunoscreening
Mikrochimica Acta, 2014Co-Authors: Lena Danckert, Sebastian Hoppe, Frank F Bier, Markus Von Nickisch-rosenegkAbstract:We report on an approach to rapidly screen thousands of Salmonella Enteritidis proteins with the goal of identifying novel immunodominant proteins. We used a microarray-based system that warrants high throughput and easy handling. Seven immunogenic candidates were selected after screening. Comparative analyses by ELISA and microarrays manifested their immunodominant character. The large repetitive protein (SEN4030) that plays a role as a putative adhesin in initial cell surface interaction and is highly specific to Salmonella is considered to be the most suitable protein for a diagnostic approach. The results further demonstrate that the strategy applied herein is convenient for specifically identifying immunogenic proteins of pathogenic microorganisms. Consequently, it enables a sound assessment of promising candidates for diagnostic applications and vaccine development. Moreover, the elucidation of immunogenic proteins may assist in unveiling unknown virulence-associated factors, thus furthering the understanding of the underlying pathogenicity of Salmonella in general, and of S. Enteritidis, one of the most frequently detected serovars of this pathogen, in particular.
Erlend B. Smeland - One of the best experts on this subject based on the ideXlab platform.
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Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display
Cancer Immunology Immunotherapy, 2004Co-Authors: Alexander Fosså, Lene Alsøe, Steinar Funderud, Gustav Gaudernack, Reto Crameri, Erlend B. SmelandAbstract:Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and Immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection Immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.
Tsengyuan Chang - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression of a cysteine rich venom protein from trimeresurus mucrosquamatus taiwan habu
Toxicon, 1997Co-Authors: Tsengyuan ChangAbstract:A full-length cDNA for cysteine-rich venom protein (CRVP) was constructed by Immunoscreening and 5′-rapid amplification of cDNA ends from a cDNA library of venom gland of Trimeresurus mucrosquamatus. The predicted CRVP consisted of 183 amino acid residues including a putative signal peptide of 21 residues. Northern blot hybridization suggested the tissue-specific expression in venom gland and its corresponding length of cDNA. The predicted amino acid sequence of CRVP was homologous to a rat epididymal metalloprotein and a lizard helothermine. Amino acid sequence analysis suggested that CRVP may be a venom metallo-protein targeted against ryanodine receptors and Ca2+ release. Moreover, CRVP expressed in Escherichia coli exhibited the same antigenicity as their native venom forms of T. mucrosquamatus. This is the first report in the cloning and expression of a CRVP from the venom gland of T. mucrosquamatus.
