scholarly journals Characterization of the pscC (Type III secretion) gene of Pseudomonas aeruginosa (PA01) and assessment of immunogenicity of pscC protein in rats

2016 ◽  
Author(s):  
SA. Bhuiyan ◽  
DJ. Vanitha ◽  
H. Sultana ◽  
F. Opook ◽  
KF. Rodrigues
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Multiple Cloning Site ◽  
Sprague Dawley ◽  
Type Iii ◽  
Antigenic Properties

ABSTRACTProteins associated with the bacterial membrane can be recruited for application as antigens for the development of vaccines. This preliminary study was directed towards evaluating the antigenic properties of the Pseudomonas aeruginosa (PA01) pscC protein which is a component of the Type III secretion system. Gene specific primers were designed to isolate the pscC gene which was isolated, ligated onto the multiple cloning site of vector pGS21(a), cloned and expressed in Escherichia coli (BL21). The molecular weight of the expressed pscC protein was determined by SDS-PAGE (10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and was found to be around 57 KDa and purified by the size exclusion chromatography. Finally, the purified pscC protein was injected subcutaneously into adult Sprague Dawley® rats with a range of concentrations (50, 100 and 150 µg per rat) respectively. Recombinant pscC antigen induced a specific humoral immune response against the antigen, which was validated by Enzyme-linked immunosorbent assay (ELISA). The results concluded that anti-pscC antibody was elicited in the animal model.

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization

1982 ◽  
Vol 152 (1) ◽  
pp. 239-245
Author(s):  
R M Berka ◽  
M L Vasil
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phospholipase C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Tetradecyltrimethylammonium Bromide ◽  
Heat Labile ◽  
Hydrophobic Moiety ◽  
Culture Supernatants

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.

scholarly journals Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

2006 ◽  
Vol 13 (10) ◽  
pp. 1155-1161 ◽  
Author(s):  
Donghee Cho ◽  
Michael T. Collins
Keyword(s):  
Solid Phase ◽  
Expression Profiles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Cattle ◽  
Mycobacterium Paratuberculosis ◽  
Serologic Tests ◽  
Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

Effects of Gamma Radiation on the Allergenic and Antigenic Properties of Milk Proteins

2001 ◽  
Vol 64 (2) ◽  
pp. 272-276 ◽  
Author(s):  
JU-WOON LEE ◽  
JAE-HUN KIM ◽  
HONG-SUN YOOK ◽  
KUN-OK KANG ◽  
SOO-YOUNG LEE ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Immunoglobulin E ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Milk Proteins ◽  
Food Irradiation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigenic Properties ◽  
Β Lactoglobulin ◽  
Irradiation Technology

This study was carried out to evaluate the application of food irradiation technology as a method for reducing milk allergies. Bovine α-casein (ACA) and β-lactoglobulin (BLG) were used as milk proteins. Using milk-hypersensitive patients' immunoglobulin E (IgE) and rabbit IgGs individually produced to ACA and BLG, the changes of allergenicity and antigenicity of irradiated proteins were observed by competitive indirect enzyme-linked immunosorbent assay. Allergenicity and antigenicity of the irradiated proteins were changed with different slopes of the inhibition curves. The disappearance of the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increase of the turbidity showed that solubility of the proteins decreased by radiation, and this decrease might be caused by agglomeration of the proteins. These results indicated that epitopes on milk allergens were structurally altered by gamma irradiation.

Impact of γ-Radiation on Antigenic Properties of Cow's Milk β-Lactoglobulin

2008 ◽  
Vol 71 (6) ◽  
pp. 1270-1272 ◽  
Author(s):  
H. KADDOURI ◽  
S. MIMOUN ◽  
K. E. EL-MECHERFI ◽  
A. CHEKROUN ◽  
O. KHEROUA ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cow’S Milk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Γ Radiation ◽  
Cow's Milk ◽  
Molecular Weight Distributions ◽  
Antigenic Properties ◽  
Γ Rays ◽  
Β Lactoglobulin

This study evaluated the effects of γ-radiation on the antigenic properties of β-lactoglobulin in cow's milk. Liquid and lyophilized samples of cow's milk and whey were irradiated with γ-cells (60Co) at dose levels of 3, 5, and 10 kGy, at room temperature in the presence of air. Effects of treatment on proteins were monitored by Lowry's method, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. Radiation did not affect the molecular-weight distributions of proteins, but it did reduce their solubility. Furthermore, results showed that irradiation at 10 kGy increased the recognition of milk and whey powders by anti–β-lactoglobulin (β-Lg) rabbit immunoglobulin G, with the other samples remaining antigenically stable. These results indicate that γ-rays do not reduce cow's milk β-lactoglobulin antigenicity.

Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Phage Display ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Library ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Phage Display Antibody

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

scholarly journals Diversity of Virulence Phenotypes among Type III Secretion Negative Pseudomonas aeruginosa Clinical Isolates

PLoS ONE ◽  
2014 ◽  
Vol 9 (1) ◽  
pp. e86829 ◽  
Author(s):  
Jonida Toska ◽  
Yan Sun ◽  
Dalina Alvarez Carbonell ◽  
Altreisha N. -S. Foster ◽  
Michael R. Jacobs ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Clinical Isolates ◽  
Type Iii
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