polyclonal antibodies against c trachomatis momp  (Biosynth Carbosynth)

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 1 Agarose gel electrophoresis analysis of DMOMP. Notes: Molecular weight (1 kb) marker (lane 1), native phCMV1 vector (lane 2), native phCMV1 MOMP (lane 3), phCMV1 vector (4239 bp) restricted BamH 1 and Not 1 (lane 4), phCMV1 MOMP positive clones (4212 bp and 1134 bp) restricted BamH 1 and Not 1 (lanes 5 and 6) and phCMV1 MOMP clones (lanes 7 and 8) restricted Pst 1 (4823 bp and 531 bp).

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Agarose Gel Electrophoresis, Molecular Weight, Marker, Plasmid Preparation, Clone Assay

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 3 Size and morphological assessments of nanoparticles by SEM and TEM. SEM analyses of CNP (A), DMCNP (B), and TEM analysis of CNP (C) and DMCNP (D). Note: A drop of the nanoparticles was deposited on a copper grid for TEM, or a glass slide mounted on a stub for SEM. Abbreviations: CNP, phosphate buffered saline encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis DMOMP; DMCNP, DMOMP encapsulated in chitosan nanoparticles.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Saline, Membrane

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 4 Cumulative DNA release and electrophoretic analysis of released DMOMP. (A) Analysis of encapsulated DMOMP released from DMCNP was evaluated using similar intestinal fluid (SIF, pH 7.0) and similar gastrointestinal fluid (SGF, pH 2.0). Samples were collected at each designated time-interval (2 hours, day 1, 2, 4, 5 and 7) and the released DNA measured using NanoDrop at 260 nm. Each bar represents the mean ± standard deviation of triplicate samples. (B) The released products from each time- point were precipitated with 5 M NaCl and amplified by PCR and amplicons subjected to agarose gel electrophoresis for verification of the specific DMOMP product. Notes: Lanes indicate the sequential collection of amplicons as follows: lane 1 (1 kb marker), lane 2 (2 hours), lane 3 (day 1), lane 4 (day 2), lane 5 (day 4), lane 6 (day 5), lane 7 (day 7) and lane 8 (positive DMOMP clone) with an expected size of 1154 bp. Abbreviations: DMOMP, DNA of the major outer membrane protein of C. trachomatis; DMCNP, DMOMP encapsulated in chitosan nanoparticles; hrs, hours; PBS, phosphate buffered saline; PCR, polymerase chain reaction; SGF, similar gastrointestinal fluid; SIF, similar intestinal fluid.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Standard Deviation, Amplification, Agarose Gel Electrophoresis, Marker, Membrane, Saline, Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 5 Stability studies of DMOMP in chitosan nanoparticles. (A) Electrophoretic analysis of CNP protection of encapsulated DMOMP after incubation with chitosanase, DNAase I, and restriction enzymes. Both DMCNP and DMOMP (1 mg/mL) were subjected to enzymatic digestion with NdeI and SalI in combination with, or without chitosanase or with DNAase 1. Lane 1, (1 kb molecular marker), lane 2 (trapped nanoparticle in well), lane 3 (released DMOMP in red box), lanes 4 and 5 (degraded released DMOMP), lane 6 (blank), lane 7 (1 kb marker), and lane 8 (DMOMP positive clone). (B) pH stability of encapsulated DMOMP. Notes: DMCNP was added to individual micro-centrifuge tubes and adjusted to various pH values (2, 4, 6, 8, 9, 10, 10.5 and 12) followed by incubation at 37°C on a shaker for 30 minutes. Lanes 1 and 10 are 1 kb marker. All samples were analyzed by agarose gel electrophoresis and visualized using the ChemiImager gel documentation system. Abbreviations: CNP, phosphate buffered saline encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis; DMCNP, DMOMP encapsulated in chitosan nanoparticles; RE, restriction.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Incubation, Marker, Agarose Gel Electrophoresis, Saline, Membrane

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 7 In vitro expression of MOMP protein in transfected Cos-7 cells. Cos-7 cells (1 × 106 cells/well) were transfected by electroporation with DMOMP (A) and DMCNP (B) at concentrations of 2, 5 and 10 µg. Notes: Transfected cells were incubated for 48 hrs at 37°C, fixed and blocked prior to incubation with goat anti-C. trachomatis polyclonal antibodies followed by a secondary FITC rabbit anti-Goat IgG (H+L) antibody. Immunofluorescence of cells were visualized using a Nikon Eclipse Ti-U microscope. Abbreviations: DMOMP, DNA of the major outer membrane protein of C. trachomatis; DMCNP, DMOMP encapsulated in chitosan nanoparticles; MOMP, major outer membrane protein of C. trachomatis.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: In Vitro, Expressing, Transfection, Electroporation, Incubation, Immunofluorescence, Microscopy, Membrane

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 8 Expression of MOMP at the protein and gene transcript levels. (A) Cos- 7 cells were transfected as described in Figure 7, immunostained (positive MOMP fluorescence cells) and then mounted with DAPI (blue nuclei stain) combined with an anti-fade mounting solution. (B) Bright-field visualization of Cos-7 cell monolayer showing the MOMP expressed protein. Red circle (positive MOMP fluorescence cells) shows expression of the MOMP protein. (C) Confirmation of expressed MOMP protein by western blot. Cos-7 cells (4 × 105 cells/well) were transfected with DMCNP or phCMV1 vector using Lipofectamine and incubated at 37°C for 48 hours. Cell lysates were collected, run on an SDS-PAGE gel, transferred onto a PVDF membrane and probed using anti-MOMP polyclonal antibodies followed by an Alexa fluor 680 secondary antibody. The bound antibody was viewed using LI-COR Odyssey imaging apparatus. (D) In vitro expression of MOMP gene transcript. Notes: RNA samples were extracted from mouse thigh muscles and spleens reversed transcribed to cDNA and then subjected to RT-PCR amplification of the MOMP gene transcript using MOMP specific primers. Lane 1 (MW marker), lanes 2 and 7 (DMOMP positive clones), lane 3 (thigh muscle of DMCNP mice), lane 4 (thigh muscle of PBS mice), lane 5 (spleen from DMCNP mice), and lane 6 (spleen from PBS mice). Red rectangle shows positive MOMP gene transcripts. Abbreviations: DMCNP, DMOMP encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis; MOMP, major outer membrane protein of C. trachomatis; PVDF, polyvinylidene difluoride.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Expressing, Transfection, Fluorescence, Staining, Western Blot, Plasmid Preparation, Incubation, SDS Page, Membrane, Imaging, In Vitro, Muscles, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Clone Assay

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 1. Schematic view of the constructs used in this study: (a and b) denote full- length MOMP and chimeric MOMP, respectively, expressed in E. coli (pET101/D- TOPO vector), (c and d) denote full-length MOMP and chimeric MOMP, respectively, expressed in plants (pGreen0229 vector).

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Construct, Plasmid Preparation

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 2. Northern blot analysis of plants transformed with the full-length MOMP construct. Plants 1 and 2 show the presence of MOMP mRNA transcripts. WT denotes untransformed wild type plant. All three tested transgenic plants were PCR positive.

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Northern Blot, Transformation Assay, Construct, Transgenic Assay

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 3. (a) Topology and primary structure of the Ct serovar E MOMP as adopted from Findlay et al. [26]: squares, amino acids residues found in membrane spanning helices; circles, amino acid residues found in extramembraneous parts of the protein. The domains selected for design of the chimeric MOMP are shown in red; (b) the putative flexible conformation that can be obtained using the (Gly4Ser)2Gly4 linker (shown in black). The amino acid residues that differ between MOMP serovar E (shown) and serovar D in the VS2 and VS4 loops are given in blue; (c) the more rigid conformation that can be obtained using the (Gly4Ser)2Gly4 linker (shown in black). The amino acid residues that differ between MOMP serovar E (shown) and serovar D in the VS2 and VS4 loops are given in blue. The green C-terminal tag contain a V5 epitope and a His6 purification tag, as expressed in Escherichia coli but not in plants (see Fig. 1). Fig. 4. (a) PCR analysis of the assembled MOMP chimeric construct. Ch denotes PCR product from a vector containing the assembled chimera, N denotes the PCR negative control, L denotes the DNA size marker. The amplified product has the expected size of 351 bp. (b) Western blot analysis of recombinant His-tagged chimeric MOMP protein expressed in Escherichia coli and purified using Ni–NTA chromatography. A band of the expected size (17 kDa) was detected using mouse monoclonal antibodies to Chlamydia trachomatis MOMP (Acris Antibodies). Ch denotes the chimeric MOMP protein, L denotes the protein size marker.

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Membrane, Construct, Plasmid Preparation, Negative Control, Marker, Western Blot, Recombinant, Chromatography, Bioprocessing

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 5. Evaluation of the anti-chimeric MOMP antiserum produced in rabbits. The purified recombinant MOMP chimera was analyzed by immunoblotting using anti- chimeric MOMP serum (S), affinity purified anti-chimeric MOMP antibodies (A) and pre-serum (P). L denotes the protein size marker.

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Produced, Recombinant, Western Blot, Marker

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 6. Immunofluorescence slides demonstrating antibody reactivity toward Chlamydia trachomatis elementary bodies and its full-length MOMP protein (bright fluorescent dots). (a) Anti-MOMP chimera antibodies (post-serum), produced in rabbits injected with MOMP chimera, showing high specific reactivity against inactivated Ct elementary bodies. (b) Rabbit pre-serum lacking MOMP reactivity. (c) Minimal fluorescence of the secondary anti-rabbit IgG antibody conjugate itself in the absence of rabbit serum. Magnification was 400 in (a) and 200 in (b and c), respectively.

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Produced, Injection

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 8. (a) Semiquantitative analysis of the content of chimeric MOMP in transformed carrots. Kar and 313 denote two different transgenic lines in cultivars Karotan and Napoli, respectively. Comparison of the intensity of the stained bands in the transgenic plants and controls (purified and accurately quantified chimeric MOMP) allowed the estimation of the approximate MOMP chimera protein concentration in the carrots. (b) Immunoblot showing the specificity of the antiserum raised against E. coli-produced chimeric Ct MOMP protein when used for probing extracts from carrot lines 350 and 604 (in the Karotan background) expressing the same protein. L denotes the molecular weight standards, WT are extract from wild type Karotan carrots, and PC are E. coli-produced positive controls (2.5 and 7.5 lg protein, respectively). The asterisks indicate the MOMP chimera dimer.

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Transformation Assay, Transgenic Assay, Comparison, Staining, Protein Concentration, Western Blot, Produced, Expressing, Molecular Weight

Journal: Protein expression and purification

Article Title: A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

doi: 10.1016/j.pep.2011.08.010

Figure Lengend Snippet: Fig. 7. (a) Western blot detection of constitutively expressed chimeric MOMP in Arabidopsis leaf extracts from T6 generation plants using polyclonal antibody against full-length C. trachomatis MOMP (Acris Antibodies). L denotes the protein size marker; 9, 15, and 25 denote three different transgenic lines of Arabidopsis; WT denotes non-transformed wild type Arabidopsis; A corresponds to 5 ll unfraction- ated plant extract and B corresponds to 15 ll unfractionated plant extract; (b) Southern blot analysis of four Arabidopsis lines transformed with the chimeric MOMP construct (lines 9, 12, 15, and 25). Two different DNA digests of each line were produced by using the Dra I and Nde I restriction enzymes and probed with random primer 32P-labeled chimera MOMP oligonucleotides. The restriction enzymes chosen did not digest the MOMP chimera transgene itself. The number of observed bands corresponds to the copy number of the transgene.

Article Snippet: Three selected transgenic lines (numbers 9, 15, and 25) were used in further analysis and stable integration of the transgene in these lines was demonstrated for up to six generations using the polyclonal antibody against C. trachomatis MOMP (Acris Antibodies; Fig. 7a).

Techniques: Western Blot, Marker, Transgenic Assay, Transformation Assay, Plant Extract, Southern Blot, Construct, Produced, Labeling