The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Gerardo Corzo - One of the best experts on this subject based on the ideXlab platform.
-
A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
2018Co-Authors: Herlinda Clement, Elba Villegas, Vianey Flores, Elia Diego-garcia, Ligia Corrales-garcia, Gerardo CorzoAbstract:AbstractBackground:The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.Methods:The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with Isopropyl Thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.Results:Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.Conclusions:The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.
-
Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.
Protein expression and purification, 2017Co-Authors: Francisco Guillén-chable, Iván Arenas-sosa, Ignacio Islas-flores, Gerardo Corzo, Cynthia Martinez-liu, Georgina EstradaAbstract:The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with Isopropyl Thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa.
-
Heterologous expression of five disulfide-bonded insecticidal spider peptides.
Toxicon : official journal of the International Society on Toxinology, 2016Co-Authors: Georgina Estrada, Anita O. Silva, Elba Villegas, Ernesto Ortiz, Paulo S.l. Beirão, Gerardo CorzoAbstract:The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with Isopropyl Thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.
-
heterologous expressed toxic and non toxic peptide variants of toxin cssii are capable to produce neutralizing antibodies against the venom of the scorpion centruroides suffusus suffusus
Immunology Letters, 2009Co-Authors: Kenya Hernandezsalgado, Georgina Estrada, Alejandro Olvera, Fredy I V Coronas, Lourival D Possani, Gerardo CorzoAbstract:Abstract Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with Isopropyl Thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro . The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30 μg/20 g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus . These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.
-
Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro.
Biochimica et Biophysica Acta - Molecular Cell Research, 2007Co-Authors: Georgina Estrada, Ernesto Ortiz, Blanca Garcia, Emanuele Schiavon, Sandrine Cestèle, Enzo Wanke, Lourival Possani, Gerardo CorzoAbstract:The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with Isopropyl Thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.
Georgina Estrada - One of the best experts on this subject based on the ideXlab platform.
-
Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.
Protein expression and purification, 2017Co-Authors: Francisco Guillén-chable, Iván Arenas-sosa, Ignacio Islas-flores, Gerardo Corzo, Cynthia Martinez-liu, Georgina EstradaAbstract:The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with Isopropyl Thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa.
-
Heterologous expression of five disulfide-bonded insecticidal spider peptides.
Toxicon : official journal of the International Society on Toxinology, 2016Co-Authors: Georgina Estrada, Anita O. Silva, Elba Villegas, Ernesto Ortiz, Paulo S.l. Beirão, Gerardo CorzoAbstract:The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with Isopropyl Thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.
-
heterologous expressed toxic and non toxic peptide variants of toxin cssii are capable to produce neutralizing antibodies against the venom of the scorpion centruroides suffusus suffusus
Immunology Letters, 2009Co-Authors: Kenya Hernandezsalgado, Georgina Estrada, Alejandro Olvera, Fredy I V Coronas, Lourival D Possani, Gerardo CorzoAbstract:Abstract Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with Isopropyl Thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro . The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30 μg/20 g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus . These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.
-
Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro.
Biochimica et Biophysica Acta - Molecular Cell Research, 2007Co-Authors: Georgina Estrada, Ernesto Ortiz, Blanca Garcia, Emanuele Schiavon, Sandrine Cestèle, Enzo Wanke, Lourival Possani, Gerardo CorzoAbstract:The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with Isopropyl Thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.
Manoj K Chakrabarti - One of the best experts on this subject based on the ideXlab platform.
-
outer membrane protein a ompa of shigella flexneri 2a induces protective immune response in a mouse model
PLOS ONE, 2011Co-Authors: Debasis Pore, Nibedita Mahata, Amit Pal, Manoj K ChakrabartiAbstract:Background: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPOH vector and expressed using induction with Isopropyl Thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.
-
Outer Membrane Protein A (OmpA) of Shigella flexneri 2a, Induces Protective Immune Response in a Mouse Model
2011Co-Authors: Debasis Pore, Nibedita Mahata, Amit Pal, Manoj K ChakrabartiAbstract:Background: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPOH vector and expressed using induction with Isopropyl Thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associate
Zili Yan - One of the best experts on this subject based on the ideXlab platform.
-
Quantitative Proteomics Reveals Significant Changes in Cell Shape and an Energy Shift after IPTG Induction via an Optimized SILAC Approach for Escherichia coli
Journal of proteome research, 2013Co-Authors: Lingyan Ping, Heng Zhang, Linhui Zhai, Eric B. Dammer, Duc M. Duong, Zili YanAbstract:Stable isotope labeling by amino acids in cell culture (SILAC) has been widely used in yeast, mammalian cells, and even some multicellular organisms. However, the lack of optimized SILAC media limits its application in Escherichia coli, the most commonly used model organism. We optimized SILACE medium (SILAC medium created in this study for E. coli) for nonauxotrophic E. coli with high growth speed and complete labeling efficiency of the whole proteome in 12 generations. We applied a swapped SILAC workflow and pure null experiment with the SILACE medium using E. coli BL21 (DE3) cells hosting a recombinant plasmid coding for glutathione-S-transferase (GST) and ubiquitin binding domain before and after Isopropyl Thiogalactoside (IPTG) induction. Finally, we identified 1251 proteins with a significant change in abundance. Pathway analysis suggested that cell growth and fissiparism were inhibited accompanied by the down-regulation of proteins related to energy and metabolism, cell division, and the cell cycle, resulting in the size and shape change of the induced cells. Taken together, the results confirm the development of SILACE medium suitable for efficient and complete labeling of E. coli cells and a data filtering strategy for SILAC-based quantitative proteomics studies of E. coli.
-
Quantitative Proteomics Reveals Significant Changes in Cell Shape and an Energy Shift after IPTG Induction via an Optimized SILAC Approach for Escherichia coli
2013Co-Authors: Lingyan Ping, Heng Zhang, Linhui Zhai, Eric B. Dammer, Duc M. Duong, Zili YanAbstract:Stable isotope labeling by amino acids in cell culture (SILAC) has been widely used in yeast, mammalian cells, and even some multicellular organisms. However, the lack of optimized SILAC media limits its application in Escherichia coli, the most commonly used model organism. We optimized SILACE medium (SILAC medium created in this study for E. coli) for nonauxotrophic E. coli with high growth speed and complete labeling efficiency of the whole proteome in 12 generations. We applied a swapped SILAC workflow and pure null experiment with the SILACE medium using E. coli BL21 (DE3) cells hosting a recombinant plasmid coding for glutathione-S-transferase (GST) and ubiquitin binding domain before and after Isopropyl Thiogalactoside (IPTG) induction. Finally, we identified 1251 proteins with a significant change in abundance. Pathway analysis suggested that cell growth and fissiparism were inhibited accompanied by the down-regulation of proteins related to energy and metabolism, cell division, and the cell cycle, resulting in the size and shape change of the induced cells. Taken together, the results confirm the development of SILACE medium suitable for efficient and complete labeling of E. coli cells and a data filtering strategy for SILAC-based quantitative proteomics studies of E. coli
Debasis Pore - One of the best experts on this subject based on the ideXlab platform.
-
outer membrane protein a ompa of shigella flexneri 2a induces protective immune response in a mouse model
PLOS ONE, 2011Co-Authors: Debasis Pore, Nibedita Mahata, Amit Pal, Manoj K ChakrabartiAbstract:Background: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPOH vector and expressed using induction with Isopropyl Thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.
-
Outer Membrane Protein A (OmpA) of Shigella flexneri 2a, Induces Protective Immune Response in a Mouse Model
2011Co-Authors: Debasis Pore, Nibedita Mahata, Amit Pal, Manoj K ChakrabartiAbstract:Background: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPOH vector and expressed using induction with Isopropyl Thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associate
