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Cloning and expression of Neisseria meningitides luxS gene

Asian Pacific Journal of Tropical Medicine 2008年第2期1卷 16-20页

作者： Bahram Kazemi Nazila Baghalzadeh-Mohammadi Reza Hadavi Mojgan Bandehpour Elham Ghayour Majid Moghbeli Kazem Parivar Cellular and Molecular Biology Research Center Shahid Beheshti UniversityM.C.TehranI.R.Iran Parasitology Department Shahid Beheshti UniversityM.C.TehranI.R.Iran TehranI.R.Iran Islamic Azad University Jahrom BranchJahromI.R.Iran

Neisseria meningitides is a Gram-negative bacterium which is an important causative agent of septicemia and meningitis.Numerous pathogenic bacteria contain luxS,which is required for autoinducer-2 production.Neisseria meningitides contains a functional copy of luxS that is necessary for full meningococcal virulence.Neisseria meningitides DNA was extracted and its luxS gene was amplified by nested PCR.PCR product was purified and cloned in to pQE-30 expression vector.Recombinant plasmid was transformed and mass cultured.luxS was expressed in E.coli and confirmed by western blot analysis.In this study,N.meningitides luxS was amplified, cloned and expressed successfully.The sequencing of PCR product confirmed that amplified gene was luxS. Gene was expressed and observed in SDS-PAGE.Protein was reacted by his tag monoclonal antibody through western blot analysis.

关键词： Neisseria meningitides luxS auto inducer 2 cloning

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Oral <i>Lactobacillus plantarum</i>NCIMB 8825 Inhibits Adhesion, Invasion and Metabolism of <i>Neisseria meningitidis</i>Serogroup B and Affords Anti-Inflammatory and Cytotoxic Protection to Nasopharyngeal Epithelial Cells

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Advances in Microbiology 2014年第2期4卷 81-93页

作者： Tezera Liku Bekele Page Keith Rhakimova Adelina Salisbury Vyvyan Davenport Victoria University of Southampton Southampton UK University of the West of England Bristol UK

In this study, we investigate the potential for oral Lactobacilli (LB) to afford innate protection against nasopharyngeal coloniser Neisseria meningitidis serogroup B (NmB), which causes the bulk of UK meningococcal disease. Oral isolates of L. plantarum, L. salivarious, L. casei, L. rhamnosus, L. gasseri and gut probiotic L. rhamnosus GG were assessed for their ability to suppress nasopharyngeal epithelial inflammatory responses to pathogenic NmB. The specificity of attenuation was examined using TLR 2 ligand, Pam3Cys, and early response cytokine IL1β;and the mechanism of attenuation was explored using heat-killed organisms and conditioned medium. Pro-inflammatory IL-6 and TNFα cytokine secretion was quantified by ELISA and associated cell death was quantified by PI staining and LDH release. NmB adhesion, invasion and metabolism were determined using standard gentamicin protection with viable counts, and bioluminescence, respectively. L. plantarum and L. salivarious suppressed IL-6 and TNFα secretions from NmB-infected epithelial cells. LB did not need to be alive and could suppress using secretions, which were independent of TLR2 or IL1β receptor signalling. L. plantarum, in particular, reduced NmB-induced necrotic cell death of epithelial monolayers. Like L. salivarious, it significantly inhibited NmB adhesion but uniquely L. plantarum abolished NmB invasion. Using bioluminescence as a reporter of pathogen metabolism, L. plantarum and its secretions were found to inhibit NmB metabolism during cell invasion assays. We conclude that oral L. plantarum and its secretions could be used to help reduce the burden of meningococcal disease by removing the intracellular nasopharyngeal reservoir of NmB.

关键词： Epithelial Protection Neisseria meningitides Lactobacillus Innate Lux

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Validation of Automated Dilution and Transfer Methods for Rapid Assessment of Functional Immune Responses to Meningococcal Vaccines

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World Journal of Vaccines 2013年第3期3卷 98-101页

作者： Angela M. Payne Xu Liu Michael J. Caulfield Jon H. Heinrichs Vaccines Basic Research Merck Sharp and Dohme Corp. West Point USA Vaccines Basic Research Merck Sharp and Dohme Corp. West Point USA.

A high-throughput Serum Bactericidal Assay (SBA) was developed to monitor the functional antibody responses to capsular polysaccharide antigens from multiple serotypes of Neisseria meningitidis. This assay measures the ability of an antibody, aided by complement, to mediate killing of bacteria. Functional assays of this type are increasingly used in the quality control arena as potency release tests. Consequently, there has been an enhanced requirement for reproducibility in the performance of this type of assay. Assay validation is, therefore, important to facilitate understanding of the significance of values obtained, and to enable appropriate and informed use of the assay. This study involved the evaluation of the high-throughput serum bactericidal assay including the effects of different dilution and transfer techniques on assay performance. The results presented here demonstrate the repeatability, and the precision and ruggedness of the assay.

关键词： Serum Bactericidal Assay SBA Neisseria meningitides Automation Repeatability

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