Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli

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BackgroundOuter membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains.ObjectivesThe aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate.MethodsProtein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines.ResultsAnti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD + Freund’s and those administered PD + OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice.ConclusionsWe conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity.

Section: Discussionmentioning

confidence: 99%

Immunization with Protein D from Non-Typeable Haemophilus influenzae (NTHi) Induced Cytokine Responses and Bioactive Antibody Production

Motlagh

Siadat

Abediankenari

et al. 2016

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“…The recombinant plasmids were transformed into E. coli Rosetta (DE3) cells, and E. coli cells were grown in shaker flasks with LB broth medium containing 100 μg/mL ampicillin at 37°C until optical density (OD) of 0.6 was reached, and then recombinant bacteria were induced using 0.1 M of isopropy-Β-D-thiogalactoside (IPTG) at 37°C for four hours. The induced recombinant bacteria were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( 27 ). Four hours after induction, the cells were harvested and centrifuged at 9,000 rpm for ten minutes and washed once with phosphate buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli, and Production of Its Monoclonal Antibodies

Jin

Sun

Xia

et al. 2016

BackgroundHerpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus.ObjectivesThis study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research.Materials and MethodsThe gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid.ResultsThe optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass.ConclusionsThe cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.

“…The type b strain of typeable H. influenzae (Hib) causes serious invasive infections such as bacteremia, pneumonia, and acute bacterial meningitis (3). The pathogenesis of diseases related to NTHi begins with bacterial colonization of the nasopharynx after spreading the organism into the middle ear, the sinuses, and the lungs resulting in otitis media, sinusitis, bronchitis, and pneumonia (4,5).…”

Section: Introductionmentioning

confidence: 99%

HMW1555-914, HMW2553-916, and Hia585-705 as Subunit Vaccine Candidates of Nontypeable Haemophilus influenzae Induce Specific Antibody Responses with Bactericidal Activity in Balb/c

Abbaszadeh‐Goudarzi

Abbaszadeh-Goudarzi

Khorramizadeh

et al. 2017

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Background: Nontypeable Haemophilus influenzae (NTHi) is responsible for diseases such as otitis media, sinusitis, bronchitis, and pneumonia. Unlike H. influenzae Serotype b (Hib), there is no vaccine against diseases induced by NTHi. High molecular weight1 (HMW1), high molecular weight 2 (HMW2), and H. influenzae type a (Hia) proteins are critical protein adhesions of NTHi with the potentiality to provide the protection. Objectives: The current study aimed at investigating the potential of recombinant HMW1555-914, HMW2553-916, and Hia585-705 proteins as subunit vaccines to induce immune responses after active immunization in Bagg albino (inbred research mouse strain) or Balb/c mice. Methods: HMW1555-914, HMW2553-916, and Hia585-705 amplified by polymerase chain reaction (PCR) were cloned into pET28a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blotting analysis were carried out to investigate the characteristics of proteins. Proteins were purified by Ni-nitrilotriacetic acid (Ni-NTA) as a gel matrix. Mice (Balb/c) were immunized subcutaneously with HMW1555-914, HMW2553-916, and Hia585-705 proteins, alone or in binary and ternary combinations. Eventually, specific antibody responses were evaluated by the enzyme-linked immunosorbent assay (ELISA) and serum bactericidal assay (SBA). Results: Antibody responses significantly increased in Balb/c immunized by ternary combination compared with the control group. IgG1/IgG2a ratio indicated that proteins directed immune responses toward T-helper 2. Antisera produced against purified proteins in ternary combination showed the highest bactericidal activity against NTHi strains with the titer of 1:32. Conclusions: The obtained results suggested that HMW1555-914, HMW2553-916, and Hia585-705 adhesins were the potential subunit vaccine candidates of NTHi and after further investigation could be considered for protection against NTHi infections.