Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts – a delivery system for the nontypeableHaemophilus influenzaeantigenOmp26

Abstract

This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

“…[21,29] Concerning periplasmic targeting of S-layer proteins in general, MBP has been reported to export the S-layer protein SbsA of G. stearothermophilus PV/72 to the periplasm of E. coli. [32] The different S-layer fusion proteins were expressed in E. coli BL21 Star (DE3) carrying the respective expression plasmids. Based on the masses of A_SgsE and G_SgsE of 93.7 and 61.0 kDa, respectively, the masses of the different fusion proteins were calculated to be 95.9 and 63.2 kDa for the ssPelB constructs, 139.2 and 106.5 kDa for the MBP constructs, and 98.4 and 65.7 kDa for the ssTor constructs.…”

The bacterial ghost platform system

“…54,55 Linking MalE to SbsA the protein subunits can also be exported to the PPS prior to S-layer formation. 56 All different options of AG presentation in BG envelopes are summarized in Figure 2A as a schematic drawing.…”

Preparation and Evaluation of Chemically Inactivated Salmonella Enteritidis Vaccine in Chickens

“…The maximum antibody responses were observed on week 3 post-vaccination period in Group B and C when compared to group A. It is well-documented that the SEG vaccines induce protective immunity [15]. Another study [27] demonstrated that the outer membrane proteins are important to induce protective immunity against Salmonella infections.…”

“…About 2 mL of this suspension was added to 1 mL of MIC of NaOH and was incubated at 37°C for different time points (15,30,45,60, and 70 minutes). About 25 µL of each incubated time points were cultured on LB agar plates and incubated for 24 hrs at 37°C.…”

“…The mucosal immunizations with Escherichia coli ghosts were reported to induce protective immunity in an SPF chicken model [14]. Furthermore, the oral vaccination of Edwardsiella tarda ghosts induced higher protection against infection in mice [15]. Moreover, BGs were able to target-specific organ or tissue, indicating that they are a powerful system for drug and DNA delivery.…”