RESULTS

Helicobacter pylori (H. pylori) represent one of the most widespread bacterial infections globally. Current evidence indicates that nearly half of the global population is chronically infected with H. pylori [1, 2] and current clinical management relies primarily on antibiotic therapy. Nevertheless, the escalating antibiotic resistance resulting from overuse has led to an increasing rate of treatment failure. Urease is a critical factor for the colonization and pathogenicity of H. pylori in the stomach and H. pylori possesses one of the highest levels of urease content and enzymatic activity among all known bacteria[3]. Urease is formed as a heterodimer composed of two distinct subunits, UreA and UreB, with the UreB subunit containing the catalytic active site of H. pylori[4]. UreB, as a key subunit of urease, facilitates the efficient incorporation of Ni²⁺ into the enzyme's active site and stabilizes the site against metal chelation[5]. In this study, we constructed a genetically engineered expression system for a recombinant nanobody specifically targeting the urease subunit (UreB) of H. pylori. However, due to the limitation of biological safety, we can't use Ureb of Helicobacter pylori for experiments, so we use Ureb of Bacillus subtilis instead in this experiment. First, the recombinant nanobody was produced using an Escherichia coli expression system. The binding effect of our antibody to Bacillus subtilis was explored through experiments, and then the binding effect of antibody to Helicobacter pylori Ureb was simulated by computer. Finally, we investigated the expression of the recombinant nanobody in food-grade probiotic bacteria, aiming to utilize food-grade probiotics as delivery vehicles to administer the antibody in the form of live bacteria.

Figure 1. Overall experimental design concept
1.1. Acquisition of target gene fragment
      Based on the literature, we choose the genes (Nb-human, UreB-Nb6, and Nb-scFv) as the target genes. The target gene sequence was synthesized by the biotech company. The amplified target fragment using the synthesized target gene fragment as the template and the vector (pET28a-1) were identified by agarose gel electrophoresis after enzymatic digestion by NheI and HindⅢ, respectively. The Gene fragment lengths of Sumo-Nb-SCFV, Sumo-UreB-Nb6, Sumo-Nb-human and UreB were about 807bp, 655bp, 936bp,375bp, respectively (Figure 2A). Electrophoresis results showed that the band size was consistent with the expected size. The size of plasmid skeleton after digestion (pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB) was consistent with the expectation (Figure 2B). It indicates that the target gene has been successfully amplified. Subsequently, we used T4 DNA ligase to link the linearized plasmids to the target gene fragment.

Figure 2. Agarose gel electrophoresis of the target gene fragment and Skeleton. A.Target gene. B.Skeleton.
1.2. Construction of pET28a-Sumo-Nb-human
      Through antibiotic screening, colony PCR, and DNA sequencing verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-human in E.Coil DH5α and E.Coil BL21). Figure 3A depict the growth of transformed colonies on Kanamycin-resistant plates post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed. The colonies selected from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was used to verify the correctness of PCR products and the results show that the range of the target size is consistent with the expectation (Figure 3B). Subsequently, the target-sized amplicons were sent for sequencing. According to the sequence results shown in Figure 3C, the target gene was successfully ligated with the vector without obvious mutations, confirming the successful construction of the pET28a-Sumo-Nb-human plasmid.


Figure 3. The validation and sequencing of the plasmid pET28a-Sumo-Nb-human
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map; C. Sequencing comparison results.
1.3. Construction of pET28a-Sumo-Nb-SCFV
      Through antibiotic screening, colony PCR, and DNA sequencing verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-SCFV in E.Coil DH5α and E.Coil BL21). Figure 4A depicts the growth of transformed colonies on Kanamycin-resistant plates post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed. The colonies selected from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was used to verify the correctness of PCR products and the results show that the range of the target size is consistent with the expectation (Figure 4B). Subsequently, the target-sized amplicons were sent for sequencing. According to the sequence results shown in Figure 4C, the target gene was successfully ligated with the vector without obvious mutations, confirming the successful construction of the pET28a-Sumo-Nb-SCFV plasmid.

Figure 4. The validation and sequencing of the plasmid pET28a-Sumo-Nb-SCFV
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map; C. Sequencing comparison results.
1.4. Construction of pET28a-Sumo-Nb-6
      Through antibiotic screening, colony PCR, and DNA sequencing verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-6 in E.Coil DH5α and E.Coil BL21). Figure 5A depicts the growth of transformed colonies on Kanamycin-resistant plates post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed. The colonies selected from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was used to verify the correctness of PCR products, and the results show that the range of the target size is consistent with the expectation (Figure 5B). Subsequently, the target-sized amplicons were sent for sequencing. According to the sequence results shown in the Figure 5C, the target gene was successfully ligated with the vector without obvious mutations, confirming the successful construction of the pET28a-Sumo-Nb-6 plasmid.

Figure 5. The validation and sequencing of the plasmid pET28a-Sumo -Nb6
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map; C. Sequencing comparison results.
1.5. Construction of pET28a-Sumo-UreB
      Through antibiotic screening and DNA sequencing verification, we verified the recombinant plasmids (pET28a-Sumo-UreB in E.Coil DH5α and E.Coil BL21). Figure 6A depicts the growth of transformed colonies on Kanamycin-resistant plates post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed. Subsequently, We selected Single clony for sequencing. According to the sequence results shown in Figure 6B, the target gene was successfully ligated with the vector without obvious mutations, confirming the successful construction of the pET28a-Sumo-UreB.

Figure 6. The validation and sequencing of the plasmid pET28a-Sumo-UreB.
A. Cloned strains on resistant plates; B. Sequencing comparison results.

2.1. SDS-PAGE was used to verify the protein expression level
      After confirming that the strain was constructed correctly, we used IPTG as inducer to induce the expression of the target protein in the next step. After protein induction, we broke the cell by physical and chemical methods, then extracted、separated and purified the protein inside the cell.
      Finally, we verified whether the target protein was successfully expressed by SDS-PAGE. The molecular weights of the corresponding target proteins (Nb-6, Nb-human Nb-scfv, UreB) are 27.5 kDa, 34.1 kDa, 44.8 kDa and 14 kDa, respectively. After coomassie brilliant blue staining, we found that the results of SDS-PAGE were completely consistent with expectations. All four target proteins(Nb-6(Figure 7A), Nb-human (Figure 7B), Nb-scfv(Figure 7C), UreB(Figure 7D)) were successfully induced to express.

Figure 7. SDS-PAGE was used to verify the protein expression. A. The SDS-PAGE result of Nb-6;
B. The SDS-PAGE result of Nb-human; C. The SDS-PAGE result of Nb-scfv; D. The SDS-PAGE result of UreB.
2.2. Protein concentration was determined by BCA method
      Using the BCA (Bicinchoninic Acid Assay ) method, we determined the concentration of the purified protein mentioned above. We applied the microplate reader to measure the absorbance at a wavelength of 562 nm during this process and calculated the protein concentration of the sample based on the standard curve. (Figure 8)

Figure 8. The BCA result of the target proteins
      By substituting the measured absorbance values of the samples (y) using this fitting equation, the concentrations of each protein (x) were calculated as follows:
Table 1. The concentration of the target protein

2.3. Western blot was used to verify whether the protein was expressed
      This SDS-PAGE result shows the protein which has the same weight as the target protein, but we still can't be sure the corresponding strip is the target protein. Our target protein has his tag; therefore we conducted western blot experiments using anti-his antibodies to further verify to confirm. Through western blot we found that the bands were indeed the target proteins and not any other non-specific expression product (Figure 9). Experiments have proved that our antibody protein has been successfully expressed. In this experiment, in order to improve the solubility of Ureb protein, we introduced several of his tags into Ureb protein, so we did not verify the protein expression of Ureb by WB. However, based on the previous SDS-PAGE experiment, we basically confirmed that the Ureb protein was successfully expressed.

Figure 9. The Western blot result of Nb-human, Nb6, Nb-SCFV.

3.1 Immunodiffusion assay of three recombinant nanobodies
      We used the Oxford cup diffusion experiment to qualitatively evaluate the binding specificity of the expressed antibody to the homologous antigen. As shown in Figure.10 (A, B, C), after 36 hours of incubation, a slight white precipitation ring (protein stability may be impaired) was formed around the well plates loaded with three antibody preparations (Nb-6, Nb-SCFV, Nb-human), indicating that there was an interaction between urease and antibody. However, it can be found that the range of our precipitation ring is very narrow, which also means that the binding effect of our antibody against Bacillus subtilis Ureb is not very significant. This may be because the antibody we designed is specific to Ureb of Helicobacter pylori, and the homology of Ureb protein between Helicobacter pylori and Bacillus subtilis is not particularly high, so our antibody may not have a particularly good binding effect on Ureb of Bacillus subtilis.

Figure 10. Oxford Cup test results. A: Nb-6; B: Nb-SCFV;C: Nb-human.
(The number means control and different concentration of the recombinant nanobody：1：Blank 2：Negative control 3：25μg/ml antibody 4：200μg/mL antibody)

3.2 Nanobody-mediated urease inhibition assay of three recombinant nanobodies
      We assessed the inhibitory potency of recombinant nanobodies against urease. After incubation, urea and phenol red were added, and the extent of urease inhibition was quantified colorimetrically.
      The experimental results show that the rate at which urea is decomposed by urease in the experimental group with antibody is almost the same as that in the control group. This shows that our antibody binding effect on Ureb of Bacillus subtilis is poor. Consistent with the above analysis, our antibody is specific to Helicobacter pylori, so its inhibitory effect on Bacillus subtilis is not good. This means that if our antibody is used to target Ureb of Helicobacter pylori, it may have a better experimental effect. Unfortunately, due to the limitation of biological safety, we can only simulate the binding of our antibodies to Helicobacter pylori by computer.

Figure 11. Colorimetric changes and quantitative analysis of urease-recombinant nanobody interaction

4.1. The mimic antibody binds to Helicobacter pylori Ureb by molecular docking
      In this experiment, the model is a very important part of our experiment. Due to the consideration of biological safety, we can't use Ureb of Helicobacter pylori in practical experiments, so we tested the binding effect of nano-antibody and Ureb of Helicobacter pylori by molecular docking. Through computer simulation, we deeply analyzed the potential inhibitory effect of our nano-antibody on Helicobacter pylori and established a good model foundation for subsequent experiments.
      The 3D position of Ureb protein after interaction with three antibodies is shown in the figure. Among them, the green protein is UreB protein, the blue protein is Nb6 protein (Figure12A), the pink protein is Nb-Human protein (Figure12B) and the purple protein is Nb-ScFv protein (Figure12C), and the bonding situation among the residues has been marked in the figure, in which the red dotted line represents hydrogen bond interaction, the yellow dotted line represents salt bridge interaction, and the number near the dotted line of the interaction bond is the bond length of the interaction bond in Amy. Through the comprehensive analysis of these interactions, we can screen out the nano-antibody protein with the strongest binding ability.

Figure 12 Interaction between Ureb protein and nano-antibody protein. A. Schematic diagram of the interaction between UreB protein and Nb-6 protein, in which UreB protein is shown in green and NB6 protein is shown in blue. B. Schematic diagram of interaction between UreB protein and Nb-Human protein, in which UreB protein is shown in green and NB-human protein is shown in pink. C. Schematic diagram of interaction between UreB protein and Nb-ScFv protein, in which UreB protein is shown in green and Nb-ScFv protein is shown in purple.
      Using the Interface analyzer module of FoldX, the binding energies of UreB protein with three proteins are shown in the following table(Table 2), among which UreB protein has a strong binding tendency with Nb-SvFc protein and Nb-Human protein (protein-protein binding energy is less than -25 kcal/mol, which means that there is a strong binding tendency). The interaction of three pairs of proteins can be further observed in combination with subsequent molecular dynamics simulation.
Table 2 The binding energies of UreB protein with three proteins


4.2. The mimic antibody binds to Helicobacter pylori Ureb by molecule dynamics
      RMSD curve shows the changes of roots mean square displacement of three proteins relative to UreB protein in the simulation process and is used to evaluate the stability of binding between proteins in this study. From the graph analysis, it can be seen that the RMSD curve of Nb6 protein relative to UreB protein shows a certain fluctuation trend in the first 80ns of the simulation. After 80 ns, the RMSD curve gradually converges and vibrates around 0.8nm until the end of the simulation. The RMSD curve of Nb-ScFv protein relative to UreB protein experienced a certain fluctuation trend in the first 60ns of the simulation, and gradually converged after 60ns, vibrating around 0.5nm until the end of the simulation. The RMSD curve of Nb-Human protein relative to UreB protein showed a rapid increase in the first 20ns of the simulation, and then gradually converged, vibrating around 1.2nm until the end of the simulation.

Figure 13 RMSD Curve of Ureb and Three Proteins in Simulated Docking Process.

      Nb-6 protein is composed of more flexible random curls at the interaction interface in the first 80ns of simulation, and the interface is pulled by UreB protein in the process of dynamic simulation, so the protein interaction interface maintains a certain conformational adjustment in the first 80 ns of simulation, which leads to a certain fluctuation trend of RMSD curve. After 80 ns, with the interaction between proteins gradually stabilized, and there was no obvious conformational change again, the skeleton structure of the two proteins gradually stabilized, only maintaining a certain change in the mutual orientation of side chains. Therefore, after 80 ns, the RMSD curve gradually converged and vibrated around 0.8 nm until the end of the simulation. It can be considered that Nb6 protein and UreB protein formed a stable complex after 100ns molecular dynamics simulation.

Figure 14 Schematic Diagram of Conformation Rendering of UreB-Nb6 Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and blue shows Nb6 protein.
      Nb-ScFv protein changed little relative to UreB protein in the whole simulation process, so the RMSD value was low in the whole kinetic simulation process. In the first 60 ns of the simulation, the RMSD curve kept a certain fluctuation trend, which was due to the fact that although the skeleton atoms of the two proteins did not change obviously at the interaction interface, the orientation of the side chains changed to some extent, so the RMSD curve still showed a certain fluctuation trend. After 60ns, as the interaction mode between the two proteins tends to be stable, the RMSD curve also tends to converge and vibrates around 0.5nm until the end of the simulation.

Figure 15 Schematic Diagram of Conformation Rendering of UreB-Nb-ScFv Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and purple shows Nb-ScFv protein.
      In the first 20 ns of the simulation, Nb-Human protein appears to be close to UreB protein, which is most obviously marked in the above figure. In the first 20ns of the simulation, Nb-Human protein tends to twist and fold spontaneously under the action of UreB protein, and this conformation is maintained until the end of the simulation. After 20ns, the interaction between the two proteins tends to be stable, and the skeleton atoms do not change obviously again, so the RMSD curve tends to converge gradually. During this period, the vibration of the RMSD curve still comes from the mutual orientation change of the side chains of the two proteins, and the whole two proteins tend to form stable complexes.

Figure 16 Schematic Diagram of Conformation Rendering of UreB-Nb-Human Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and pink shows Nb-Human protein.
      Combined with the analysis of the movement trajectory of the protein complex, it can be seen that the three proteins have no obvious displacement relative to the UreB protein during the whole simulation process, so the RMSD value is relatively stable. Similarly, in the early stage of the simulation, the structure of the interaction between the three proteins and UreB protein has changed to some extent, so the RMSD curve shows a certain fluctuation trend in the first certain period of the simulation, and then gradually converges with the dynamic simulation. On the whole, after 100ns molecular dynamics simulation, all three proteins formed a stable complex with UreB protein, which can be combined with subsequent analysis to further observe the binding behavior of the three proteins.
      RMSF analysis showed the structural adaptability of each residue in protein, which was used in this study to analyze the flexibility and movement intensity of amino acid residues in protein during the whole simulation process. It can be seen from the figure that the overall RMSF of the protein remained at a low level during the whole simulation process. The reason why the RMSF at the N-terminal and C-terminal of the protein is large is that this residue is at the head end of the protein and is less constrained by other domains in the protein, which leads to greater flexibility and has a certain impact on its own stability. In addition, the reason why the rest of the protein RMSF is large may be that there is some disturbance due to the combination of small molecules, or because the peptide itself is flexible and has some disturbance during the simulation.
      By comparing the RMSF curves of three protein complexes, it can be seen that the RMSF value of Nb-ScFv protein and Nb-Human protein is higher than that of Nb-6 protein after forming complexes with Ureb protein. Which may be due to the stronger interaction between Nb-ScFv protein and Nb-Human protein and UreB protein, so the flexible residues at the interaction interface are more affected, which leads to the more obvious flexible jitter of different residues of Ureb protein. At the same time, it was observed that the RMSF value of N-terminal of Nb-Human protein increased obviously. Combined with the above analysis, it can be seen that this is because the Nb-Human protein moved towards UreB protein in the first 20ns of simulation, so the RMSF value of this random curly segment increased, which is consistent with the above analysis.
      On the whole, the RMSF values of the three proteins are low in the simulation process, so it can be considered that the proteins have little jitter in the simulation process, keep stable vibration in the solution environment, and the sampling and analysis are reliable.


Figure 17 RMSF curve of each protein in the simulation process. A. The RMSF curve of Ureb. B. The RMSF curve of Nb-6. C. The RMSF curve of Nb-ScFv. D. The RMSF curve of Nb-Human.
      The Radius of Gyration (Rg) can be used to characterize the compactness of protein structure, and it can also be used to characterize the change of protein's peptide chain looseness in the simulation process. Combined with the above analysis and Rg diagram analysis, it can be seen that the Rg curves of the complexes formed by Nb6 protein and Nb_ScFv protein with UreB protein respectively remained stable throughout the simulation process and changed within 2.5nm and 2.8nm respectively until the end of the simulation. After the complex of Nb_Human protein and UreB protein was formed, the Rg curve showed an upward trend in the first 20ns of the simulation, and then it tended to converge, maintaining the vibration around 2.75nm until the end of the simulation.
      Combined with the above analysis, it can be seen that after Nb6 protein and Nb-ScFv protein form complexes with UreB protein, the protein complexes remain relatively stable during the whole dynamic simulation process. As mentioned in the aforementioned RMSD analysis, only the mutual orientation of the residues at the interaction interface of the two proteins changes during the simulation process, so the RMSD curve remains in a low state, showing a certain fluctuation trend. As the skeleton atoms of the proteins do not change obviously as a whole, the Rg curve does not change significantly, but only keeps a certain vibration until the end of the simulation. After the complex of Nb-Human protein and UreB protein was formed, the random curl of Nb-Human protein was close to UreB protein in the first 20 ns of simulation, and the overall compactness became larger, so the Rg curve increased in the first 20 ns of simulation. After 20ns, with the convergence of the two protein complexes after exercise, the Rg curve also tends to be stable, vibrating around 2.75 nm until the end of the simulation.
      On the whole, the Rg curve of protein is stable in the simulation process and the sampling is reliable.

Figure 18 Radius of Gyration

      We analyzed the changes of the number of hydrogen bonds formed between proteins with the simulation time and systematically investigated the stability of protein binding from the perspective of interaction. From the graph analysis, it can be seen that the number of hydrogen bonds formed between Nb-6 protein and Nb-ScFv protein and UreB protein respectively remained relatively stable and had a certain amplitude during the whole simulation process. The number of hydrogen bonds formed between Nb-Human protein and UreB protein showed a certain fluctuation trend in the first 40ns of the simulation, and gradually increased as a whole, and stabilized after 40ns until the end of the simulation.

      Based on the above analysis and the analysis of the protein complex's trajectory, it can be seen that although Nb-6 protein and Nb-ScFv protein have no obvious displacement relative to UreB protein during the whole simulation process, due to the conformational adjustment between the residues at the interaction interface of the two proteins, accompanied by the residue change and flexible jitter of the protein flexible binding cavity, this movement leads to the continuous formation and destruction of hydrogen bonds between the residues at the protein interaction interface, which leads to a certain vibration of the hydrogen bond number curve with the change of simulation time, and the whole is relatively stable; Nb-Human protein appeared close to UreB protein in the first 20ns of the simulation, and maintained this conformation until the end of the simulation, so the number of hydrogen bonds formed with UreB protein showed a certain fluctuation trend in the first 40ns of the simulation, and the whole showed an upward trend due to the increase of protein interaction interface. After 40ns, with the gradual convergence of the residues at the interaction interface between the two proteins, the number curve of hydrogen bonds tends to be stable until the end of the simulation.
      In addition, it can be noted that although the number curve of hydrogen bonds changes obviously with the simulation, there are hydrogen bonds between the two proteins at all times, which explains that the binding between proteins is stable from the perspective of force.


Figure 19 The change of the number of hydrogen bonds formed between proteins with the simulation time.

      After eliminating the cycle of the simulated trajectory, combined with the aforementioned RMSD analysis, the movement trajectory of protein complex in the period of 90ns-100ns was extracted for binding free energy (MM/GBSA) analysis, and the energy term was decomposed.
      The results showed that the binding energy between protein and UreB was mainly van der Waals energy (Nb6 protein was -88.53 kcal/mol, Nb-ScFv protein was -109.01 kcal/mol, Nb-Human protein was -139 kcal/mol), and the electrostatic energy of Nb-6 protein was -26.58 kcal/mol, Nb-ScFv protein is -40.13 kcal/mol and Nb-Human protein is -43.19 kcal/mol), which indicates that electrostatic interaction is beneficial to protein binding, and proteins interact mainly through van der Waals interaction. The aforementioned hydrogen bond analysis and force analysis show that there are certain van der Waals interactions and hydrophobic interactions between different amino acids of each protein, so van der Waals can significantly facilitate the binding between proteins and occupy a dominant position; At the same time, there is a certain electrical property at the protein-protein interaction interface, so there is a certain electrostatic interaction at the protein-protein interaction interface, which leads to the contribution of electrostatic energy to the binding. In addition, we noticed that the solvation energies of Nb6 protein, Nb-ScFv protein and Nb-Human protein were 26.53 kcal/mol, 41.46 kcal/mol and 42.60 kcal/mol respectively in this simulation, suggesting that the addition of solvents was not conducive to the binding between proteins.
      The overall binding energy of Nb-6 protein is -88.59 kcal/mol, Nb-ScFv protein is -107.68 kcal/mol, and Nb-Human protein is -139.59 kcal/mol), which indicates that the binding tendency of proteins is relatively large, and the relative order of binding tendency is Nb-Human > Nb-ScFv > Nb-6, which further explains the binding between proteins from the perspective of energy.

      Based on the above computer simulation analysis, our nano-antibody has a good binding effect on Ureb protein of Helicobacter pylori in theory, among which Nb-Human has the best binding effect. Unfortunately, due to the limitation of biological safety, we can't directly verify the inhibitory effect of antibodies on Helicobacter pylori. Later, we may consider cooperating with more professional research institutions to verify the actual inhibitory effect of our antibodies on Helicobacter pylori.


Figure 20 Analysis of binding free energy (MM/GBSA)

Table 3 Energy analysis in simulation process

       5.1. Construction of the pGEX4T-1-Nb-human plasmid
      Based on preliminary experimental results, among the three recombinant nanobodies, the antibody encoded by the Nb-human gene exhibited the strongest urease inhibitory activity. Given this finding, we selected this gene for cloning into the pGEX4T-1 expression vector to achieve optimal expression in Escherichia coli Nissle 1917(probiotic).

      Following the same procedure as the construction of pET28a-Sumo-Nb-human, we digested the target gene amplification product and the pGEX-4T-1 plasmid with restriction enzymes, followed by ligation using T4 DNA ligase. The ligation product was then transfered into DH5α competent cells, and through DNA sequencing，we verified the success of the construction of the pGEX4T-1-Nb-human plasmid(Figure 21).


Figure 21. The validation and sequencing of the plasmid pGEX4T-1-Nb-human. A: The transformed strain growing on the resistant plate; B: The comparison results of the target genes of the positive cloned strains after sequencing.

5.2. Protein expression in Escherichia coli Nissle 1917
      Then, we transferred the pGEX4T-1-Nb-human expression plasmid into Escherichia coli Nissle 1917 competent cells, induced the expression of the target protein by 1mM IPTG and purified the acquired Nb-human protein using nickel affinity purification. SDS-PAGE results (Figure 22) indicated that the antibody protein (34.1 kDa) encoded by Nb-human was successfully expressed in the EcN 1917.


Figure 22. SDS-PAGE electrophoresis gel of Nb-human protein expressed in EcN 1917.

1. In this study, recombinant antibodies were induced for expression with IPTG at an appropriate temperature. However, the obtained recombinant nanobodies exhibited lower purity and concentration than expected. Although the urease inhibitory activity of these nanobodies was confirmed, the suboptimal yield may affect subsequent antibody potency assessments. To obtain high-quality antibodies, a systematic purification strategy must be implemented.
2. In the experiment, due to the limitation of biological safety, we can't use Ureb of Helicobacter pylori for the experiment, so we use Ureb of Bacillus subtilis as a substitute. However, because our antibody is specific to Helicobacter pylori, even in computer simulation, it has a good binding effect on Ureb of Helicobacter pylori. But in order to draw an accurate conclusion, we may consider cooperating with more professional research institutions to verify the actual inhibitory effect of our antibodies on Helicobacter pylori. In this experiment, we utilized purified UreB as the substrate to assess the activity of the recombinant nanobodies. Subsequent studies should apply these nanobodies directly to Helicobacter pylori to evaluate their ability to inhibit bacterial growth and proliferation, thereby strengthening the credibility of our findings.

[1] Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global Prevalence of Helicobacter pylori Infection: Systematic Review and Meta-Analysis. Gastroenterology. 2017;153:420-9.
[2] Malfertheiner P, Camargo MC, El-Omar E, Liou J-M, Peek R, Schulz C, et al. Helicobacter pylori infection. Nature Reviews Disease Primers. 2023;9.
[3] Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1991;59:2470-5.
[4] Almarmouri C, El-Gamal MI, Haider M, Hamad M, Qumar S, Sebastian M, et al. Anti-urease therapy: a targeted approach to mitigating antibiotic resistance in Helicobacter pylori while preserving the gut microflora. Gut Pathogens. 2025;17.
[5] Mobley HL. The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration. Aliment Pharmacol Ther. 1996;10 Suppl 1:57-64.