CONTRIBUTION

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.
      During our experiment, we added some new parts for iGEM part and new information to an existing part (Table 1).
Table 1. Part contributions

1. Add new basic part
1.1 UreB, BBa_25ZPXIXD
      Name: UreB
      Base Pairs: 375bp
      Origin: Bacillus subtilis.168; BSU_36650; 936967
      Usage and Biology
      UreB urease (beta subunit) [ Bacillus subtilis subsp. subtilis str. 168 ].
1.2 Nb-human, BBa_25UR7GES
      Name: Nb-human
      Base Pairs: 507bp
      Origin: Artificial sequences; GenBank: LC375193.1
      Usage and Biology
      Nb-human is a nanobody that specifically binds to the UreB site of Helicobacter pylori urease.
1.3 Nb-SCFV, BBa_25QIUV2A
      Name: Nb-SCFV
      Base Pairs: 807bp
      Origin: Synthetic construct; GenBank: LC373564.1
      Usage and Biology
      Nb-SCFV is a nanobody targeting the UreB subunit of Helicobacter pylori urease.
1.4 Nb6, BBa_25JBNCR9
      Name: Nb6
      Base Pairs: 258bp
      Origin: synthetic construct
      Usage and Biology
      Nb-6 is a nanobody targeting the UreB subunit of Helicobacter pylori urease.

Figure 1 Agarose gel electrophoresis of the target gene fragment
2. Add new Composite Part
      Name: pET28a-SUMO-Nb-human, pET28a-SUMO-Nb-scFv, pET28a-SUMO-UreB-Nb6, and pET28a-UreB
      Plasmid design
      Based on literatures, the three genes (Nb-human, UreB-Nb6, and Nb-scFv) targeting the urease subunit B (UreB) of H. pylori were chosen to be constructed the recombined nano-antibody. This program employed the pET28a expression system to construct four optimized plasmids: pET28a-SUMO-Nb-human, pET28a-SUMO-Nb-scFv, pET28a-SUMO-UreB-Nb6, and pET28a-UreB, aiming to aquire the recombinant protein and urease unit UreB(Bacillus subtilis). In the plasmid design, all target genes were fused with a SUMO tag (6×His tag) to enhance protein solubility and expression efficiency. Additionally, a 6×His tag on both the N-terminal and C-terminal can be used to facilitate subsequent protein purification via nickel-affinity chromatography. The LacI gene on the plasmid provides an inducible and controllable switch for gene expression through a dynamic regulatory mechanism of "repression-release". We can use IPTG to induce the expression of the protein. Furthermore, a signal peptide sequence (MKYLLPTAAAGLLLLAAQPA) was integrated into the vector to optimize the production yield of antibody proteins (Nb-human, UreB-Nb6, and Nb-scFv) by regulating their secretion pathway, thereby improving their therapeutic potential. This plasmid system fully leverages the strong T7 promoter and prokaryotic expression advantages of the pET28a vector, while combining the synergistic effects of the SUMO tag, His tag, and signal peptide, thereby establishing a robust foundation for the efficient expression and study of nanobodies (Figure 2).

Figure 2. The design of pET28a-SUMO-Nb-human, pET28a-SUMO-Nb-scFv, pET28a-SUMO-UreB-Nb6, and pET28a-UreB
Experiment
Plasmid transformation and verification
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.
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.
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 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.
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.
Protein expression
      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.
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 8). 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.

FFigure 8. The Western blot result of Nb-human, UreB-Nb6, Nb-SCFV. Immunodiffusion assay of three recombinant nanobodies
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.9 (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 9. 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)
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 10. Colorimetric changes and quantitative analysis of urease-recombinant nanobody interaction

This study provides an important reference for other iGEM teams. Other teams can carry out further exploration based on our research results. Our team Use food-grade probiotics as a carrier; the antibody was delivered into the body in the form of live bacteria. Oral delivery of antibodies by probiotics can directly fight urease, which has the advantages of convenience, high efficiency and high specificity compared with traditional antibiotic treatment. In addition, compared with vaccines that require the body to produce active antibodies first and then fight against antigens, passive antibodies that directly enter the body will neutralize urease more directly and shorten the treatment cycle. In addition, probiotics will also play a beneficial role in the gastrointestinal tract, thus "combining two swords" to quickly kill Helicobacter pylori. Antibodies that specifically recognize Helicobacter pylori antigen can not only cope with infection but also overcome the development of bacterial drug resistance. In our experiment, the binding of antibody to Helicobacter pylori Ureb was simulated by computer, and the possible mechanism of antibody binding to antigen was deeply analyzed, which laid an important foundation for the subsequent analysis of antibody action mechanism. Through computer simulation, we also preliminarily determined that our antibody may have a certain effect on inhibiting Helicobacter pylori infection. Under the premise of biosafety, other teams can use our antibodies to test and develop safer and more effective drugs against Helicobacter pylori infection.

[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.