EcN can be used as a probiotic for the treatment of intestinal related diseases due to its good ability to instantly implant into the intestine and non-pathogenicity. According to recent research, targeted drugs such as monoclonal antibodies have greatly improved the clinical efficacy of diseases such as intestinal inflammation, but there are also problems such as high prices, ineffective treatment for patients, and infections. The design of our experiment employs a comprehensive approach to treating the disease through a combination of therapeutic agents, including A-nanobody, a nanobody derived from Ozoralizumab, and B-nanobody, a nanobody associated with V565, in conjunction with Escherichia coli Nissle 1917. And we added a signal peptide to the front of the antibody, which can release the nanobody to the extracellular. The engineered EcN 1917 could secret the nanobody A and B, which binds to the natural TNFα and block the inflammatory reaction(Figure 1).

Figure 1: Summary of the application of engineered EcN 1917 bacterial for treatment of TNFα associated inflammatory bowel disease.

1.1 Design the PCR primer according to plasmid diagram

Figure 2: The plasmid maps of p15A-A and p15A-B.

We identified the gene sequences of target gene A and target gene B, followed by the selection of the p15A vector for obtaining their plasmid profiles. Subsequently, homologous recombination was utilized to merge them( Figure 2).

1.2 Amplification of target fragment and vector backbone

We utilized polymerase chain reaction (PCR) to amplify the target DNA and prepared an agarose gel for electrophoresis to separate the amplified DNA fragments. The final results were analyzed using ultraviolet imaging to visualize the DNA bands.

Figure 3: PCR amplification of target A/B and p15A vector backbone

Figure 3A displays the electrophoresis image of the p15A vector, with molecular markers and corresponding values shown on the left. The lengths of p15A-1 and p15A-2 fall between 5000 bp and 7500 bp, with the p15A vector having a length of 6535 bp. The target band aligns with the expected size of the target gene.

Figure 3B presents the electrophoresis image of target gene A/B, with markers and values on the left. The length of target gene A/B ranges from 500 bp to 1000 bp, and the specific length is 622 bp. The target band is consistent with the expected size of the target gene.

To ensure that the DNA is ready for use in subsequent experiments, we removed redundant agents, substrates, and Taq polymerase through solubilization and centrifugation, ultimately isolating the target fragment.

1.3 Transformation of plasmid into E. coli DH5α

1.3.1 Homologous recombination

Untreated receptor cells are generally not receptive to recombinant molecules, making transformation inefficient. To address this, we employed methods to induce the cells into an optimal receptive state, significantly improving transformation efficiency. This was achieved by adjusting and mixing calcium chloride, culture medium, and regulating the temperature to prepare the cells.

For the transformation process, we mixed agar, plasmids, and other necessary reagents in specific proportions, followed by ice bath incubation and temperature regulation. The final transformation step was performed using a shaking table to ensure even mixing and proper conditions.

We first prepared the cells, bringing them into the most suitable receptive state, and then carried out the plasmid transformation. Using the nonpathogenic Escherichia coli bacterial transformation method, we successfully amplified the target plasmid DNA, providing essential raw materials for subsequent experiments.

1.3.2 Transformation of p15A-GEx-A/B into E. coli DH5α via heat-shock

Preparation of competent cells: Untreated receptor cells are insensitive to recombinant molecules and difficult to achieve conversion. We use methods to induce cells to be in the most suitable receptive state with a high conversion frequency. Prepare cells by adjusting and mixing calcium chloride, culture medium, and temperature. For the transformation process, we combined the plasmid with competent cells and incubated the mixture in an ice bath for a minimum of 30 minutes. This was followed by procedures including heat shock and subsequent ice stimulation.

1.3.3 Verification of monoclonal colonies

Figure 4: Colony culture of DH5α-p15A-A, DH5α-p15A-B and Sanger sequencing result.

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 4 A and C).

The results in the following two images were obtained (Figure 4 B and D). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

1.4 Transformation of p15A-GEx-A/B into EcN 1917

Figure 5: Colony culture of transformed EcN1917 and colony PCR verification

This image depicts a culture dish in which the target genes A and B were successfully transformed into EcN 1917. The bacteria labeled with -1 on the left are diluted by a factor of 100, whereas those on the right remain undiluted. Figure 5 illustrates that the colony count of p15A-A bacteria significantly decreased upon 100-fold dilution, while an increased number of colonies was observed in the undiluted sample. In contrast, for the p15A-B gene subjected to 100-fold dilution, there was virtually no observable growth of colonies.

On the right side is the validation of monoclonal colonies. According to the image, the length is between 800-1200bp, with a specific length of 1088bp. The length is consistent with the target gene, indicating successful transformation.

1.5 Culture of EcN1917- A/B at LB/SB at 16/37 Celsius and collect the supernatant of medium

1. The preceding night, the bacteria were inoculated into LB liquid culture medium and incubated overnight. Chloramphenicol was subsequently added to achieve a final concentration of 25 micrograms per milliliter, specifically for Nissle1917 expressing antibodies A and B.
2. On the following evening, measure the optical density (OD) and select the strain with the highest OD value. Inoculate this strain at a concentration of 1% of its volume into SB medium containing chloramphenicol and adjust the volumes of the remaining strains to match this maximum OD value. Based on the added volume, proportionally increase the inoculation volume in LB medium while incubating at 16°C and shaking at 220 rpm for 12 hours; prior to incubation at 37°C, measure the OD to ensure that all inoculated liquid starts from an equivalent initial volume.
3. Centrifuge at 3000 g for 5 minutes at 4°C, collect the supernatant, filter it through a 0.22-micron filter, and store the resulting solution at -80°C.

2.1 Validation of the existence and expression of nanoAb-A/B by Western Blot

Figure 6: Nano-antibody expression in the supernatant verified by Western blot assay

We cultured Escherichia coli Nissle 1917 (EcN 1917) transformed with A/B plasmids and optimized the cultivation conditions at both 16°C and 37°C, using LB and SB media. After the cultivation process, we collected the supernatant, centrifuged it, and filtered out bacterial cells to obtain the final product. We then selected the antibody products A-LB and B-LB for further analysis.

To confirm the successful expression of nanoAbs, we performed Western blotting to detect the His tag. As shown in the figure, a band corresponding to the His tag appears around 15 kDa, which aligns with molecular weight predictions and the theoretical sequence of the nanoAbs. Additionally, the expression level of nanoAb-B is slightly higher compared to nanoAb-A, as indicated by the intensity of the bands in the Western blot in the Figure 6.

2.2 Validation of nano-antibody expression by ELISA

Figure 7: Expression of Nano-antibody A and B by ELISA(coated with TNFα).

The ELISA verified the relationship between A/B proteins and TNF-a binding ability. The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.

The antibody binding signal obtained at 37°C is significantly higher than that obtained at 16°C, and the B antibody signal is higher than the A antibody signal, indicating that the expression level of antibody B is higher, which is consistent with the Western blot results shown in Figure 7. The results presented above demonstrate that the nanobodies we engineered have been successfully expressed in the supernatant of the harvested EcN1917 bacterial suspension.

Figure 8: Color diagram of the enzyme plate: Add TMB for color development (left) and after termination (right).

Additionally, we established a standard curve utilizing a commercial kit for TNF-α to function as a reference standard for our detection system and its corresponding results in the Figure 8and Figure 9.

Figure 9: Standard curve of ELISA.

3.1 Validation of nano-antibody function by molecular assay

Figure 10: The ELISA verified the blocking effect of A/B proteins on the interaction of TNFα-TNFR.

Initially, we coated the ELISA plate with TNFR, followed by the addition of various antibody sera mixed with TNFα at four distinct concentrations. Finally, we quantified the resultant light signal by detecting antibodies of anti-TNFα-HRP. We find that blank control group need to higher than positive control group in the Figure 10. So, we found that only in the concentration of 1ng/ml and 0.5ng/ml TNFα, the nano-antibody A shows block effect on the binding of TNFα-TNFR.

3.2 Validation of nano-antibody function by cell viability assay

Figure 11: The luminescence-based cell viability assay for antibody A and B under different temperatures and growth conditions.

After TNF-α binds to the TNF receptor (TNFR) on the cell surface, it triggers apoptosis and necrosis pathways, ultimately leading to cell death. However, when TNF-α binds to a nanoantibody, it is prevented from binding to TNFR, thereby inhibiting the cell death process. To further explore the effect of nanoantibodies in inhibiting inflammatory factors, we conducted cell experiments.

TNF-α was mixed with a series of nanoantibodies at different concentrations (five nanoantibodies obtained from culture, each diluted stepwise by half), and TNF-α mixed with PBS was used as a negative control. This mixture was incubated with U937 cells for 6 hours. Cell viability in each group was then assessed using the CellTiter-Lumi kit.

The results (Figure 11) demonstrated that, compared to the control group, the nanoantibodies produced by the five culture methods exhibited strong inhibition of cell death. Furthermore, this inhibitory effect was positively correlated with the concentration of the nanoantibody—meaning that as the concentration of the nanoantibody increased, the ability to prevent cell death also improved.