Part:BBa_K5522002

pET28a-IL18-BPc

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction Design

The modified human BPc gene was designed as described previously in the "Add a new basic part" section. The pET-28alpha containing kanamycin resistance and an additional His tag at the N-terminal was obtained from GenScript. Both the plasmid and gene fragment were cut using restriction enzymes NheI and XhoI, then recombined using ligation enzymes to form the recombinant plasmid (Figure 1).

Figure 1: The plasmid map of pET28a-IL18-SUMO-BPc-Fc.

Engineering Principle

The spread of Inflammatory Bowel Disease (IBD) in Asian countries due to the adoption of Western dietary habits has become a severe healthcare problem in recent years [1]. Target therapies that turn off specific inflammation-causing genes have been proven to be a major treatment in IBD [2]. In this project, a drug that interferes with Interleukin-18 (IL-18) was designed and tested as a potential solution for IBD treatment. IL-18 binding protein (IL-18BP) binds to IL-18 to stop its function [3].

In humans, four types of IL-18BP exist (IL-18BPa, Pb, Pc, Pd). Among these, IL-18BPc has been shown to antagonize IL-18 activity [4-5]. The plasmid containing IL-18BP with SUMO for better stability, Fc for potential targeted drug delivery, and His tag for easier purification was constructed and tested. The protein was expressed and purified through BL21 E. coli strains. Purified protein was validated using Western Blotting, and its activity was confirmed through T cell activation inhibition experiments.

Figure 2: The IL18 signal pathway diagram.

Experimental Approach

The PET-28α blank plasmid was cut by restriction enzymes to make it linear. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. After the DNA was recovered from gel, the concentration and purity of the samples were measured.

The length of the target gene, SUMO-IL-18BPc-Fc, is 1641bp. Figure 3A shows that the length of the target gene was consistent with the electrophoresis results, indicating successful amplification. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Figure 3B shows that the plasmid was successfully digested.

Figure 3: Identification of PCR amplified gene and enzyme digested pET-28α vector.

After restriction enzyme digestion and ligation of pET-28a with Sumo-IL-18BP-Pc-Fc, the recombinant plasmids were transformed into E. coli DH5α competent cells (Figure 4A). Transformants were identified by colony PCR, and agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating successful construction (Figure 4B).

Figure 4: The transformants form colonies on solid LB medium and colony PCR amplification of IL-18-BPc.

The plasmid pET-28a with Sumo-IL-18BP-Pc-Fc was sent for sequencing, and comparison of the sequencing results showed that the target gene sequence was consistent with expectations (Figure 5).

Figure 5: Gene sequencing of IL-18-BPc.

Cultivation, Purification and SDS-PAGE

The purified IL18-BPc protein was 61kDa. SDS-PAGE verified the extracted and purified IL18-BPc proteins from E. coli BL21 (Figure 6).

Figure 6: SDS-PAGE verification of extracted proteins.

The proteins expressed carried a His tag, and His antibodies were used to detect purified proteins using Western blot. The protein size was consistent with the expected size, demonstrating successful protein expression (Figure 7).

Figure 7: Detection of recombination protein expression by Western blot.

Characterization/Measurement

The activity of the protein was characterized by its ability to inhibit T cell activation. Mice abdominal T-cells were stimulated by TNF-alpha and IL18-BPc. The recombinant protein was added, and the production of IFN-gamma was measured by ELISA. The results showed that the recombinant protein was able to inhibit IFN-gamma production, indicating an anti-inflammatory effect (Figure 8).

Figure 8: The influence of storage temperature on protein activity.

Summary

Testing in all three stages showed positive results, indicating that our model design was reasonable and accurate. Both IL-18BPa and IL -18BPc exhibited significant anti-inflammatory effects comparable to IL-10. This suggests that these two proteins can serve as a basis for further research in anti-inflammatory therapies.

During the process, we encountered challenges, such as using an inefficient primer for the IL-18BPc gene. After redesigning the primers and performing further experiments, we successfully constructed the recombinant plasmids. Additionally, to serve as a positive control, we synthesized and transformed the pET28a-SUMO-IL-10-Fc plasmid into BL21 cells alongside our constructs.

The final results demonstrated that SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-Fc have potential to reduce inflammation. This opens up future directions for research, such as testing the stability of the protein in the intestinal environment, optimizing doses for different levels of inflammation, and investigating other potential therapeutic applications.

References

• [1] Piersiala K, Hjalmarsson E, da Silva PFN, Lagebro V, Kolev A, Starkhammar M, Elliot A, Marklund L, Munck-Wikland E, Margolin G, Georén SK, Cardell LO. Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes. Int J Cancer. 2023 Aug 15;153(4):854-866. doi: 10.1002/ijc.34555. Epub 2023 May 5. PMID: 37144812.
• [2] Liang X, Fan Y. Bidirectional two-sample Mendelian randomization analysis reveals a causal effect of interleukin-18 levels on postherpetic neuralgia risk. Front Immunol. 2023 May 25;14:1183378. doi: 10.3389/fimmu.2023.1183378. PMID: 37304287; PMCID: PMC10247971.
• [3] Menachem A, Alteber Z, Cojocaru G, et al. Unleashing Natural IL18 Activity Using an Anti-IL18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects. Cancer Immunol Res. 2024. OOF1-OF17.
• [4] Yamanishi K, Hata M, Gamachi N, et al. Molecular Mechanisms of IL18 in Disease. Int J Mol Sci. 2023;24(24).
• [5] Ihim, S. A., Abubakar, S. D., Zian, Z., Sasaki, T., Saffarioun, M., Maleknia, S., & Azizi, G. Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment. Front Immunol. 2022;13:919973. doi:10.3389/fimmu.2022.919973.