The spread of Inflammatory Bowel Disease (IBD) in Asian countries due to the adoption of Western dietary habits have become a severe healthcare problem in recent years[1]. Target therapies that turn off specific inflammation-causing genes have been proven as a major treatment in IBD[2]. In this project, a drug that interferes with Interleukin-18 is designed and tested to provide a new possible solution of IBD treatment IL-18 binding protein (IL-18BP) could bind to IL-18 to stop its function[3] (Figure 1). In humans, while there are four types of IL-18BP (IL-18BPa, Pb, Pc, Pd), only IL-18BPa and IL-18BPc could antagonize IL-18 activity[4,5]. Plasmid containing IL-18 BP with SUMO for better stability, Fc for potential targeted drug delivery, and His tag for easier purification is constructed and tested, the protein is then expressed and purified through BL21 E.coli strands. The purified protein was validated using Western Blotting and its activity was confirmed through T cell activation inhibition experiments.

1.1 Double enzyme digestion of pET-28a vector and PCR amplified the target gene
The pET-28α blank plasmid was cut by restriction enzymes to make them 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 then measured (Tab 1).

The length of target genes SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-F are 1542bp and 1641bp, respectively. Figure 2 A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the pET-28α blank plasmid. Figure 2.B shows that the plasmid was successfully digested.
1.2 Construction and Extraction of Recombinant plasmids
After restriction enzyme digest and ligation of pET-28a with Sumo-IL-18BP-Pa-Fc and Sumo-IL-18BP-Pc-Fc , the recombinant plasmids were transformed into E.coli DH5α competent (Fig 3A and B). The transformants were then identified by colony PCR. The agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating the construction were successfully completed (Fig3 C).
1.3 Plasmid extraction and sequencing
The correct clones were cultured in LB medium, then we extracted plasmids for sequencing. The concentration and purity of plasmids were also measured (Tab 2).
Then the plasmid pET-28a with Sumo-IL-18BP-Pa-Fc and Sumo-IL-18BP-Pc-Fc were sent to the biological company for sequencing. The comparison of the sequencing results showed that the target gene sequence was consistent with the sequencing results (Fig 4A and B ), indicating that the plasmid was successfully constructed.

2.1 protein induced expression
We transformed the correct recombinant plasmids into E.coli BL21 competent cells to promote protein expression. The positive clones were selected out by kanamycin resistance in the solid LB medium.The BL21 colony cultures were expanded into large flasks for growth. After IPTG induction, the colonies were shaken overnight for protein production. The proteins were extracted then purified use the His tag.
At the same time, we transformed the pET28a-Sumo-IL10-Fc synthesized by the company into E.coli BL21 to promote protein expression as our subsequent positive control. In the fig 5 A, the agarose gel electrophoresis results showed that we obtained the expected length of sumo-IL10-Fc, indicating the construction were successfully completed. And the target gene sequence was consistent with the sequencing results(Fig. 5 B).
2.2 Protein verification after purification
The purified IL18-BPa, IL18-BPc, and IL10-BP proteins were 56.9kDa, 61kDa, and 62.1kDa, respectively. The SDS-PAGE successfully verified the IL18-BPa, IL18-BPc, and IL10 proteins extracted and purified from E. coli BL21 (Fig 6).
2.3 Western Blot verification of extracted protein
Compared to coomassie Brilliant Blue staining, the principle of Western detection is antibody antigen specific reaction, with high detection specificity. The proteins we expressed all carrying His tag, and specific His antibodies can be used to detect purified proteins. As shown in Figure 7, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.
2.3 Use BCA colorimetric method to determine the protein concentration
The concentration of the samples was measured using the BCA calorimetric method. We use standard concentration protein solution and BCA colorimetric method to determine the protein concentration. The absorbance value was measured by the enzyme-linked immunosorbent assay (ELISA) reader. We draw a scatter plot, insert a standard curve, and calculate the R-squared value.In the Fig.8, the fitting coefficient is greater than 0.99, indicating that the fitting effect is perfect.
The protein concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc were calculated according to the standard curve, as shown in Tab 3.

3.1 Measuring the standard curve using an INF-γ standard.
In the experiment, we need the concentration of INF-γ, and we establish the standard curve of INF-γ.The R squared value is larger than 0.99, which indicates that the curve is accurate (Fig. 9 ).
3.2 The influence of dose and storage conditions on protein activity
Store the three proteins (Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10) at different temperatures (-80, -20, 4, 37 ℃) for 24 hours and incubate them with mouse primary T lymphocytes. Stimulate the cells with IL-18 and detect the IFN-γ in the cell supernatant.
In Figure 10, the concentration of IFN-γ gradually decreased with the increase of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 protein concentrations. It illustrates the inhibitory effect of recombinant Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ production.
The results are showed in Fig 10,the activity of protein is affected by temperature. The inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 on IFN-γwas not affected after 24 hours at -80 °C. After 24 hours at -20 °C and 4 °C, the inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ decreased. After 24 hours at -37°C, the inhibitory effect of Sumo-IL-BP-18-BPc, Sumo-IL-BP-18-BPa and Sumo-IL-10 protein on IFN-γ was significantly decreased. Storing at low-temperature preservation is beneficial for the stability of recombinant proteins. Furthermore, the anti-inflammatory effect of recombinant proteins is dose-dependent.
In the fig 11, it can be seen that after storage at 4°C for 24 hours, IL-18BPc's effects are still comparable to that of IL-10 in the 2.0 ug/ml, while IL-18BPa is less stable.

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