The number of monkeypox infections worldwide has increased significantly during the COVID-19 epidemic1. Due to there has been no effective treatment for monkeypox, we think it could help if we could develop a kit that can effectively screen for effective compounds against monkeypox.

According to recent research, it is found that I7L and ASFV-I7L are highly conserved in Orthopoxvirus and play a key role in virus assembly23. Therefore, drug design and development targeting I7L and ASFV-I7L are of great value for controlling monkeypox virus infection.

In this project, we plan to obtain and purify I7L protein and ASFV-I7L protein by the construction of recombinant E. coli and then further develop a kit for screening potential monkeypox inhibitors.

We designed two genes, I7L and ASFV-I7L, and extracted the plasmid pET28a from Escherichia coli.We then amplified the I7L and ASFV-I7L genes and the target DNA was obtained by PCR (figure 3). The I7L and ASFV-I7L genes are inserted into pET28a to get a recombinant plasmid. Then, the I7L and ASFV-I7L-pET28a plasmid were transformed into BL21 (DE3) and DH5α by heat shock method. .

As shown in Figure 4, I7L and ASFV-I7L obtained the correct genes we needed by confirming the length of bp which is 1269bp and 723bp around , respectively. So we can make this a prerequisite for furtheraction.

To insure over I7L and ASFV-I7L DNA fragment have inset into the plasmid in a right direction, and there is no mutation during the heat shock process, we sent the plasmids constructed successfully in PCR identification for sequencing, and sequencing using template primers showed that the I7L and ASFV fragments were inserted into the plasmid in the correct orientation. No mutations were found in the sequencing files (figure 5), and the sequences were consistent with the sequences of Monkeypox virus (I7L) and African Swine Fever (ASFV) by NCBI blast comparison2.

We validated protein purification production from the recombinant plasmid by Western Blot. As shown in Figure 4, two pure proteins (I7L and ASFV-I7L proteins) with correct molecular weight (47kD) were obtained by purification of the two proteins with His-Tag. The protein markers used were Beyotime 14.4-116 protein markers.

In order to evaluate the activity of our purified proteins, we applied the FRET assay for function validation. After the purified protein was configured, 0.4 mM EDTA,4 mM DTT and 20% glycerol were added into the reaction buffer containing 20 mM HEPES,120 mM NaCl and pH 6.5, and then 100 nM I7L and ASFV-I7L were added into two parts, respectively. Finally, the blank control group was set up with BSA protein.

Table 1. FRET data of I7L and I7L-ASAV against the control group (BSA protein)

The linear curves in figure 7 show that the fluorescence values of the two groups of target proteins decreased with the increase of time, and the control protein (BSA protein) remained unchanged with the increase of time, demonstrating that I7L and ASFV-I7L were biologically active3.

In summary, we successfully designed and validated I7L and ASFV-I7L dna parts, and inserted these two genes into plasmid pET28a from Escherichia coli by enzyme digestion, enzyme-linked plasmid construction and thermal excitation. We then transferred recombinant plasmids I7L and ASFV-I7L-pET28a into BL21, and the protein expression of the recombinant plasmid was induced by 0.4 mM IPTG, and the two target proteins were purified by His-Tag to obtain two pure proteins. Finally, the I7L and ASFV-I7L biological activity were confirmed by fluorescence assay.

We have successfully obtained the target proteins in the experiment, and in the future, we can use our proteins to screen some compounds that may have potential therapeutic effects on monkeypox. In addition, we are still looking for positive controls that could supplement our kit. We will also optimize our protein and try different preparation processes to reduce the cost of our kit, improve the accuracy and efficiency of our kits by trying different compounds to find more efficient and accurate ones.

1. Lum FM, Torres-Ruesta A, Tay MZ, et al. Monkeypox: disease epidemiology, host immunity and clinical interventions. Nat Rev Immunol. Oct 2022;22(10):597-613. doi:10.1038/s41577-022-00775-4

2. Aleshin AE, Drag M, Gombosuren N, et al. Activity, specificity, and probe design for the smallpox virus protease K7L. J Biol Chem. Nov 16 2012;287(47):39470-9. doi:10.1074/jbc.M112.388678

3. Katritch V, Byrd CM, Tseitin V, et al. Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches. J Comput Aided Mol Des. Oct-Nov 2007;21(10-11):549-58. doi:10.1007/s10822-007-9138-7