We aim to develop a method for screening other proteins or potential compounds that might possess the ability to inhibit the monkeypox virus.

First, we optimized the monkeypox virus I7L protein sequence, constructed a cloning recombinant vector, and transferred it into E. coli for expression. We isolated the I7L-His tag protein in vitro and finally verified its activity through the principle of fluorescence quenching.

By considering that monkeypox virus protease I7L is also recognized as an ideal target against monkeypox for inhibiting virus replication. A recombinant plasmid containing I7L sequence was planned to be structured to purify I7L protein. The correction and biological activity of our purified protein would be confirmed and the protein I7L could be used to discovery the novel lead compounds with inhibitory activity against I7L which has the therapeutic potential against monkeypox.

1.Recombinant plasmid construction

(1)Target genes I7L and ASFV-I7L extraction

(2)Vector plasmid PET28a extraction

(3)Double enzyme cleavage by Nhel+Xhol

(4)Target DNA fragments recycle

(5)Ligation by T4 ligase

2.Preparation of protein expression engineering bacteria

(1)Recombinant plasmid verification by DNA agarose gel electrophoresis

(2)Transformation of recombinant plasmid into Escherichia coli

(3)Achievement of monoclonal colony expressing I7L and ASFV-I7L

(4)Amplification of Escherichia coli expressing I7L and ASFV-I7L

3.Plasmid sequencing

No mutations were found in the sequencing files and the sequences were consistent with the sequences of Monkeypox virus (I7L) and African Swine Fever (ASFV) by NCBI blast comparison. Thus we could confirm the plamid construction is successful.

1.Purification of target protein

(1)Protein expression induced by IPTG

(2)fragmentation and lysis of Escherichia coli

(3)Purification of protein by nickel column

(4)Collect the target protein by elution

2.Protein Validation - Western Blot

Two pure proteins (I7L and ASFV-I7L proteins) with correct molecular weight (47kD) were observed which confirmed the successful engineering of these two plasmids in E. coli BL21.

3.Biological Activity Validation - FRET

(1)Construction of reaction system

Detection of fluorescence intensity

The linear curves 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 active.

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 according to the existing literature. and we can also optimize our protein and try different preparation processes to reduce the cost of our kit, and improve the accuracy and efficiency of our kits by trying different compounds to find more efficient and accurate ones. All in all, I think that we achieved success on various different levels. The most obvious one is being able to test the protein and gain positive results. But far from that, I think that during this project, the success that we have gained is that we united together as a team, solved one problem after another, and gained friendship through the entire process. The success itself has elevated the platform and taken a step closer to biological engineering for the cure of monkeypox. I’m extremely glad that I participated in this iGEM competition, because it not only made me less narrow-minded and broadened my horizon, but all the same, it has opened up an irresistible world to me that I’ve never dabbled in.