CN116854817B - Nano antibody targeting eIF4E2, E3 ligase chimeric and application thereof in ISGylation activation - Google Patents

Abstract

The invention provides a nano antibody targeting eIF4E2, an E3 ligase chimeric body and application thereof in ISGylation activation, wherein the nano antibody takes eukaryotic translation initiation factor eIF4E2 of hypoxia response as a target, nano antibodies Nb.30C7 and Nb.28E11 targeting eIF4E 2N-terminal and C-terminal are screened through a nano antibody yeast display library, and specific activation of eIF4E2 ISGlytion in cells is realized by constructing a nano antibody-E3 ligase chimeric body; the activation of the site-specific ISGylation is beneficial to revealing the functions of the ISGylation, and provides a new idea for the research of single-target ISGylation in the future.

Description

Nano antibody targeting eIF4E2, E3 ligase chimeric and application thereof in ISGylation activation

Technical Field

The invention belongs to the technical field of application of eukaryotic translation initiation factor eIF4E2, and particularly relates to a nano antibody targeting eIF4E2, an E3 ligase chimera and application thereof in ISGylation activation.

Background

Regulation of protein synthesis is important for cellular homeostasis, survival, differentiation, and many other functions. The protein synthesis process is "very expensive" and consumes a large portion of the energy of the cell. Therefore, the cells need to produce an appropriate amount of protein at the correct time and place (Hershey et al 2019). Translational control is a critical step in regulating protein expression, and abnormal translational regulation leads to the occurrence of many diseases. Eukaryotic translation initiation factor eIF4E (eukaryotic translation initiation factor E, eIF 4E) plays a critical role in the regulation of protein synthesis by recognizing the cap structure present 5' to eukaryotic mRNA to initiate cap-dependent translation. eIF4E2is a homologous protein to eIF4E and also modulates translation initiation by cap-dependent translation. eIF4E2is not expressed in high amounts, but is ubiquitously expressed in various tissues, and can form a plurality of regulatory complexes with different binding partners, thereby coping with different physiological environment regulation translations (Christie et al 2021). eIF4E2 undergoes ISG (international-stimulated gene) post-translational modification that enhances its cap binding capacity, thereby functioning as a translational inhibitor and thus inhibiting translation of certain viral mrnas (Okumura et al 2007). However, whether the modified eIF4E2 modulates certain physiological processes and is related to the occurrence and development of certain diseases is yet to be studied.

Like ubiquitination, the ubiquitin-like modified ISGylation modification is an important post-translational modification that is widely present in eukaryotes. Ubiquitin-like protein ISG15 undergoes covalent attachment to a target protein mediated by a cascade of specific E1 activating enzyme, E2 coupling enzyme and E3 ligase upon stimulation of cells by type I interferon or the like to effect modification of ISGylation (Villarroya-Belri et al 2017). ISGylation is closely related to the regulation of physiological processes and diseases such as protein translation, autophagy, innate immunity, neoplasms, stroke and the like (Mirzalieva et al 2022). Although the activation of ISGylation modification can be accomplished by drugs such as LPS or co-transformation of ISGylation modification system plasmids (including E1 activating enzyme, E2 binding enzyme, E3 ligase and ISG15 plasmids), these methods activate ISGylation as global and multi-target activation, which cannot be done if one wants to study the effect of ISGylation modification of a certain target alone. Therefore, we want to develop a novel E3 ligase through nanobody to target and activate ISGylation modification, thereby helping us to conduct relevant research on specific target ISGylation.

Disclosure of Invention

A first object of the present invention is to propose a nanobody targeting eIF4E2, which can specifically bind to eIF4E2 protein; the nano antibody targeting eIF4E2 comprises the amino acid sequence shown in SEQ ID NO: 2-12.

Further, the amino acid sequence of the nano antibody is SEQ ID NO:2 and/or SEQ ID NO:11.

a second object of the present invention is to provide a nucleotide sequence encoding the above nanobody; the nucleotide sequence.

A third object of the present invention is to provide an eIF4E2 ISGylation-modified E3 ligase chimera comprising the nanobody described above, and a linking domain fragment linked to the nanobody; the connecting domain fragment is a domain for catalyzing ISG15 to transfer to target protein; the chimera can utilize the high specificity and high affinity of the nanobody to realize the ISGylation activation specific to the target protein.

Further, in the E3 ligase chimeric, the enzyme is ISGlyation E3 ligase and/or ubiquitin E3 ligase.

Preferably, the isglytion E3 ligase is HERC5 and the ubiquitin E3 ligase is HHARI.

More preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO:11, said linking domain fragment is selected from the group consisting of isglytion E3 ligase.

A fourth object of the present invention is to provide an expression vector comprising the above E3 ligase chimera; the expression vector can realize ISGylation activation specific to target proteins by utilizing high specificity and high affinity of the chimeric body.

A fifth object of the present invention is to provide a cell transfected with the above expression vector; the high specificity and high affinity of the expression vector can be exerted in cells, and the specific ISGylation activation of the target protein can be completed.

The fifth object of the invention is to provide the application of the expression vector in eIF4E2 ISGlytion activation, which can realize specific ISGylation modification for activating eIF4E2 and provide a new guide for independently researching the effect of ISGylation modification of a certain target point.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a method for screening the nano antibody for recognizing eIF4E2, and realizes specific activation of eIF4E2 ISGlytion in cells by constructing a nano antibody-E3 ligase chimera, thereby providing a new method and thinking for relevant research of ISGlytion.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of screening for antibodies targeting eIF4E2 nanobodies using the yeast surface display technology of the invention. (A) Results of eIF4E 2C-terminal (28 library-eye) nanobody 3 in-turn sorting. Three rounds of antigen concentrations were 1. Mu.M, 100nM,1nM, respectively; quadrant Q2 is the double positive region; the X-axis represents the fluorescence signal of the APC channel corresponding to Alexa647 and the Y-axis represents the fluorescence signal of the FITC channel corresponding to the antigen moiety. (B) Results of the round robin sorting of eIF4E 2N-terminal (30 library-eye) nanobody 3. Three rounds of antigen concentrations were 500nM,50nM,1nM, respectively; quadrant Q2 is the double positive region; the X-axis represents the fluorescence signal of the APC channel corresponding to Alexa647 and the Y-axis represents the fluorescence signal of the FITC channel corresponding to the antigen moiety.

FIG. 2is a graph showing the results of the verification of eIF4E2ISGylation modification of the present invention. (A) site of modification of eIF4E2 by ISGylation. (B) LPS treatment activated eIF4E2ISGylation, si-eIF4E2 in 293T cells reduced modification. After 24h treatment of 293T cells with si-eIF4E2 or Scramble, LPS (10 Mm) was added for 6h, protein samples were collected and Western felt detection was performed using eIF4E2 antibody detection. (C) LPS treatment activated eIF4E2ISGylation, PROTAC-eIF4E2 in BT549 cells reduced modification. pcDNA3.1-Flag-PROTAC-30C7 and pcDNA3.1-Flag-RPOTAC-28E11 were transfected into BT549 cells for 48h, and then treated with LPS (10 Mm) for 6h, and protein samples were collected and subjected to Western bolt assay with eIF4E2 antibody assay.

FIG. 3 shows the principle and results related to targeting eIF4E2ISGylation modified E3 ligase chimera of the present invention. (A) The E3 ligase HERC5 and HHARI are schematic structural representations in which the HECT domain and the RING1-IBR-RING2 (RBR) domain are functional domains that deliver ISG15, respectively. (B) Overlap PCR was performed to link E3 ligase with nanobody. (C) chimera-mediated ISGylation mechanism. The chimera is specifically combined with the target protein through the nano antibody, and the positions of the E3 ligase and the target protein are spatially pulled up, so that ISGylation modification is specifically activated. (D) Chimeric HECT-30C7 may be more effective in activating eIF4E2 ISGylation. 4 sets of chimeras (pcDNA3.1-HA-HECT-30C 7, pcDNA3.1-HA-RBR-30C7, pcDNA3.1-HA-HECT-28E11, pcDNA3.1-HA-RBR-28E 11) were co-transfected with pcDNA3.1-Flag-ISG15, respectively, into HeLa cells, protein samples were harvested after 48h and Western bolt assays were performed using eIF4E2 antibody assays.

FIG. 4 shows the results of the chimeric HECT-30C7 activation of eIF4E2ISGylation according to the present invention. (A) The HECT domain of E3 ligase HERC and its active center Cys994. Mutation of Cys to Ala inhibits its ability to transmit ISG 15. (B) Chimeric HECT-30C7 activates the ISGylation modification of eIF4E 2. The chimeras pcDNA3.1-HA-HECT-30C7, pcDNA3.1-HA-HECT (C994A) -30C7 and pcDNA3.1-HA-HECT-BV025 were co-transformed or single-transformed with pcDNA3.1-Flag-ISG15, respectively, into HeLa cells, and after 48 hours protein samples were collected and Western felt detection was performed using eIF4E2 antibody detection. (C) pcDNA3.1-HECT-30C7, pcDNA3.1-HA-eIF4E2 and pcDNA3.1-Flag-ISG15 were co-transfected into 293T cells, anti-Flag co-immunoprecipitation was performed on the cells after 48h, western-felt detection was performed on the obtained samples, and HECT-BV025 was used as a control.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Proteolysis targeting chimera (PROTAC) is a technology developed in recent years to target protein degradation. The PROTAC molecule consists essentially of two parts, a ligand (mainly a small molecule inhibitor) capable of binding to the target protein and E3 ubiquitin ligase, and when the ligand binds to the target protein, ubiquitination of the target protein is induced by recruiting ubiquitin by the E3 ubiquitin ligase linked to the ligand, thereby mediating degradation of the target protein (Li et al 2020). In addition, researchers have further developed the Bio-PROTAC technology by directly replacing the substrate recognition domain of the E3 ligase with a small peptide fragment or small molecule protein that specifically binds to the target protein, thereby expanding the range of applications of the PROTAC technology (Lim et al 2020). ISGylation is similar to the ubiquitination modification process in that isgyl modification covalently binds ISG15 to the target protein via E3 ligase. Since engineering an E3 ubiquitin ligase is capable of mediating specific site ubiquitination, engineering the substrate recognition domain of an E3 ligase for ISGylation based on similar principles should also be possible to target activation of ISGylation modification. We have therefore engineered the ISGylation E3 ligase: novel E3 chimeric enzyme is formed by fusion expression of a functional domain (a domain catalyzing transfer of ISG15 to a target protein) in an ISGylating E3 ligase and a nanobody, and the chimeric body can realize ISGylating activation specific to the target protein by utilizing high specificity and high affinity of the nanobody.

In the following examples, the biological materials involved are as follows:

1) Experimental strains

Coli DH5a and EBY100 were laboratory-preserved strains.

2) Cell lines

3) Vectors and plasmids

Eukaryotic expression vectors pcDNA3.1, pcDNA3.1-3 xFlag, pcDNA3.1-HA and yeast protein expression vector pNACP are all preserved in a laboratory, and other plasmids are all self-constructed.

Example 1 Targeted screening of eIF4E2 nanobodies

Construction of nanobody library: at the beginning of the experiment we obtained the eIF4E2 targeting nanobodies 28A1 and 30G11 by phage display technology. On this basis, we designed NNK degenerate primers and introduced saturation mutagenesis into the CDR regions by PCR techniques. And then, by utilizing the high homologous recombination property of the yeast, the mutation library and the linearized surface display carrier pNACP are jointly and electrically transformed into Saccharomyces cerevisiae EBY100 to obtain two small yeast display libraries (28 library-yeast and 30 library-yeast) containing CDR region mutations, so that yeast display screening is performed.

Screening of yeast libraries first the library is induced to express so that nanobodies can be displayed on the surface of yeast cells. The antigen BSA-peptide (BSA coupled eIF4E2N, C end polypeptide sequence) was then mixed with anti-BSA (murine) and incubated with rotation at room temperature. After the incubation of the antigen part is completed, the yeast cells after induced expression and anti-Myc (rabbit source) are added, and the yeast cells with eIF4E2 nanobody are rotated and incubated at room temperature, so that the yeast cells with eIF4E2 nanobody are fully combined with the antigen. The BSA was then incubated with FITC (showing the binding efficiency of antigen to nanobody) and Myc-tagged Alexa647 (showing the display efficiency of nanobody). After the secondary antibody incubation is completed, yeast cells with double fluorescence can be selected through a flow cell sorter, and the nano antibody carried by the obtained double fluorescence yeast cells is the nano antibody capable of being specifically combined with the antigen.

The enrichment rates of 28 library-yeast and 30 library-yeast were continuously increased in 3 rounds of sorting to 43% and 60.9% respectively (A, B in FIG. 1) using CDR region saturation mutagenesis nanobody yeast display libraries. The sequencing of the nano antibody with the positive rate of ten in 30 library-yeast has 5 successful sequencing, wherein 3 of the 5 sequences are the same sequence, namely the sequence of the 30C7 clone is enriched, and the 30C7 clone with the highest positive rate in 30 library-yeast is also obtained. 9 sequences were successfully sequenced in 28 library-year, with the highest positive rate for the 28E11 clone, and the results after sequence deduplication are shown in Table 1, where WT represents the wild type. We therefore selected nanobodies 28E11 (targeting eIF4E 2C-terminus) and 30C7 (targeting eIF4E 2N-terminus) for subsequent experiments. Clones with a positive rate of 10 a in the 28 library-year and 30 library-year 96 Kong Banliu format were selected for sequencing and aligned with the WT sequence, and the sequencing results showed that mutations occurred predominantly in the CDR3 region.

Table 1: positive rate

The sequences shown in Table 1 are as follows.

>28WT(SEQ ID NO：1):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISSGGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYPHRYGHYWGQGTQVTVSS

>28E11(SEQ ID NO：2):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISSGGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYHSLYYGYWGQGTQVTVSS

>28B1(SEQ ID NO：3):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISSGGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYPPLYCHYWGQGTQVTVSS

>28C4(SEQ ID NO：4):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISCGGAITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYLPRYGSYWGQGTQVTVSS

>28F12(SEQ ID NO：5):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISCGAGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYYSRHYGYWGQGTQVTVSS

>28F11(SEQ ID NO：6):

QVQLQESGGGLVQAGGSLRLSCAASGRTSSSYAMGWFRQAPGKEREFVAAISCGAAITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYYSRHYGYWGHGTQGTVSS

>28F5(SEQ ID NO：7):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISCAGAITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYLRRYGSSYDYWGQGTQVTVSS

>28A9(SEQ ID NO：8):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISCGGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAHYLPHHYDYWGQGTQVTVSS

>28E10(SEQ ID NO：9):

QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISCAGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAHSPDLYYDYWGHGTQGTVSS

>28E3(SEQ ID NO：10)：

QVQLQESGGGLVQAGGSLRLSCAASGCTSSSYAMGWFRQAPGKEREFVAAISCDSGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARYYSNPYGYWGQGTQVTVSS

>30C7(SEQ ID NO：11):

QVQLQESGGGLVQAGGSLRLSCAASGYTFSSNVMGWFRQAPGKEREFVAAINSGGGRTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYGRANSSYNYGYWGQGTQVTVSS

>30F8(SEQ ID NO：12):

QVQLQESGGGLVQAGGSLRLSCAASGYTNSRYYMGWFRQAPGKEGEFVAAISRSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAASRSSSSSLYDYWGQGTQVTVSS

>30WT(SEQ ID NO：13)：

QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAAISRGGGRTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARRSGRRGSSLLRYD YWGQGTQVTVSS

Example 2eIF4E2 ISGylation modified verification

1) Constructing a bio-PROTAC system by replacing a substrate recognition domain of E3 ubiquitin ligase with a high-affinity eIF4E2 nanobody, degrading eIF4E2 in cells, adding LPS to activate eIF4E2ISGylation modification after 48h transfection, and verifying migration bands after eIF4E2ISGylation modification;

2) And (3) transferring siRNA targeting eIF4E2 into cells, and adding LPS to activate eIF4E2ISGylation modification after 36h transfection to verify migration bands after eIF4E2ISGylation modification occurs.

ISGylation may occur at the K134 and K222 sites of eIF4E2 (FIG. 2A). LPS (lipopolysaccharide) is a strong activator of immune cells and can be used to treat cells that stimulate IFN expression, thereby activating the onset of modification of the related protein ISGylation. To verify ISGylation modification of eIF4E2 we treated 293T cells with LPS, SDS dissociated the cells and Western felt detection with antibodies to eIF4E 2. We found that the LPS treated group showed a clearly activated band at approximately 45kd compared to the control group (fig. 2B lane 2). To further demonstrate that this activated band may be a migration band after modification of eIF4E2, we designed siRNA to lower its mRNA level for eIF4E2 and added LPS 48h after eIF4E2 transfection of siRNA. Protein samples were harvested 6h after LPS treatment and Western felt detection with an antibody to eIF4E 2. The results are shown in FIG. 2B: the siRNA targeting eIF4E2 reduces the expression level of the migration bands occurring after eIF4E2 background and LPS treatment.

We also performed similar experiments in BT549 cells (breast cancer cells), applying the Bio-PROTAC technique to target degradation of eIF4E2 at the protein level, and adding LPS 48h after transfection of PROTAC. Protein samples were harvested 6h after LPS treatment and Western felt detection with an antibody to eIF4E 2. We found that the activated bands appeared at the same positions in the LPS-treated BT549 cells (FIG. 2C lane 2), and that PROTAC-30C7 and PROTAC-28E11, which target degradation of eIF4E2 protein levels, reduced levels of eIF4E2 background and migration bands that appeared after LPS treatment, with higher degradation efficiency of PROTAC-30C7 (FIG. 2C), indicating that nanobody 30C7 may be more suitable for targeting the native conformation of eIF4E 2. In different cell lines, the migration band of eIF4E2 activated by LPS could be reduced by siRNA and PROTAC targeting eIF4E2, both different ways yielding the same result. We therefore speculate that eIF4E2 migrates after ISGylation modification, with a migration band occurring at about 45 kd.

Example 3 construction of E3 ligase chimeras targeting eIF4E2ISGylation modification

The ISG 15-transmitting domain in the ISGylation E3 ligase was linked by molecular cloning to a nanobody that specifically recognized eIF4E 2.

HERC5 is a relatively common ISGylation E3 ligase. HHARI is the ubiquitin E3 ligase of eIF4E 2. The structure of these two E3 ligases mainly includes: the functional domain, substrate recognition domain and other ancillary domains of ISG15 are transferred. The HECT domain functions to deliver ISG15 in HERC5 and the RBR domain functions to deliver ISG15 in HHARI (FIG. 3A). For construction of eIF4E 2-targeting E3 ligase chimera we retained HECT in the HERC5 and HHARI E3 ligase with the RING1-IBR-RING2 (RBR) domain and linked the HECT domain to nanobody 30C7 targeting eIF4E 2N-terminus and nanobody 28E11 targeting eIF4E 2C-terminus, respectively, by a short linker, as well as the RBR domain to nanobodies 30C7 and 28E11, respectively (fig. 3B). The 4 sets of E3 chimeric fragments were then constructed onto mammalian cell expression vector pcdna3.1, yielding 4 sets of plasmids: pcDNA3.1-HA-HECT-30C7, pcDNA3.1-HA-RBR-30C7, pcDNA3.1-HA-HECT-28E11, pcDNA3.1-HA-RBR-28E11.

The working principle of the novel E3 ligase chimera is as follows: the chimera can utilize nanobody specific targeting to bring the E3 ligase of ISGylation into proximity with the target protein, facilitating covalent attachment of ISG15 to the target protein, thereby specifically mediating ISGylation modification of the protein of interest (fig. 3C). To initially verify the feasibility of activation of eIF4E2ISGylation modification of 4 sets of nanobody-E3 ligase chimeras, we transfected pcdna3.1-Flag-ISG15 with plasmids of these four sets of chimeras, respectively, in Hela cells, and after 48h of transfection, cell harvest samples were lysed with SDS for Western felt detection with antibodies to eIF4E 2. The results show that chimeric HECT-30C7 may be effective in activating the ISGylation modification of eIF4E2 (FIG. 3D).

Example 4 chimeric activation of eIF4E2ISGylation

The activation of eIF4E2ISGylation by the chimera was further verified by constructing the E3 ligase mutant with negative nanobody control.

Cys994 located in the HECT domain is the E3 ligase HERC5 active site (FIG. 4A). Cys994 is critical for the delivery of ISG15, and after mutation to alanine, its function of delivering ISG15 is reduced or lost. In addition BV025 is a nanobody reported in the literature that does not recognize any target protein, and is often used as a negative control for nanobodies. We have thus constructed HECT (C994A) mutant and BV 025E 3 chimeric as controls to further verify HECT-30C7 activation of eIF4E2ISGylation modification. And respectively transferring the constructed pcDNA3.1-HA-HECT-30C7, pcDNA3.1-HA-HECT (C994A) -30C7 and pcDNA3.1-HA-HECT-BV025 plasmids and pcDNA3.1-Flag-ISG15 into Hela cells, and collecting protein samples after 48 hours for Western felt detection. The results are shown in fig. 4C: the C994A mutation of HECT inhibited activation of the migration band after ISGylation modification of eIF4E2 relative to HECT-30C7 of WT, and the activation of the eIF4E2ISGylation modification by either chimeric HECT-BV025 or singly-transferred Flag-ISG15 was not apparent, indicating that chimeric HECT-30C7 might be specific for ISGylation modification of eIF4E 2.

We also performed CO-immunoprecipitation (CO-IP) to verify the activation of the eIF4E2ISGylation modification by the chimera HECT-30C 7. For IP experiments we constructed the sequences HECT-30C7 and HECT-BV025 on the mammalian expression vector pcDNA3.1, respectively, along with the HA-tagged eIF4E2 and Flag-tagged ISG15 pcDNA3.1 plasmids. The pcDNA3.1-HECT-30C7 was then co-transferred into 293T cells with pcDNA3.1-HA-eIF4E2 and pcDNA3.1-Flag-ISG15 plasmids, and the cell lysates were subjected to anti-Flag co-immunoprecipitation after 48h of transfection. If the chimeric HECT-30C7 activates the ISGylation modification of eIF4E2, flag-ISG15 will be covalently bound to the modification site of HA-eIF4E2, then when we pull Flag-ISG15 with the tags of Flag, we will pull HA-eIF4E2 down together, at which time we can detect the HA signal in IP samples by Western felt. The experimental results are shown in fig. 4C: HA signal was detected in IP samples co-transfected HECT-30C7 with HA-eIF4E2 and Flag-ISG15, and the band size of HA was also around 45kd, consistent with the eIF4E2 migration band size detected in the previous LPS-treated group, while no HA signal was detected in control HECT-BV 025.

Conclusion: among the four groups of chimeras, chimera HECT-30C7 activated eIF4E2ISGylation modification was the best. Neither the C994A mutant of HECT nor the nanobody negative control BV025 activated eIF4E2ISGylation relative to the chimeric HECT-30C 7. IP results showed that the chimeric HECT-30C7 activated eIF4E2 ISGylation.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (4)

1. The nano antibody targeting eIF4E2is characterized by having an amino acid sequence shown in SEQ ID NO:11.

2. The E3 ligase chimera modified by targeting eIF4E2ISGylation sequentially comprises the nanobody of claim 1, a linker and a connecting domain fragment connected with the nanobody; the linking domain fragment is the HECT domain in HERC5 that delivers ISG15 function.

3. An expression vector comprising the E3 ligase chimera of claim 2.

4. A cell transfected with the expression vector of claim 3.