CN115948353B

CN115948353B - Application of E3 ubiquitin ligase TRIM21 in preparation of medicines for preventing or treating novel coronaviruses

Info

Publication number: CN115948353B
Application number: CN202211442146.2A
Authority: CN
Inventors: 汪德强; 毛胜蓝; 张绍城; 王雯; 蔡雪飞; 黄爱龙; 张津; 马园艳; 沈仕梅
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2022-11-17
Filing date: 2022-11-17
Publication date: 2024-05-31
Anticipated expiration: 2042-11-17
Also published as: CN115948353A

Abstract

The invention provides an application of E3 ubiquitin ligase TRIM21 in preparing a medicament for preventing or treating novel coronavirus (SARS-CoV-2). The research shows that the E3 ubiquitin ligase TRIM21 has degradation effect on SARS-CoV-2N protein in vitro and in vivo, and has potential to be developed into a medicine for preventing or treating novel coronavirus (SARS-CoV-2).

Description

Application of E3 ubiquitin ligase TRIM21 in preparation of medicines for preventing or treating novel coronaviruses

Technical Field

The invention belongs to the technical field of biological medicines, relates to prevention or treatment of novel coronavirus (SARS-CoV-2), and in particular relates to application of E3 ubiquitin ligase TRIM21 in preparation of a medicament for preventing or treating novel coronavirus (SARS-CoV-2).

Background

The innate immune system (innate immune system) is the first line of defense of hosts against pathogens including SARS-CoV-2. The innate immune response limits the entry, transcription, replication and assembly of viruses, helps identify and act on infected cells, coordinates and accelerates the development of adaptive immunity. Cell surface, endosome and cytoplasmic pattern recognition receptors (pattern recognition receptors, PRRs) respond to pathogen-associated molecular patterns (pathogen-associated molecular patterns, PAMPs) triggering inflammatory responses and programmed cell death, limiting viral infection and facilitating clearance. However, excessive immune activation can lead to systemic inflammation and severe disease. In response to innate immune-dependent viral clearance mechanisms, coronaviruses (CoVs) have evolved escape strategies to limit host control and enhance their own replication and transmission. It was found that the timely response of IFN-I inhibited viral replication, thereby enhancing its antiviral capacity, while SARS-CoV-2 can evade IFN-I response by a different mechanism. The innate immune response of viruses is related to the life cycle of viral replication and the like. The coronavirus nucleocapsid protein (N protein) plays a key role in the assembly and replication process, and the function of the coronavirus nucleocapsid protein (N protein) is to wrap the single-stranded RNA virus genome to form a ribonucleoprotein complex, thereby protecting the viral RNA from degradation by host factors, and N is also involved in regulating the synthesis of the viral RNA. Thus, if the translation or degradation of viral N protein can be regulated, it is possible to inhibit viral assembly and replication, thereby treating novel coronavirus (SARS-CoV-2) infection.

Disclosure of Invention

The invention aims to provide an application of E3 ubiquitin ligase TRIM21 in preparing a medicament for preventing or treating novel coronavirus (SARS-CoV-2).

Except for special descriptions, the parts are parts by weight, and the percentages are mass percentages.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

Application of E3 ubiquitin ligase TRIM21 in preparing medicaments for preventing or treating novel coronavirus (SARS-CoV-2); the amino acid sequence of the E3 ubiquitin ligase TRIM21 is as follows (SEQ ID NO. 1):

Advantageous effects

The invention provides an application of E3 ubiquitin ligase TRIM21 in a medicine for preventing or treating novel coronavirus (SARS-CoV-2). The research shows that the E3 ubiquitin ligase TRIM21 has degradation effect on SARS-CoV-2N protein in vitro and in vivo, and has potential of being developed into a novel coronavirus (SARS-CoV-2) prevention or treatment drug.

Drawings

FIG. 1 is a diagram showing successful enzyme digestion verification of adenovirus Ad-N recombinant plasmid construction;

FIG. 2 is a graph showing the results of WB validation of N protein expression in HEK293 cells;

FIG. 3 is a host factor graph of interactions with N protein in four cell lines;

FIG. 4 is a graph of the interaction results of N with TRIM 21;

FIG. 5 is a graph showing the negative regulatory effect of TRIM21 on N;

FIG. 6 is a graph showing the degradation results of TRIM21 on N protein in mice.

Detailed Description

The present invention is described in detail below by way of specific examples, which are given herein for the purpose of further illustration only and are not to be construed as limiting the scope of the present invention, as many insubstantial modifications and variations of the present invention will become apparent to those skilled in the art in light of the foregoing disclosure. The raw materials and the reagents used in the invention are all commercial products.

Example 1

Materials and methods:

cell culture: 50mL of fetal bovine serum (Clark) was added to 500mL of DMEM medium (Gibco) to a final concentration of about 10%, and 5mL of diabody (Biyun) was added for subsequent cultivation. Human embryonic kidney cells HEK293 (ATCC), human lung epithelial cells BEAS-2B (Qiao Xinzhou in) and human hepatic stellate cell line LX-2 (Procell) and human cardiac muscle cells AC16 (Qiao Xinzhou) were placed in the complete medium prepared as described above and incubated at 37℃under 5% CO ₂.

Cell transfection:

Appropriate amounts of cells were seeded in appropriate dishes and transfected at cell densities of around 70% -90% using Lipo8000 transfection reagent (bi yun day), as required for the experiment. After 8 hours of transfection, the cells were replaced with fresh complete medium and continued to be cultured for a period of time as required by the experiment.

Protein extraction:

1. removing the culture dish from the incubator, discarding the culture medium, and washing off the residual culture medium for 3 times by using sterile PBS;

2. Adding proper volume of protein lysate (RIPA and PMSF are mixed uniformly according to a ratio of 100:1), cracking for 15mins on ice, transferring completely cracked cells into a 1.5mL EP tube, and transferring 12000g of cells to 15mins at 4 ℃;

3. the supernatant was collected and transferred to a new 1.5mL EP tube;

4. measuring the concentration: 200. Mu.L BCA solution was added to 10. Mu.L of the sample to be measured, incubated at 37℃for 30 minutes, absorbance measured at 562nm and the sample concentration was calculated.

Immunoprecipitation (Immunoprecipitation):

1. antigen sample preparation: the medium was removed and the 10cm cell culture dish was washed 3 times with 1mL of 1 XPBS (pH 7.4); scraping cells with a cell scraper, collecting in a 1.5mL EP tube, adding 1mL cell lysis buffer and protease inhibitor into a10 cm cell culture dish, mixing, and treating on ice for 10

Mins, centrifuging (4 ℃,14000g,10 mins), collecting supernatant, and placing on ice for use (or placing at-20℃)

Long-term preservation);

2. Antigen-antibody reaction: 1mg of the protein sample prepared in the step 1 is added with 1 mu L of antibody, and the mixture is placed in a tumbling mixer for rotary incubation at 4 ℃ for overnight;

3. pretreatment of magnetic beads: the beads were suspended thoroughly, 30. Mu.L of beads were placed in a 1.5mL EP tube, 500 was added

Mu L of binding/washing buffer solution, fully suspending, placing the mixture in a magnetic rack, magnetically separating, discarding supernatant, and repeating the above washing steps for 3 times;

4. Binding of magnetic beads to antigen-antibody complexes: adding the antigen-antibody complex in the step 2, fully suspending, incubating in a tumbling mixer (4 ℃,4 hours), magnetically separating, and discarding the supernatant.

5. Washing: the beads were resuspended thoroughly using 500 μl binding/washing buffer, magnetically separated, the supernatant discarded, washed repeatedly 4 times, and finally transferred into a new 1.5mL EP tube;

6. Antigen elution: the beads were separated, the supernatant was discarded, 30. Mu.L of 1 XSDS-PAGE Loading Buffer was added to the beads and mixed well, and heated at 95℃for 5 minutes. The beads were separated and the supernatant was collected and subjected to SDS-PAGE.

Immunoblotting experiments (Western blotting):

First day

1. Preparing 12% protein electrophoresis gel, and loading 20 μg;

2. preparing 1x electrophoresis liquid, carrying out electrophoresis for 30mins at a constant voltage of 90V and a constant voltage of 150V for 1h;

3. Cutting PVDF film with proper size (30 s activated by methanol in advance), preparing 1x electric transfer liquid, and transferring film in water bath;

4. Constant current 300mA1h 30mins;

5. preparing 5% skimmed milk for sealing the PVDF membrane after the electric transfer, and incubating for 2 hours at room temperature;

6. Discarding the sealing liquid, and washing off residual skim milk with the prepared sterilized 1 XTBST;

7. primary antibody was incubated, dilution ratio 1: the mixture was shaken overnight at 1000,4 ℃.

The next day

8. Recovering the primary antibody, washing the residual primary antibody by 1 XTBE for 3 times each time for 10 mins;

9. secondary antibody was incubated, dilution ratio 1:4000, room temperature, shaking table 1h;

10. discarding the secondary antibody, washing off residual secondary antibody by 1 XTBE for 10mins each time, and washing for 3 times;

11. ECL solution was used for exposure on a gel imager.

Total RNA extraction:

Immediately after the 1.6-well petri dish was removed from the incubator, 1mL TRIzol reagent was added after 3 washes with sterile PBS;

2. cells were transferred to 1.5mL RNase-free EP tube. Placing on ice, and standing for 5 minutes;

3. 200 mu L of chloroform was added to each tube, and the mixture was left to stand on ice for 10 minutes after thorough mixing;

4. Centrifugation at 13000rpm at 4℃for 15 min; during this period, a new EP tube was taken, 500. Mu.L of isopropanol was added, and pre-chilled on ice;

5. After centrifugation, the upper aqueous phase (about 500 μl) was transferred to the new EP tube;

6. Standing on ice, and precipitating with ethanol for 10 min;

7. Centrifuging at 13000rpm for 10 minutes;

8. The supernatant was removed and the RNA pellet was washed once with 1mL of freshly prepared 75% ethanol;

centrifuging at 9.12000rpm for 5 min;

10. removing the supernatant, and air-drying the RNA precipitate for 5-10mins;

11. RNA was dissolved in 30. Mu.L DEPC water;

12. spectrophotometric analysis was performed to determine sample concentration and purity.

Specific operations and results:

① Construction of adenovirus over-expressing N protein

Based on the gene sequence of the new coronavirus, synthesizing new coronavirus N gene through codon optimization, constructing corresponding eukaryotic cell expression plasmid, and cloning N gene onto adenovirus system shuttle vector (pAdTrack). The amino acid sequence of the novel coronavirus N protein is as follows (SEQ ID NO. 2):

Linearizing the shuttle plasmid carrying the target gene fragment, transferring into BJ5183-AD-1 competence for homologous recombination, picking the recombinants for enzyme digestion identification, picking the recombinant plasmid with correct enzyme digestion identification, and carrying out the next packaging (figure 1). The recombinant plasmid obtained by screening is transfected into 293A cells by a liposome method, and the preparation of recombinant adenovirus for expressing N protein is completed by amplification and purification, and the expression of the N protein is verified in HEK293 cells by immunoblotting experiments (figure 2).

② Screening for N-interacting host proteins

The recombinant adenovirus is respectively infected with four cell lines of human embryo kidney cell HEK293, human bronchial epithelial-like cell BEAS-2B, human hepatic stellate cell LX-2 and human cardiac muscle cell AC-16, and the expression level of N protein is detected by utilizing N monoclonal antibody. After cell lysis, capturing by using N monoclonal antibody, enriching Protein-antibody complex by using Protein A/G magnetic beads, then performing SDS-PAGE electrophoresis, and screening host proteins interacted with virus N proteins by using mass spectrometry high-flux detection after gel cutting.

③ Data analysis

Analysis found that three genes (TRIM 21, LSG1 and VCP) were present simultaneously in the four sets of cell mass spectrometry identified interacting proteins (fig. 3), TRIM21 being an E3 ubiquitin ligase which was related to the ubiquitin protease system and therefore focused mainly on TRIM21 targeting novel coronavirus N protein related studies thereafter.

④ Interaction between N protein and TRIM21

The domains of the N protein and TRIM21, respectively, are shown briefly in fig. 4A. The interaction between the N protein and TRIM21 was first verified by immunoblotting experiments. The plasmid encoding the N-Myc protein was transfected into HEK293 cells and N-Myc was found to interact with endogenous TRIM21 (FIG. 4B). In order to find the interaction region of TRIM21 with N protein, TRIM21 plasmid with Flag tag, and its truncations were constructed in vitro: RING & B-box (amino acid residues 1-129), SMC (amino acid residues 122-270), and SPRY (amino acid residues 287-465). They were co-transfected into HEK293 cells with plasmids expressing the N protein. As a result, it was found that the N protein interacted with full-length TRIM21 (WT) and SPRY (FIG. 4C). The SPRY domain generally serves as a platform for protein interactions and exhibits significant protein binding specificity. Truncations NTD (amino acid residues 1-176), LKR (amino acid residues 173-256) and CTD (amino acid residues 253-419) were constructed on pEGFP-N1 vector and co-transfected with Flag-tagged TRIM21 plasmid in HEK293 cells, only CTD interaction with TRIM21 was observed (FIG. 4D). To further verify the respective interaction regions of TRIM21 and N proteins, the plasmid encoding Flag-tagged SPRY was co-transfected with the plasmid encoding GFP-tagged CTD in HEK293, observing that SPRY interacted with CTD (fig. 4E), which further underscores that TRIM21 interacted with CTD domain of N through SPRY domain. Taken together, these observations ultimately indicate that the N protein interacts intimately with TRIM21, their interacting domains being CTD and SPRY, respectively.

RING & B-box amino acid sequence (SEQ ID NO. 3):

SMC amino acid sequence (SEQ ID NO. 4):

SPRY amino acid sequence (SEQ ID No. 5):

NTD amino acid sequence (SEQ ID No. 6):

LKR amino acid sequence (SEQ ID No. 7):

CTD amino acid sequence (SEQ ID No. 8):

⑤ Negative regulation of N by TRIM21

The inventors performed the following experiments in HEK293, BEAS-2B, AC-16 and LX-2 cell lines: the same amount of plasmid expressing N protein and an incremental amount of plasmid expressing Flag-TRIM21 were co-transfected in these four cells. The inventors found that TRIM21 overexpression reduced N protein levels in a dose dependent manner (fig. 5A-5D). To further verify the inhibition of the N protein by TRIM21, the inventors first tried to down-regulate the expression of endogenous TRIM21 in HEK293 cells by 4 specific small interfering RNAs (siRNAs) directed against TRIM 21. Of these 4 specific siRNAs, siTRIM, ① and siTRIM, 21 ② showed significant inhibition of TRIM21 gene expression, with inhibition rates of over 50% (fig. 5E). Consistent with the results of western blot experiments, the inventors found that both sirnas (selected for use in subsequent experiments) were able to effectively inhibit the level of TRIM21 mRNA in HEK293 cells by qRT-PCR (fig. 5F). Inhibition of TRIM21 significantly enhanced N protein expression when HEK293 cells were co-transfected with siTRIM a ① and siTRIM a ② (fig. 5G). Rescue by overexpression of TRIM21, the inventors found that expression of N protein in HEK293 cells was again reduced (fig. 5H).

Knocking down siRNA sequence of TRIM21 and primer sequence for evaluating expression level of TRIM21 mRNA

⑥ TRIM21 also has degradation effect on N protein in mice

Genes expressing TRIM21 and N proteins were successfully integrated into AAV virus targeting lung, AAVs were injected into BALB/c mice at a certain titer by tail vein, and lung tissues of the mice were taken 2 weeks after injection for immunoblotting experiments. The results showed that TRIM21 also had an effect of degrading N protein in mice (FIG. 6). These observations indicate that TRIM21 has a degrading effect on SARS-CoV-2N protein in vitro and in vivo.

Claims

Application of E3 ubiquitin ligase TRIM21 in preparing medicaments for treating novel coronaviruses (SARS-CoV-2); the amino acid sequence of the E3 ubiquitin ligase TRIM21 is shown as SEQ ID NO. 1.