Interferon gene stimulator mutant and its application

Abstract

The present invention relates to an interferon gene stimulating factor mutant and its application, belonging to the field of biomedicine technology. The interferon gene stimulating factor mutant of the present invention is obtained by mutating the cysteine ​​at position 292 of the interferon gene stimulating factor to O-(3-bromopropyl)-L-tyrosine. The interferon gene stimulating factor mutant of the present invention can be cross-linked with the interferon gene stimulating factor to form a spheroid and enhance the downstream TBK1-IRF3 signaling pathway, thereby promoting the large-scale expression and secretion of type I interferon and other immunomodulatory factors, and enhancing the intensity and persistence of the immune response. At the same time, the interferon gene stimulating factor mutant of the present invention can greatly improve the recognition ability and affinity of the interferon gene stimulating factor for pathogens after combining with the interferon gene stimulating factor, effectively solving the problem of insufficient sensitivity of the natural interferon gene stimulating factor to low-dose pathogen recognition.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an interferon gene stimulator mutant and application thereof. Background Art

[0002] The stimulator of interferon genes (STING protein), a core component of the body's immune response, plays a key role in recognizing pathogen-associated molecular patterns (PAMPs), such as bacterial cyclic dinucleotides (CDNs) and viral double-stranded DNA. Upon recognition, STING rapidly activates the downstream serine-threonine kinase 1-interferon regulatory factor 3 (TBK1-IRF3) signaling pathway, promoting the expression and secretion of interferons (IFNs) and various other immune regulatory factors, thereby initiating the body's immune defense mechanisms. STING plays an indispensable role in antiviral, antibacterial, and anti-tumor immunity.

[0003] Despite its important functions, the STING protein has numerous shortcomings in its natural state. For example, its signal activation efficiency is low when exposed to low doses or mutated pathogens, resulting in a slow and insufficient immune response, making it difficult to effectively defend against pathogen invasion. Furthermore, the STING protein itself has limited stability and is susceptible to degradation pathways such as the proteasome within the cell, limiting its ability to function sustainably.

[0004] To improve the performance of the STING protein, researchers have conducted extensive research. Early attempts to activate the STING protein involved small molecule chemical agonists. However, these agonists often suffer from poor specificity and can easily trigger nonspecific immune activation, leading to serious side effects such as systemic inflammatory response syndrome. In genetic engineering, attempts have been made to enhance the activity of the STING protein by performing point mutations, but some mutants have failed to significantly improve signal transduction efficiency, and some have been unable to fold normally within the cell, resulting in loss of protein function. Other attempts have involved fusion tags, hoping to alter the localization or stability of the STING protein. However, the introduction of tags often interferes with the normal structure and function of the protein itself, and the fusion process is complex and costly, making large-scale application difficult.

[0005] In summary, the current modification methods for STING protein have shortcomings in terms of effectiveness, safety, and ease of operation. An innovative and effective strategy is urgently needed to break through these bottlenecks. Summary of the Invention

[0006] To this end, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a STING protein activator with good stability and high activation efficiency.

[0007] To address the above-mentioned technical problems, the present invention provides a mutant stimulator of interferon genes and its use. The present invention utilizes the unnatural amino acid O-(3-bromopropyl)-L-tyrosine (BprY), which has suitable chemical activity and structural characteristics. This unnatural amino acid specifically reacts with the cysteine ​​residue at position 292 of the STING protein to form a stable disulfide bond. Using site-directed mutagenesis, the cysteine ​​residue at position 292 of the STING protein was mutated to BprY to generate the STING mutant. When the STING mutant was mixed and incubated with STING protein, it was found that the STING mutant could cross-link with STING to form spheroids and enhance the downstream TBK1-IRF3 signaling pathway. Furthermore, when the STING mutant was injected into virus-infected mice, it was found that the STING mutant enhanced the function of STING by binding to the STING protein, thereby enhancing the mice's immune response to viral infection and inhibiting viral replication.

[0008] The first object of the present invention is to provide a STING protein mutant, wherein the cysteine ​​at position 292 of the STING protein is mutated to O-(3-bromopropyl)-L-tyrosine, wherein the amino acid sequence of the STING protein is shown in SEQ ID NO.1.

[0009] Further, SEQ ID NO.1:

[0010] MPHSSLHPSIPCPRGHGAQKAALVLLSAACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLL RGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS.

[0011] Furthermore, the present invention utilizes an orthogonal translation system to mutate the cysteine ​​at position 292 of the STING protein to O-(3-bromopropyl)-L-tyrosine. Specifically, the codon corresponding to position C292 in the gene encoding the STING protein is replaced with a codon encoding a selected unnatural amino acid. By introducing an orthogonal translation system, including an orthogonal aminoacyl-tRNA synthetase (aaRS) and an orthogonal tRNA pair, the TGC codon for cysteine ​​at position 292 of the STING protein is replaced with TAG. Under the action of the orthogonal aminoacyl-tRNA synthetase, the tRNA recognizes TAG, allowing the unnatural amino acid carried on the tRNA to attach to the amino acid at position 291. Consequently, during protein translation, the selected unnatural amino acid is accurately incorporated into the C292 position of the STING protein. During protein folding, the newly incorporated unnatural amino acid spontaneously forms a stable disulfide bond with the cysteine ​​residue at position 292, thereby achieving cross-linking modification of the STING protein.

[0012] The second object of the present invention is to provide a use of the above-mentioned STING protein mutant in the preparation of a STING protein activator.

[0013] Furthermore, the STING protein activator also includes 2'3'-cyclic GMP. 2'3'-cGAMP is a direct ligand of the STING protein. Upon binding to the CTD region of the STING protein, it induces conformational changes in the STING protein, thereby activating the downstream TBK1-IRF3 signaling pathway and ultimately promoting the production of type I interferon. The present invention discovered that when a STING protein mutant is cross-linked with the STING protein, it can enhance activation of the TBK1-IRF3 signaling pathway, and the addition of 2'3'-cGAMP can strengthen the cross-linking between the STING protein mutant and the STING protein.

[0014] Furthermore, the STING protein activator promotes the oligomerization of the STING protein.

[0015] The third object of the present invention is to provide a drug for promoting the polymerization of stimulator of interferon genes, wherein the drug comprises the above-mentioned stimulator of interferon genes mutant.

[0016] The fourth object of the present invention is to provide a use of the above-mentioned STING protein mutant in the preparation of a herpes simplex virus type 1 prevention or treatment product, wherein the herpes simplex virus type 1 prevention or treatment product includes a fusion protein, which contains the above-mentioned interferon gene stimulator mutant and a membrane-penetrating polypeptide connected and arranged.

[0017] Furthermore, the STING protein comprises a transmembrane domain (TMD) consisting of amino acids 1-153 and a soluble intracellular domain (CTD) consisting of amino acids 154-379. Preferably, to enable prokaryotic synthesis of the STING protein, the TMD of the STING protein can be truncated, leaving only the CTD.

[0018] Furthermore, the amino acid sequence of the membrane-penetrating polypeptide is shown in SEQ ID NO.2.

[0019] Further, SEQ ID NO. 2: YGRKKRRQRRR.

[0020] Furthermore, the interferon gene stimulator mutant and the membrane-penetrating polypeptide are connected via a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.3.

[0021] Further, SEQ ID NO. 3: GGGGS.

[0022] Furthermore, the amino acid sequence of the fusion protein is: NVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLF(BprY)RTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSGGGGSYGRKKRRQRRR, wherein (BprY) represents O-(3-bromopropyl)-L-tyrosine.

[0023] Furthermore, the fusion protein includes the CTD region of the STING protein mutant, a connecting peptide, and a transmembrane polypeptide.

[0024] Furthermore, the herpes simplex virus type 1 prevention or treatment product promotes the expression of interferon β1 gene.

[0025] Furthermore, the herpes simplex virus type 1 prevention or treatment product inhibits the replication of herpes simplex virus type 1.

[0026] Furthermore, the herpes simplex virus type 1 prevention or treatment product inhibits the expression of the herpes simplex virus type 1 tegument protein UL46. The tegument protein UL46 of herpes simplex virus type 1 (HSV-1) is an important viral protein that HSV-1 uses to antagonize the host's antiviral immune response. The present invention discovered that cross-linking a STING protein mutant with the STING protein can significantly inhibit the expression of the herpes simplex virus type 1 tegument protein UL46, thereby preventing the virus from escaping the host's immune response.

[0027] A fifth object of the present invention is to provide a drug for preventing or treating viral infection, which comprises the above-mentioned mutant of the stimulator of interferon genes.

[0028] Furthermore, the virus comprises herpes simplex virus type 1.

[0029] Beneficial effects of the present invention:

[0030] The STING protein mutants of the present invention can cross-link with the STING protein to form spheroids and enhance the downstream TBK1-IRF3 signaling pathway, thereby promoting the expression and secretion of type I interferon and other immune regulatory factors, thereby enhancing the intensity and persistence of the immune response. At the same time, the STING protein mutants of the present invention, after binding to the STING protein, can greatly improve the STING protein's recognition ability and affinity for pathogen-associated molecular patterns. Compared with the native STING protein, the STING protein mutants can bind to the STING protein more quickly and sensitively and initiate an immune response under low-dose pathogen stimulation, effectively solving the problem of the native STING protein's insufficient sensitivity to low-dose pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram showing the validation results of STING protein mutants;

[0032] Figure 2 This is a graph showing the cross-linking detection results between STING protein mutants and STING protein;

[0033] Figure 3 This is a graph showing the results of the in vitro spheroidization ability test of STING protein mutants and STING protein;

[0034] Figure 4 This is a schematic diagram showing the effects of STING protein mutants and cross-linking with STING protein on STING protein function and downstream signal transduction capabilities;

[0035] Figure 5 This is a schematic diagram of the test results of STING protein mutants on STING protein liquid-liquid phase separation (LLPS) and downstream signal transduction capabilities;

[0036] Figure 6 This is a schematic diagram of the detection results of IFN-β1 and HSV-gRNA mRNA transcription in mouse spleen, liver, and brain tissues;

[0037] Figure 7 This is a schematic diagram of the results of detecting the IFN-β1 content in mouse plasma through ELISA experiment;

[0038] Figure 8 This is a schematic diagram of the immunohistochemistry (IHC) experimental results of HSV-UL46 protein expression in mouse brain tissue;

[0039] Figure 9 This is a schematic diagram of the detection results of the distribution and metabolism of SCT protein and SCBT protein in mice. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0041] Example 1

[0042] 1. Experimental Materials

[0043] STING expression plasmid, STING protein mutation primers (STING-C292-(TAG)-F / R, primer sequences are shown in Table 1), high-fidelity polymerase (TAKARA), plasmid extraction kit (from Quanshi Gold), unnatural amino acid O-(3-bromopropyl)-L-tyrosine (BprY), unnatural amino acid synthase orthogonal system (tRNA PylCUA / MmXYRS, hereinafter abbreviated as "MmXYRS"), fetal bovine serum (from Gibco), modified ketone essential medium (DMEM), penicillin / streptomycin solution (from Gibco), 293T cell line (from ATCC), transfection reagent polyethyleneimine (PEI), Flag tag antibody, glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH) and related secondary antibodies (from CST).

[0044] Table 1 Sequences of SEQ ID NO. 1-3 and STING protein mutation primers and their sequences

[0045]

[0046] 2. Experimental Methods

[0047] This example uses site-directed mutagenesis to replace the codon corresponding to position C292 in the gene encoding the STING protein with a codon encoding the selected unnatural amino acid, BprY. By introducing an orthogonal translation system, including an orthogonal aminoacyl-tRNA synthetase (aaRS) and an orthogonal tRNA pair, into the cell expression system, the selected unnatural amino acid, BprY, is accurately incorporated into the C292 position of the STING protein during protein translation, thereby mutating the cysteine ​​residue at position 292 to BprY and generating a STING protein mutant. During protein folding, the newly incorporated unnatural amino acid spontaneously forms stable disulfide bonds with the cysteine ​​residue near position 292, thereby cross-linking the STING protein. Co-expressing the gene encoding STING-C292-TAG with the orthogonal unnatural amino acid synthetase system in the presence of the unnatural amino acid BprY allows for site-specific production of STING-C292-BprY in eukaryotic or prokaryotic expression systems.

[0048] The specific method involved extracting a plasmid containing the STING protein-encoding gene. Using a site-directed mutagenesis kit according to the kit's instructions, the codon encoding the C292 position was mutated to TAG, encoding the selected unnatural amino acid BprY. A Flag tag was then introduced to construct the STING-C292(TAG)-Flag plasmid. STING-C292(TAG)-Flag and MmXYRS were co-transfected into 293T cells. After 24 hours, 10 μM BprY was added, and the cells were harvested 12 hours later. Synthesis of the STING mutant protein was assessed by western blotting.

[0049] 3. Experimental Results

[0050] like Figure 1 The model diagram depicts the working principle of this unnatural amino acid: the unnatural amino acid BprY, under the action of unnatural amino acid synthase, specifically mutates the cysteine ​​at position 292 of the STING protein expressed from the STING-C292(TAG)-Flag plasmid to BprY, generating a STING protein mutant. When either the unnatural amino acid BprY or MmXYRS was added alone, the expected STING protein mutant band was not observed in the western blot analysis, indicating that the system was not functioning properly. Only when BprY and MmXYRS were added simultaneously did a clear STING protein mutant band be detected, confirming that the system was functioning properly and that the unnatural amino acid BprY had successfully mutated the STING protein C292 position.

[0051] Example 2

[0052] 1. Experimental Materials

[0053] Empty plasmid, STING-Myc-WT plasmid (a plasmid containing a Myc tag and the wild-type STING protein gene), STING-C292A-Myc plasmid (a plasmid containing a Myc tag and the STING protein gene with cysteine ​​at position 292 mutated to alanine), STING-C292 (TAG)-Flag plasmid constructed in Example 1, BprY, MmXYRS, and cell culture-related products, please refer to Example 1.

[0054] 2. Experimental Methods

[0055] 293T cells were divided into six groups. The first and second groups were transfected with empty plasmid + STING-C292 (TAG)-Flag plasmid and MmXYRS. The third and fourth groups were transfected with STING-C292 (TAG)-Flag plasmid, STING-Myc-WT plasmid and MmXYRS. The fifth and sixth groups were transfected with STING-C292A-Myc plasmid, STING-C292 (TAG)-Flag plasmid and MmXYRS. After 24 hours, 10 μM BprY was added to the second, fourth and sixth groups, respectively, and the cells were cultured continuously. After 12 hours of culture, the cells were harvested and the cross-linking was detected by western blotting.

[0056] 3. Experimental Results

[0057] like Figure 2 As shown in the Western blotting results, no clear crosslinking band was detected in the sixth sample at the expected molecular weight of the crosslinked protein, indicating that the STING protein with the cysteine ​​292 mutated to alanine (STING-C292A protein) cannot form crosslinks with the STING mutant. However, the fourth sample showed a clear band at the molecular weight corresponding to the crosslinked protein (around 75 kDa), indicating that the STING mutant can form stable crosslinks with the STING protein. Taken together, these results indicate that the STING mutant can form stable covalent crosslinks with the wild-type STING protein.

[0058] Example 3

[0059] 1. Experimental Materials

[0060] Highly purified STING protein was produced via a eukaryotic expression system and purified using an AKTA molecular sieve system, ensuring a purity exceeding 95% to eliminate impurities that could interfere with experimental results. The STING mutant and STING-C292A protein, in which cysteine ​​292 is mutated to alanine, also underwent rigorous purification procedures to ensure their quality and activity. AF488 fluorescent dye, a laser confocal microscope (Zeiss), and slides and coverslips (Biosharp) were used.

[0061] 2. Experimental Methods

[0062] To each 100 μL solution of 1 mg / mL STING protein, STING mutant, and STING-C292A protein, 10 μL of a 10 mM AF488 fluorescent dye solution was added at a molar ratio of 10:1. The mixture was incubated for 2 hours at room temperature in the dark with gentle shaking. Unbound fluorescent dye was removed by dialysis to obtain pure AF488-labeled STING protein, STING mutant, and STING-C292A protein solutions. Fluorescently labeled STING protein and STING mutant were dripped onto a glass slide at varying concentrations, covered with a coverslip, and observed under a microscope for protein spheroidization. STING protein and STING mutant were then mixed in a 1:1 ratio and their spheroidization was again observed. Finally, STING mutant and STING-C292A were mixed in a 1:1 ratio and their spheroidization was again observed.

[0063] 3. Experimental Results

[0064] like Figure 3 As shown, in the single-protein spheroidization assay, the number of droplets formed by STING protein alone and the STING mutant alone was relatively small. At a concentration of 1.5 μM, the average number of droplets formed by STING protein in 10 fields of view was 60 ± 10, while the average number of droplets formed by the STING mutant was only 12 ± 5, indicating that the STING mutant alone has a lower in vitro spheroidization ability than STING protein. The in vitro spheroidization ability of the STING mutant and STING-C292A protein was also not significant. However, when STING protein and the STING mutant were mixed, the STING protein's spheroidization ability was significantly enhanced. This enhanced spheroidization ability is closely related to the function of the STING mutant, indicating that the STING mutant further promotes the function of STING protein by interacting with wild-type STING protein during liquid-liquid phase separation.

[0065] Example 4

[0066] 1. Experimental Materials

[0067] Empty plasmid, STING-WT-Flag plasmid, STING-C292 (TAG)-Flag plasmid, 2'3'-cGAMP, Flag tag-specific antibody (Flag antibody), serine-threonine protein kinase 1 antibody (TBK1 antibody), phosphorylated serine-threonine protein kinase 1 antibody (p-TBK1 antibody), phosphorylated interferon regulatory factor 3 antibody (p-IRF3 antibody), interferon regulatory factor 3 antibody (IRF3 antibody), actin antibody (Actin antibody) and related secondary antibodies (from CST).

[0068] 2. Experimental method.

[0069] 293T cells were plated in 12-well plates and divided into six groups when the cell density reached 80%. Groups 1 and 2 were transfected with an empty vector and MmXYRS; Groups 3 and 4 were transfected with the STING-WT-Flag plasmid and MmXYRS; and Groups 5 and 6 were transfected with the STING-WT-Flag plasmid, the STING-C292(TAG)-Flag plasmid, and MmXYRS. After 24 hours, all six groups of cells were treated with 10 μM BprY. Twelve hours later, groups 2, 4, and 6 were stimulated with 1 μg / mL 2'3'-cGAMP. Cells were harvested 1 hour later, and cross-linking of STING protein and phosphorylation of the downstream key kinases TBK1 and IRF3 were analyzed by western blotting.

[0070] 3. Experimental Results

[0071] like Figure 4 As shown, the fourth group of cells was able to induce phosphorylation of the key downstream kinase TBK1 and transcription factor IRF3. In the fifth and sixth groups of cells, not only were crosslinked bands observed, but the phosphorylation levels of the downstream kinase TBK1 and transcription factor IRF3 were significantly enhanced. Moreover, after stimulation with 2'3'-cGAMP, the phosphorylation levels of TBK1 and IRF3 in the sixth group of cells were further deepened. This fully demonstrates that after crosslinking the STING protein mutant with the wild-type STING protein, not only did it successfully promote the crosslinking activation of the STING protein, but it also significantly enhanced the phosphorylation levels of the downstream kinase TBK1 and transcription factor IRF3, strongly proving that crosslinking the STING protein mutant with the STING protein can effectively promote the activation of its own function and its ability to transduce downstream signals.

[0072] Example 5

[0073] 1. Experimental Materials

[0074] STING-WT-Flag plasmid, STING-C292 (TAG)-Flag plasmid, 2'3'-cGAMP, 4% paraformaldehyde / PBS, 0.1% triton X-100, fluorescein isothiocyanate-labeled rabbit anti-sheep immunoglobulin G, rhodamine fluorescein-labeled goat anti-mouse immunoglobulin G, STING protein antibody (from CST), TBK1 antibody (from Santa Cruz), 4',6-diamidino-2-phenylindole (DAPI).

[0075] 2. Experimental Methods

[0076] 293T cells were cultured and divided into four groups. Groups 1 and 2 were transfected with the STING-WT-Flag plasmid and MmXYRS, while groups 3 and 4 were transfected with the STING-WT-Flag plasmid and the STING-C292(TAG)-Flag plasmid and MmXYRS. After 24 hours of culture, 10 μM BprY was added to each group. 12 hours later, cells in groups 2 and 4 were stimulated with 1 μg / mL 2'3'-cGAMP, while cells in groups 1 and 3 were stimulated with phosphate-buffered saline (PBS). One hour later, cells were harvested, fixed with 4% paraformaldehyde / PBS, and perforated. Cells were then incubated with antibodies against STING and TBK1 overnight, followed by incubation with fluorescent secondary antibodies for 1 hour at room temperature. The slides were mounted and observed under a confocal microscope.

[0077] 3. Experimental Results

[0078] like Figure 5 As shown, STING protein hardly undergoes LLPS. After stimulation with 2'3'-cGAMP, STING protein undergoes LLPS in cells, forming spheroids and colocalizing with TBK1. However, STING protein mutants can undergo LLPS in cells without 2'3'-cGAMP stimulation. 2'3'-cGAMP stimulates STING protein to undergo strong LLPS, and the interaction with TBK1 is higher than that of STING protein. In summary, cross-linking of STING protein mutants with STING protein can promote the occurrence of LLPS in cells, and this promoting effect is more significant under 2'3'-cGAMP stimulation. At the same time, cross-linking of STING protein mutants with STING protein significantly enhances the interaction between STING protein and TBK1, providing strong support for the efficient transduction of downstream signals.

[0079] Example 6

[0080] 1. Experimental Materials

[0081] To verify the intracellular function of the STING protein mutant, this example used protein transduction technology to fuse the HIV-TAT transmembrane peptide (amino acid sequence shown in SEQ ID NO. 2) to the C-terminus of the STING protein mutant to facilitate its transmembrane delivery. Specifically, this example used genetic engineering methods to insert the coding sequence of the HIV-TAT transmembrane peptide before the STING protein stop codon, and a flexible linker peptide (shown in SEQ ID NO. 3) was added between the two to maintain the native conformation and function of the STING protein mutant. The STING protein consists of a transmembrane domain (TMD) consisting of amino acids 1-153 and a soluble intracellular domain (CTD) consisting of amino acids 154-379. Because the full-length protein cannot be purified in prokaryotes, the TMD region was truncated in this example, retaining only the CTD. Subsequently, proteins containing the CTD region of the STING protein mutant linked to HIV-TAT (SCBT protein) and SCT protein containing the CTD region of the STING protein linked to HIV-TAT (SCT protein) were purified through a prokaryotic expression system, and their transmembrane efficiency was verified through in vitro cellular uptake experiments. This strategy ensures that the STING protein mutant can efficiently enter cells and function intracellularly, providing a reliable tool for studying its biological effects.

[0082] 6-8 week-old specific pathogen-free (SPF) C57BL / 6 mice, purified SCBT and SCT proteins, herpes simplex virus type 1 (HSV-1) strain, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interferon β1 (IFN-β1), and HSV-1-gRNA gene primers (see Table 2, synthesized by Youkang Bio), guanidine isothiocyanate-phenol (Trizol, from TAKARA), reverse transcription kit and fluorescent DNA binding dye premix (SYBR Green MasterMix, from Novizan), fluorescent quantitative PCR instrument (from Bio-Rad), tissue grinder, electronic balance, and pipette.

[0083] Table 2 Primers and their sequences involved in Example 6

[0084]

[0085] 2. Experimental Methods

[0086] Thirty mice were randomly divided into six groups, with five mice in each group. The control groups included PBS, SCT, and SCBT, while the experimental groups included PBS+HSV-1, SCT+HSV-1, and SCBT+HSV-1. Mice in the PBS and PBS+HSV-1 groups were intraperitoneally injected with 100 μL of PBS; mice in the SCT and SCT+HSV-1 groups were intraperitoneally injected with 100 μL of a 1 mg / mL SCT protein solution; and mice in the SCBT and SCBT+HSV-1 groups were intraperitoneally injected with 100 μL of a 1 mg / mL SCBT protein solution. Two hours after injection, mice in the PBS+HSV-1, SCT+HSV-1, and SCBT+HSV-1 groups were injected through the tail vein with 100 μL of a 1×10 6 One group received 1000 PFU of HSV-1 viral suspension, while the remaining three groups received an equal volume of PBS. Twelve hours after viral infection, spleen, liver, and brain tissues were collected from the mice. Approximately 50 mg of tissue was placed in an RNase-free centrifuge tube and total RNA was extracted using Trizol reagent. Reverse transcription was then performed according to the reverse transcription kit's instructions, followed by qPCR.

[0087] 3. Experimental Results

[0088] like Figure 6 As shown, Coomassie Brilliant Blue staining clearly demonstrates the results of purified SCT and SCBT proteins. In the PBS group, IFN-β1 mRNA expression in the spleen, liver, and brain tissues of mice remained at basal levels. IFN-β1 mRNA expression in the SCT group was slightly elevated, but not significantly different from the PBS group. IFN-β1 mRNA expression in the SCBT group was significantly higher than in the PBS and SCT groups, indicating that SCBT protein can promote the expression of the mouse's own IFN-β1 gene. In the PBS + HSV-1 group, IFN-β1 mRNA expression was significantly elevated. IFN-β1 mRNA expression in the SCT + HSV-1 group was slightly higher than in the PBS + HSV-1 group. IFN-β1 mRNA expression in the SCBT + HSV-1 group was significantly higher than in the PBS + HSV-1 and SCT + HSV-1 groups. This indicates that the STING protein mutant can significantly enhance the immune response of mice infected with HSV-1 and inhibit viral replication, further demonstrating that the STING protein mutant can significantly enhance STING protein function and promote antiviral capacity in mice.

[0089] Example 7

[0090] 1. Experimental Materials

[0091] 6-8 week old SPF C57BL / 6 mice, purified SCBT and SCT proteins, herpes simplex virus type 1 (HSV-1) strain, sterile ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, interferon-β1 enzyme-linked immunosorbent assay kit (IFN-β1 ELISA kit), and a microplate reader (from Bio-Tek).

[0092] 2. Experimental Methods

[0093] Thirty mice were randomly divided into six groups, with five mice in each group. The control groups included PBS, SCT, and SCBT, while the experimental groups included PBS+HSV-1, SCT+HSV-1, and SCBT+HSV-1. Mice in the PBS and PBS+HSV-1 groups were intraperitoneally injected with 100 μL of PBS; mice in the SCT and SCT+HSV-1 groups were intraperitoneally injected with 100 μL of a 1 mg / mL SCT protein solution; and mice in the SCBT and SCBT+HSV-1 groups were intraperitoneally injected with 100 μL of a 1 mg / mL SCBT protein solution. Two hours after injection, mice in the PBS+HSV-1, SCT+HSV-1, and SCBT+HSV-1 groups were injected through the tail vein with 100 μL of a 1×10 6 The 1000 cells were injected with a 1000 mmol HSV-1 viral suspension, while the other three groups were injected with an equal volume of PBS. Twelve hours after viral infection, 1-2 mL of blood was collected from the orbital venous plexus using a capillary tube and injected into a sterile centrifuge tube containing EDTA anticoagulant. The supernatant was then centrifuged to obtain the supernatant. IFN-β1 ELISA kit was used for detection, and the absorbance of each well was measured at 450 nm on a microplate reader.

[0094] 3. Experimental Results

[0095] like Figure 7 As shown, the plasma IFN-β1 levels of mice in the PBS group were low and maintained at a basal level. There was no significant difference in the plasma IFN-β1 levels of mice in the SCT group compared with the PBS group. The plasma IFN-β1 levels of mice in the SCBT group were significantly higher than those in the PBS and SCT groups, indicating that the STING protein mutant can promote IFN-β1 secretion in mice. The plasma IFN-β1 levels of mice in the PBS + HSV-1 group were significantly higher than those in the PBS group, and the plasma IFN-β1 levels of mice in the SCT + HSV-1 group were slightly higher than those in the PBS + HSV-1 group. The plasma IFN-β1 levels of mice in the SCBT + HSV-1 group were significantly higher than those in the PBS + HSV-1 group and the SCT + HSV-1 group. This further confirms that the STING protein mutant can significantly enhance the immune response, promote the secretion of large amounts of IFN-β1, and enhance the antiviral ability of mice after HSV-1 infection, demonstrating that the STING protein mutant significantly enhances the function of the STING protein.

[0096] Example 8

[0097] 1. Experimental Materials

[0098] 6-8 week-old SPF-grade C57BL / 6 mice, purified SCBT and SCT proteins, herpes simplex virus type 1 (HSV-1) strain, rabbit anti-HSV-1 UL46 polyclonal antibody (rabbit anti-HSV-1-UL46 polyclonal antibody, from Abcam), horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (goat anti-rabbit IgG-HRP secondary antibody, from Jackson Immuno Research), diaminobenzidine colorimetric kit (DAB colorimetric kit, from Boster), PBS buffer, antigen retrieval solution, 4% paraformaldehyde / PBS, and embedding reagent (from Shanghai Bioengineering Co., Ltd.).

[0099] 2. Experimental Methods

[0100] Thirty mice were randomly divided into six groups, with five mice in each group. The control groups included PBS, SCT, and SCBT, while the experimental groups included PBS+HSV-1, SCT+HSV-1, and SCBT+HSV-1. Mice in the PBS and PBS+HSV-1 groups were intraperitoneally injected with 100 μL of PBS; mice in the SCT and SCT+HSV-1 groups were intraperitoneally injected with 100 μL of a 1 mg / mL SCT protein solution; and mice in the SCBT and SCBT+HSV-1 groups were intraperitoneally injected with 100 μL of a 1 mg / mL SCBT protein solution. Two hours after injection, mice in the PBS+HSV-1, SCT+HSV-1, and SCBT+HSV-1 groups were injected through the tail vein with 100 μL of a 1×10 6 The mice were injected with a 1000 PFU HSV-1 viral suspension, while the remaining three groups were injected with an equal volume of PBS. Twelve hours after viral infection, mice were euthanized with carbon dioxide, and brain tissue was quickly removed and fixed in 4% paraformaldehyde for 24 hours. Subsequently, the brains were fixed, sectioned, and subjected to immunohistochemistry (IHC) staining.

[0101] 3. Experimental Results

[0102] like Figure 8As shown, numerous areas of positive staining for HSV-1 UL46 (HSV-1 UL46, a key viral protein that HSV-1 uses to antagonize host antiviral immune responses, hereinafter referred to as "UL46 protein") were observed in the brain tissues of mice in the PBS+HSV-1 group, with higher average optical density values, indicating that HSV-1 replicates extensively in the mouse brains and that UL46 protein is expressed. The areas of UL46 protein-positive staining in the brain tissues of mice in the SCT+HSV-1 group were significantly less than those in the PBS+HSV-1 group, and the areas of UL46 protein-positive staining in the brain tissues of mice in the SCBT+HSV-1 group were significantly less than those in the PBS+HSV-1 and SCT+HSV-1 groups. This indicates that the STING protein mutant can significantly inhibit HSV-1 replication and UL46 protein expression in the mouse brains, further demonstrating that the STING protein mutant can significantly enhance the antiviral capacity of mice, improve the function of the STING protein, and effectively reduce viral proliferation and spread in the body.

[0103] Example 9

[0104] 1. Experimental Materials

[0105] 6-8 week old SPF-grade C57BL / 6 mice were used. Purified SCBT and SCT proteins were labeled using the Cyanidin 5.5 fluorescent dye (Cy5.5) labeling kit (MCE). Unbound Cy5.5 dye was removed by desalting columns. The concentration and labeling efficiency of the labeled proteins were measured using a UV spectrophotometer to ensure a labeling efficiency of at least 80%. The labeled proteins were adjusted to a concentration of 1 mg / mL and stored at 4°C until use. Small Animal In vivo Imaging System (IVIS Spectrum) was used.

[0106] 2. Experimental Methods

[0107] Animal Grouping and Injection: Twenty-four mice were randomly divided into three groups, each consisting of eight mice: PBS, SCT+Cy5.5, and SCBT+Cy5.5. Each mouse was accurately weighed using an electronic balance, and the injection dose was calculated based on body weight. The PBS group received PBS, the SCT+Cy5.5 group received Cy5.5-labeled SCT protein, and the SCBT+Cy5.5 group received Cy5.5-labeled SCBT protein. Images were captured using a small animal in vivo imaging system 2 and 6 hours after injection.

[0108] 3. Experimental Results

[0109] like Figure 9As shown in the in vivo imaging images 2 hours after injection, distinct fluorescence signals were observed in both the SCT+Cy5.5 and SCBT+Cy5.5 groups. The fluorescence signals were primarily distributed in organs such as the liver, spleen, and lungs, indicating that SCT and SCBT proteins rapidly enter the blood circulation and accumulate in these key immune and metabolic organs within a short period of time after injection. In the in vivo imaging images 6 hours after injection, strong fluorescence signals were still detected in both the SCT+Cy5.5 and SCBT+Cy5.5 groups, with a distribution range similar to that observed 2 hours after injection, although the overall fluorescence intensity decreased slightly. These results demonstrate that SCT and SCBT proteins are well distributed in mice and can still be detected in major organs 6 hours after injection. This provides important foundational data for further investigation of their functions and mechanisms of action in vivo and strongly supports their potential clinical applications from the perspectives of distribution and metabolism.

[0110] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A mutant of interferon gene stimulator, characterized in that The interferon gene stimulator mutant is obtained by mutating the cysteine ​​at position 292 of the interferon gene stimulator to O-(3-bromopropyl)-L-tyrosine, wherein the amino acid sequence of the interferon gene stimulator is shown in SEQ ID NO.

1.

2. Use of the interferon gene stimulator mutant according to claim 1 in the preparation of an interferon gene stimulator activator.

3. The use according to claim 2, characterized in that The interferon gene stimulator activator also includes 2'3'-cyclic guanosine monophosphate.

4. A drug for promoting the polymerization of interferon gene stimulator, characterized in that The drug comprises the interferon gene stimulator mutant according to claim 1.

5. Use of a fusion protein in preparing a product for preventing or treating herpes simplex virus type 1, characterized in that: The fusion protein contains a truncated sequence of the interferon gene stimulator mutant according to claim 1 and a membrane-penetrating polypeptide connected together. The amino acid sequence of the truncated sequence of the interferon gene stimulator mutant is NVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVS QRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLF(BprY)RT LEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSL KTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS. The amino acid sequence of the membrane-penetrating polypeptide is shown in SEQ ID NO. 2, wherein (BprY) represents O-(3-bromopropyl)-L-tyrosine.

6. The use according to claim 5, characterized in that The truncated sequence of the interferon gene stimulator mutant is connected to the membrane-penetrating polypeptide via a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.

3.

7. The use according to claim 5, characterized in that The amino acid sequence of the fusion protein is: NVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILL PLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLF(BprY)RTLEDILAD APESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPS TSTMSQEPELLISGMEKPLPLRTDFSGGGGSYGRKKRRQRRR, wherein (BprY) represents O-(3-bromopropyl)-L-tyrosine.

8. A drug for preventing or treating viral infection, characterized in that: The drug for preventing or treating viral infection comprises the fusion protein according to any one of claims 5 to 7, and the virus is herpes simplex virus type 1.

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