Interferon gene stimulating factor mutant and application thereof

Abstract

The invention relates to an interferon gene stimulating factor mutant and application thereof, and belongs to the technical field of biological medicine. The interferon gene stimulating factor mutant disclosed by the invention is obtained by mutating cysteine at the 292th site of an interferon gene stimulating factor into O-(3-bromopropyl)-L-tyrosine, and the interferon gene stimulating factor mutant disclosed by the invention can be cross-linked with the interferon gene stimulating factor to form a spheroid and enhance a downstream TBK1-IRF3 signal channel; therefore, the expression and secretion of a large amount of interferon I and other immunoregulatory factors are promoted, and the intensity and persistence of immune response are enhanced. Meanwhile, after the interferon gene stimulating factor mutant is combined with an interferon gene stimulating factor, the recognition capability and affinity of the interferon gene stimulating factor to pathogens can be greatly improved, and the problem that the recognition sensitivity of a natural interferon gene stimulating factor to low-dose pathogens is insufficient is effectively solved.

Description

Technical Field

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

[0002] Stimulator of interferon genes (STING protein), as a core element in the body's immune response, plays a key role in recognizing pathogen-associated molecular patterns (hereinafter referred to as "PAMPs"), such as cyclic dinucleotides (CDNs) of bacteria and double-stranded DNA of viruses. Once recognized, the STING protein rapidly activates the downstream serine-threonine protein kinase 1-interferon regulatory factor 3 signaling pathway (TBK1-IRF3 signaling pathway), promoting the expression and secretion of interferon (IFN) and other multiple immune regulatory factors, thereby initiating the body's immune defense mechanism and playing an indispensable role in antiviral, antibacterial, and anti-tumor immunity processes.

[0003] Although the STING protein is functionally important, it has many shortcomings in its natural state. For example, in the face of some low-dose or mutated pathogens, its signal activation efficiency is relatively low, resulting in a slow and insufficient immune response, making it difficult to effectively resist pathogen invasion. In addition, the stability of the STING protein itself is limited, and it is easily affected by degradation pathways such as proteasomes in cells, resulting in limited functional persistence.

[0004] To improve the performance of the STING protein, researchers have conducted a large number of explorations. Early on, chemical small molecule agonists were used to activate the STING protein. However, such agonists often have poor specificity and are prone to non-specific immune activation, leading to serious side effects such as systemic inflammatory response syndrome. In terms of genetic engineering modification, attempts have been made to perform point mutations on the STING protein to enhance its activity. However, some mutants have not significantly improved the signal transduction efficiency, and some cannot fold properly in cells, resulting in the loss of protein function. There has also been an attempt to change the localization or stability of the STING protein by fusing tags. 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 it difficult to achieve large-scale application.

[0005] In summary, the current methods for modifying the STING protein have deficiencies in terms of effect, safety, and operational convenience, and there is an urgent need for an innovative and effective strategy 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 that there is a lack of a STING protein activator with good stability and high activation efficiency.

[0007] To solve the above technical problem, the present invention provides a stimulator of interferon genes mutant and its application. The present invention selects the unnatural amino acid O-(3-bromopropyl)-L-tyrosine (English name "O-(3-Bromopropyl)-l-tyrosine", hereinafter referred to as BprY) with suitable chemical activity and structural characteristics. This unnatural amino acid has the ability to specifically react with the cysteine residue at position 292 of the STING protein and form a stable disulfide bond. The cysteine at position 292 of the STING protein is mutated to BprY by site-directed mutagenesis to obtain the STING protein mutant. When the STING protein mutant of the present invention is mixed and incubated with the STING protein, it is found that the STING protein mutant can crosslink with the STING protein to form spheroids and enhance the downstream TBK1-IRF3 signaling pathway. At the same time, when the STING protein mutant of the present invention is injected into virus-infected mice, it is found that the STING protein mutant of the present invention enhances the function of the STING protein by binding to the STING protein, thereby enhancing the immune response of mice under virus infection and inhibiting virus 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, and the amino acid sequence of the STING protein is as shown in SEQ ID NO.1.

[0009] Further, SEQ ID NO.1: MPHSSLHPSIPCPRGHGAQKAALVLLSACLVTLWGLGEPPEHTLRYLVLHLASLQLGLLLNGVCSLAEELRHIHSRYRGSYWRTVRACLGCPLRRGALLLLSIYFYYSLPNAVGPPFTWMLALLGLSQALNILLGLKGLAPAEISAVCEKGNFNVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFS。

[0010] Furthermore, in the present invention, the cysteine at position 292 of the STING protein is mutated to O-(3-bromopropyl)-L-tyrosine using an orthogonal translation system. Specifically, the codon corresponding to the C292 site in the gene encoding the STING protein is replaced with a codon capable of encoding the selected unnatural amino acid. By introducing an orthogonal translation system into the cell expression system, including an orthogonal aminoacyl-tRNA synthetase (aaRS) and an orthogonal tRNA pair, the cysteine codon TGC at position 292 of the STING protein is replaced with TAG. Then, under the action of the orthogonal aminoacyl-tRNA synthetase, the tRNA can recognize TAG, so the unnatural amino acid carried on the tRNA is linked to the amino acid at position 291. Therefore, during protein translation, the selected unnatural amino acid can be accurately incorporated into the C292 site 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.

[0011] The second object of the present invention is to provide the use of the above-mentioned STING protein mutant in the preparation of a STING protein activator.

[0012] Furthermore, the STING protein activator further includes 2'3'-cyclic guanosine monophosphate. 2'3'-cyclic guanosine monophosphate (2'3'-cGAMP) is a direct ligand of the STING protein. After it binds to the CTD region of the STING protein, it induces a conformational change in the STING protein, thereby activating the downstream TBK1-IRF3 signaling pathway and ultimately promoting the production of type I interferon. The present invention discovers that when the STING protein mutant is cross-linked with the STING protein, it can strengthen the activation of the TBK1-IRF3 signaling pathway, and the addition of 2'3'-cyclic guanosine monophosphate can strengthen the cross-linking of the STING protein mutant and the STING protein.

[0013] Furthermore, the STING protein activator promotes the oligomerization of the STING protein.

[0014] The third object of the present invention is to provide a drug for promoting the aggregation of stimulator of interferon genes, and the drug includes the above-mentioned stimulator of interferon genes mutant.

[0015] The fourth object of the present invention is to provide an application of the above-mentioned STING protein mutant in the preparation of a product for preventing or treating herpes simplex virus type 1. The product for preventing or treating herpes simplex virus type 1 includes a fusion protein, and the fusion protein contains the above-mentioned stimulator of interferon genes mutant and a transmembrane polypeptide connected thereto.

[0016] Furthermore, the STING protein contains a transmembrane region (TMD) composed of amino acids at positions 1-153 and a cytosolic soluble region (CTD) composed of amino acids at positions 154-379. Preferably, in order to enable the STING protein to be synthesized in a prokaryotic system, the transmembrane region TMD of the STING protein can be truncated, and only the CTD part is retained.

[0017] Furthermore, the amino acid sequence of the transmembrane polypeptide is as shown in SEQ ID NO.2.

[0018] Furthermore, SEQ ID NO.2: YGRKKRRQRRR.

[0019] Furthermore, the stimulator of interferon genes mutant and the transmembrane polypeptide are connected through a linker peptide, and the amino acid sequence of the linker peptide is as shown in SEQ ID NO.3.

[0020] Furthermore, SEQ ID NO.3: GGGGS.

[0021] Furthermore, the amino acid sequence of the fusion protein is: NVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLF(BprY)RTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSGGGGSYGRKKRRQRRR, wherein, (BprY) represents O-(3-bromopropyl)-L-tyrosine.

[0022] Furthermore, the fusion protein comprises the CTD region of the STING protein mutant, a linker peptide, and a transmembrane polypeptide.

[0023] Furthermore, the product for preventing or treating herpes simplex virus type 1 promotes the expression of the interferon-β1 gene.

[0024] Furthermore, the product for preventing or treating herpes simplex virus type 1 inhibits the replication of herpes simplex virus type 1.

[0025] Furthermore, the product for preventing or treating herpes simplex virus type 1 inhibits the expression of the tegument protein UL46 of herpes simplex virus type 1. The tegument protein UL46 of herpes simplex virus type 1 (HSV-1) is an important viral protein for HSV-1 to antagonize the host antiviral immune response. The present invention discovers that after cross-linking the STING protein mutant with the STING protein, the expression of the tegument protein UL46 of herpes simplex virus type 1 can be significantly inhibited, so that the virus cannot escape the immune killing effect of the host.

[0026] The fifth object of the present invention is to provide a drug for preventing or treating viral infection, and the drug for preventing or treating viral infection comprises the above-mentioned stimulator of interferon genes mutant.

[0027] Furthermore, the virus includes herpes simplex virus type 1.

[0028] Advantages of the present invention: The STING protein mutant of the present invention can crosslink with the STING protein to form spheroids and enhance the downstream TBK1-IRF3 signaling pathway, thereby promoting the large expression and secretion of type I interferons and other immune regulatory factors, and enhancing the intensity and persistence of the immune response. At the same time, after the STING protein mutant of the present invention binds to the STING protein, it can greatly improve the recognition ability and affinity of the STING protein for pathogen-associated molecular patterns. Compared with the natural STING protein, the STING protein mutant can bind to the STING protein and initiate an immune response more quickly and sensitively under low-dose pathogen stimulation, effectively solving the problem of insufficient recognition sensitivity of the natural STING protein to low-dose pathogens. Description of the Drawings

[0029] Figure 1 It is a verification result diagram of the STING protein mutant;

[0030] Figure 2 It is a detection result diagram of the crosslinking situation between the STING protein mutant and the STING protein;

[0031] Figure 3 It is a detection result diagram of the in vitro spheroid formation ability between the STING protein mutant and the STING protein;

[0032] Figure 4 It is a schematic diagram of the results of the influence of the crosslinking of the STING protein mutant and the STING protein on the function of the STING protein and the downstream signal transduction ability;

[0033] Figure 5 It is a schematic diagram of the detection results of the influence of the STING protein mutant on the liquid-liquid phase separation (LLPS) of the STING protein and the downstream signal transduction ability;

[0034] Figure 6 It is a schematic diagram of the detection results of IFN-β1 and HSV-gRNA mRNA transcription in mouse spleen, liver, and brain tissues;

[0035] Figure 7 It is a schematic diagram of the results of detecting the content of IFN-β1 in mouse plasma by ELISA experiment;

[0036] Figure 8 It is a schematic diagram of the results of the immunohistochemistry (IHC) experiment of HSV-UL46 protein expression in mouse brain tissues;

[0037] Figure 9 It is a schematic diagram of the detection results of the distribution and metabolism of SCT protein and SCBT protein in mice. Detailed Embodiments

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it. However, the exemplified embodiments do not limit the present invention.

[0039] Example 1

[0040] 1. Experimental materials

[0041] 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 Quanshijin Company), 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 Company), modified Eagle's basal medium (DMEM medium), penicillin / streptomycin solution (from Gibco Company), 293T cell line (from ATCC), transfection reagent polyethyleneimine (PEI), Flag-tag antibody, glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH) and related secondary antibodies (from CST Company).

[0042] Table 1 Sequences of SEQ ID NO.1-3, STING protein mutation primers and their sequences

[0043] 2. Experimental methods

[0044] In this example, site-directed mutagenesis technology was used to replace the codon corresponding to the C292 site in the gene encoding the STING protein with the codon capable of 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 can be accurately incorporated into the C292 site of the STING protein during protein translation, thereby mutating the cysteine at the 292 site to BprY to obtain a STING protein mutant. During protein folding, the newly incorporated unnatural amino acid spontaneously forms a stable disulfide bond with the cysteine residue near the 292 site, thereby realizing the cross-linking modification of the STING protein. Co-expression of the gene encoding STING-C292-TAG and the unnatural amino acid synthase orthogonal system in the presence of the unnatural amino acid BprY can generate site-specific STING-C292-BprY in eukaryotic or prokaryotic expression systems.

[0045] The specific method is as follows: Extract the plasmid containing the STING protein-coding gene. Using a site-directed mutagenesis kit, operate according to the kit instructions to mutate the codon encoding the C292 site into the codon TAG that can encode the selected unnatural amino acid BprY and introduce a Flag tag to construct the STING-C292(TAG)-Flag plasmid. Co-transfect STING-C292(TAG)-Flag and MmXYRS into 293T cells. After 24 h, add 10 μM BprY. Harvest the cells after 12 h and detect the synthesis of the STING protein mutant by Western blot assay.

[0046] 3. Experimental results

[0047] As Figure 1 shown, this model diagram describes the working principle of this unnatural amino acid, that is, under the action of the unnatural amino acid synthase, the unnatural amino acid BprY specifically mutates the cysteine at the 292nd site of the STING protein expressed by the STING-C292(TAG)-Flag plasmid to BprY, obtaining the STING protein mutant. When the unnatural amino acid BprY or MmXYRS is added alone, the expected STING protein mutant band cannot be observed in the Western blot detection result, indicating that the system cannot work properly. Only when BprY and MmXYRS are added simultaneously can an obvious STING protein mutant band be detected, proving that the system can work properly and successfully realizing the mutation of the C292 site of the STING protein by the unnatural amino acid BprY.

[0048] Example 2

[0049] 1. Experimental materials

[0050] Empty plasmid, STING-Myc-WT plasmid (a plasmid containing the Myc tag and the wild-type STING protein gene), STING-C292A-Myc plasmid (a plasmid containing the Myc tag and the STING protein gene with the cysteine at the 292nd site mutated to alanine), the STING-C292(TAG)-Flag plasmid constructed in Example 1, BprY, MmXYRS. For cell culture-related products, please refer to Example 1.

[0051] 2. Experimental methods

[0052] The 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 h, 10 μM BprY was added to the second, fourth and sixth groups respectively and the cells were further cultured. After culturing for 12 h, the cells were harvested and the crosslinking situation was detected by Western blot assay.

[0053] 3. Experimental results

[0054] As Figure 2 shown, in the Western blot detection results, no obvious crosslinking band was detected at the expected crosslinked protein molecular weight position in the sixth group of samples, indicating that the STING protein with cysteine at position 292 mutated to alanine (STING-C292A protein) could not form a crosslinking band with the STING protein mutant. However, in the fourth group of samples, there was an obvious band (about 75KDa) at the position corresponding to the crosslinked protein molecular weight, indicating that the STING protein mutant could form a stable crosslinking band with the STING protein. Based on the above results, it is shown that the STING protein mutant can form a stable covalent crosslink with the wild-type STING protein.

[0055] Example 3

[0056] 1. Experimental materials

[0057] High-purity STING protein, prepared by eukaryotic expression system and purified by AKTA (Yonglian) molecular sieve to ensure its purity is above 95% to exclude the interference of impurities on experimental results. STING protein mutants and STING-C292A protein with cysteine at position 292 mutated to alanine, which also passed through a strict purification process to ensure their quality and activity. AF488 fluorescent dye, laser confocal microscope (from Carl Zeiss), glass slides and coverslips (from Biosharp).

[0058] 2. Experimental methods

[0059] Take 100 μL of STING protein, STING protein mutant, and STING-C292A protein solutions with a concentration of 1 mg / mL respectively, and add 10 μL of AF488 fluorescent dye solution with a concentration of 10 mM according to the ratio of the molar ratio of AF488 fluorescent dye to protein being 10:1. Gently shake and incubate the mixture in the dark at room temperature for 2 hours, and remove the unbound fluorescent dye by dialysis method to obtain pure AF488-labeled STING protein, STING protein mutant, and STING-C292A protein solutions respectively. Drop the fluorescently labeled STING protein and STING protein mutant on the glass slide at different concentrations respectively, then cover with a coverslip, and observe the protein's ability to form spheres under the microscope. Then mix the STING protein and STING protein mutant at a ratio of 1:1, and observe its ability to form spheres again. Finally, mix the STING protein mutant and STING-C292A protein at a ratio of 1:1, and observe its ability to form spheres again.

[0060] 3. Experimental results

[0061] As Figure 3 shown, in the single protein sphere formation experiment, the number of droplets formed by the single STING protein and the single STING protein mutant is small. Taking the concentration of 1.5 μM as an example, the average number of droplets formed by the STING protein in 10 fields of view is 60 ± 10; while the average number of droplets formed by the STING protein mutant is only 12 ± 5, indicating that the in vitro sphere formation ability of the single STING protein mutant is lower than that of the STING protein. The in vitro sphere formation ability of the STING protein mutant and STING-C292A protein is not significant either. However, when the STING protein and the STING protein mutant are mixed, the sphere formation ability of the STING protein is significantly improved, and this enhanced sphere formation ability is closely related to the function of the STING protein mutant, indicating that the STING protein mutant promotes the function of the STING protein further in the process of promoting liquid-liquid phase separation by interacting with the wild-type STING protein.

[0062] Example 4

[0063] 1. Experimental materials

[0064] Empty plasmid, STING-WT-Flag plasmid, STING-C292(TAG)-Flag plasmid, 2'3'-cGAMP, antibody specifically recognizing Flag tag (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).

[0065] 2. Experimental methods.

[0066] Seed 293T cells in 12-well plates. When the cell density reaches 80%, divide the cells into six groups. The first and second groups are transfected with empty plasmid and MmXYRS, the third and fourth groups are transfected with STING-WT-Flag plasmid and MmXYRS, and the fifth and sixth groups are transfected with STING-WT-Flag plasmid, STING-C292(TAG)-Flag plasmid and MmXYRS. After 24 h, add 10 μM BprY to all six groups of cells. After 12 h, stimulate the cells in the second, fourth and sixth groups with 1 μg / mL of 2'3'-cGAMP. After 1 h, collect the cells and detect the cross-linking of STING protein and the phosphorylation of downstream key kinases TBK1 and IRF3 by Western blot.

[0067] 3. Experimental results

[0068] As Figure 4 shown, the cells in the fourth group can induce the phosphorylation of downstream key kinases TBK1 and transcription factor IRF3. In the fifth and sixth groups of cells, not only cross-linking bands were observed, but also the phosphorylation levels of downstream kinase TBK1 and transcription factor IRF3 were significantly enhanced. After the cells in the sixth group were stimulated with 2'3'-cGAMP, the phosphorylation levels of TBK1 and IRF3 were further deepened. This fully demonstrates that after the STING protein mutant is cross-linked with the wild-type STING protein, it not only successfully promotes the cross-linking activation of the STING protein, but also significantly enhances the phosphorylation levels of downstream kinase TBK1 and transcription factor IRF3, strongly proving that the cross-linking of the STING protein mutant with the STING protein can effectively promote the activation of its own function and the signal transduction ability to downstream signals.

[0069] Example 5

[0070] 1. Experimental materials

[0071] STING-WT-Flag plasmid, STING-C292(TAG)-Flag plasmid, 2'3'-cGAMP, 4% paraformaldehyde / PBS, 0.1% polyoxyethylene octylphenyl ether (TritonX-100), fluorescein isothiocyanate-labeled rabbit anti-sheep immunoglobulin G, rhodamine-labeled goat anti-mouse immunoglobulin G, STING protein antibody (from CST), TBK1 antibody (from Santacruz), 4',6-diamidino-2-phenylindole (DAPI).

[0072] 2. Experimental methods

[0073] Culture 293T cells and divide them into four groups. The first group and the second group are transfected with STING-WT-Flag plasmid and MmXYRS. The third group and the fourth group are transfected with STING-WT-Flag plasmid, STING-C292(TAG)-Flag plasmid and MmXYRS. After culturing for 24 h, add 10 μM BprY to each group. After 12 h, stimulate the cells in the second group and the fourth group with 1 μg / mL 2'3'-cGAMP, and use phosphate buffer solution (PBS) for the first group and the third group. After 1 h, collect the cells, fix and punch them with 4% paraformaldehyde / PBS solution, then incubate them overnight with STING protein antibody and TBK1 antibody, and then incubate them with fluorescent secondary antibody at room temperature for 1 h. Mount the slides and observe under a confocal microscope.

[0074] 3. Experimental results

[0075] As Figure 5 shown, STING protein hardly undergoes LLPS. After stimulation with 2'3'-cGAMP, STING protein undergoes LLPS in cells, forms spheroids, and co-localizes with TBK1; while the STING protein mutant can undergo LLPS in cells without 2'3'-cGAMP stimulation, and 2'3'-cGAMP stimulates the STING protein to undergo strong LLPS, and the interaction with TBK1 is higher than that of the STING protein with TBK1. In summary, the cross-linking of the STING protein mutant and the STING protein can promote the occurrence of LLPS in cells, and under the stimulation of 2'3'-cGAMP, this promotion is more significant. At the same time, the cross-linking of the STING protein mutant and the STING protein significantly enhances the interaction between the STING protein and TBK1, providing strong support for the efficient transduction of downstream signals.

[0076] Example 6

[0077] 1. Experimental materials

[0078] To verify the function of the STING protein mutant in cells, in this example, the protein transduction technology was adopted, and the HIV-TAT transmembrane polypeptide (the amino acid sequence is shown in SEQ ID NO.2) was fused to the C-terminus of the STING protein mutant to facilitate its transmembrane delivery. Specifically, in this example, by genetic engineering methods, the coding sequence of the HIV-TAT transmembrane polypeptide was inserted before the stop codon of the STING protein, and a flexible linker peptide (such as SEQ ID NO.3) was added between the two to maintain the native conformation and function of the STING protein mutant. The STING protein contains a transmembrane domain (TMD) composed of amino acids 1-153 and a cytosolic soluble domain (CTD) composed of amino acids 154-379. Since the full-length protein cannot be purified in the prokaryotic system, therefore, in this example, its transmembrane domain TMD was truncated, and only the CTD part was retained. Subsequently, the CTD region of the STING protein mutant containing the protein linked to HIV-TAT (abbreviated as SCBT protein) and the protein containing the CTD region of the STING protein linked to HIV-TAT (abbreviated as SCT protein) were purified through the prokaryotic expression system, and their transmembrane efficiency was verified through in vitro cell uptake experiments. This strategy ensured that the STING protein mutant could efficiently enter cells and play a role intracellularly, thus providing a reliable tool for studying its biological role.

[0079] 6- to 8-week-old specific pathogen-free (SPF) C57BL / 6 mice, the purified SCBT protein and SCT protein, 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 Biotech), guanidine isothiocyanate-phenol (Trizol, from TAKARA), reverse transcription kit and fluorescent DNA-binding dye premix (SYBR Green MasterMix, from Novizan), fluorescence quantitative PCR instrument (from Bio-Rad), tissue grinder, electronic balance, pipette.

[0080] Table 2 Primers and their sequences involved in Example 6

[0081] 2. Experimental methods

[0082] Thirty mice were randomly divided into 6 groups, with 5 mice in each group. The control groups were the PBS group, the SCT group, and the SCBT group; the experimental groups were the PBS+HSV-1 group, the SCT+HSV-1 group, and the SCBT+HSV-1 group. Mice in the PBS group and the PBS+HSV-1 group were intraperitoneally injected with 100 μL of PBS; mice in the SCT group and the SCT+HSV-1 group were intraperitoneally injected with 100 μL of an SCT protein solution with a concentration of 1 mg / mL; mice in the SCBT group and the SCBT+HSV-1 group were intraperitoneally injected with 100 μL of an SCBT protein solution with a concentration of 1 mg / mL. Two hours after the injection, mice in the PBS+HSV-1 group, the SCT+HSV-1 group, and the SCBT+HSV-1 group were injected with 100 μL of a virus suspension containing 1×10 6 PFU of HSV-1 via the tail vein, and the other three groups were injected with an equal volume of PBS. Twelve hours after the viral infection, the spleens, livers, and brains of the mice were taken, and about 50 mg of tissue was placed in a ribonuclease (RNase)-free centrifuge tube, and total RNA was extracted using Trizol reagent. Subsequently, reverse transcription was performed according to the instructions of the reverse transcription kit, and then qPCR experiments were carried out.

[0083] 3. Experimental results

[0084] As Figure 6 shown, Coomassie brilliant blue staining clearly demonstrated the results of the purified SCT protein and SCBT protein. In the PBS group, the expression of IFN-β1 mRNA in the spleen, liver, and brain tissues of the mice was at the basal level; the expression of IFN-β1 mRNA in the SCT group of mice increased slightly, but there was no significant difference compared with the PBS group; the expression of IFN-β1 mRNA in the SCBT group of mice was significantly higher than that in the PBS group and the SCT group, indicating that the SCBT protein could promote the expression of the mouse's own IFN-β1 gene. In the PBS+HSV-1 group, the expression of IFN-β1 mRNA increased significantly; the expression of IFN-β1 mRNA in the SCT+HSV-1 group of mice was higher than that in the PBS+HSV-1 group, and the expression of IFN-β1 mRNA in the SCBT+HSV-1 group of mice was significantly higher than that in the PBS+HSV-1 group and the SCT+HSV-1 group. It is shown that the STING protein mutant can significantly enhance the immune response of mice under HSV-1 infection, inhibit virus replication, and further prove that the STING protein mutant can significantly enhance the function of the STING protein and promote the antiviral ability of mice.

[0085] Example 7

[0086] 1. Experimental materials

[0087] 6 - 8 week - old SPF - level C57BL / 6 mice, purified SCBT protein and SCT protein, 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), microplate reader (from Bio - Tek).

[0088] 2. Experimental methods

[0089] Thirty mice were randomly divided into 6 groups, with 5 mice in each group. The control groups were the PBS group, the SCT group, and the SCBT group; the experimental groups were the PBS + HSV - 1 group, the SCT + HSV - 1 group, and the SCBT + HSV - 1 group. Mice in the PBS group and the PBS + HSV - 1 group were intraperitoneally injected with 100 μL of PBS; mice in the SCT group and the SCT + HSV - 1 group were intraperitoneally injected with 100 μL of SCT protein solution with a concentration of 1 mg / mL; mice in the SCBT group and the SCBT + HSV - 1 group were intraperitoneally injected with 100 μL of SCBT protein solution with a concentration of 1 mg / mL. Two hours after injection, mice in the PBS + HSV - 1 group, the SCT + HSV - 1 group, and the SCBT + HSV - 1 group were injected with 100 μL of virus suspension containing 1×10 6 PFU of HSV - 1 via the tail vein, and the other three groups were injected with an equal volume of PBS. Twelve hours after virus 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. Subsequently, the supernatant was obtained by centrifugation. Detection was performed using the IFN - β1 ELISA kit, and finally, the absorbance value of each well was measured on a microplate reader at a wavelength of 450 nm.

[0090] 3. Experimental results

[0091] As Figure 7 shown, the content of IFN - β1 in the plasma of mice in the PBS group was low, maintaining at the basal level. There was no significant difference in the plasma IFN - β1 content between mice in the SCT group and the PBS group. The plasma IFN - β1 content in mice in the SCBT group was significantly higher than that in the PBS group and the SCT group, indicating that the STING protein mutant could promote the secretion of IFN - β1 in mice. The plasma IFN - β1 content in mice in the PBS + HSV - 1 group was significantly higher than that in the PBS group. The plasma IFN - β1 content in mice in the SCT + HSV - 1 group was somewhat higher than that in the PBS + HSV - 1 group. The plasma IFN - β1 content in mice in the SCBT + HSV - 1 group was significantly higher than that in the PBS + HSV - 1 group and the SCT + HSV - 1 group, further confirming that after mice were infected with HSV - 1, the STING protein mutant could significantly enhance the immune response, promote the large - scale secretion of IFN - β1, enhance the antiviral ability of mice, and reflect the significant enhancement effect of the STING protein mutant on the function of the STING protein.

[0092] Example 8

[0093] 1. Experimental materials

[0094] SPF-grade C57BL / 6 mice at 6 - 8 weeks old, purified SCBT protein and SCT protein, herpes simplex virus type 1 (HSV-1) strain, rabbit anti-herpes simplex virus type 1 - cortical protein 46 polyclonal antibody (rabbit anti-HSV-1-UL46 polyclonal antibody, from Abcam), horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G secondary antibody (goat anti-rabbit IgG-HRP secondary antibody, from Jackson Immuno Research), diaminobenzidine chromogenic kit (DAB chromogenic kit, from Boster), PBS buffer, antigen retrieval solution, 4% paraformaldehyde / PBS, embedding reagent (from Shanghai Sangon).

[0095] 2. Experimental methods

[0096] Thirty mice were randomly divided into 6 groups with 5 mice in each group. The control groups were the PBS group, SCT group, and SCBT group; the experimental groups were the PBS + HSV-1 group, SCT + HSV-1 group, and SCBT + HSV-1 group. Mice in the PBS group and PBS + HSV-1 group were intraperitoneally injected with 100 μL of PBS; mice in the SCT group and SCT + HSV-1 group were intraperitoneally injected with 100 μL of SCT protein solution at a concentration of 1 mg / mL; mice in the SCBT group and SCBT + HSV-1 group were intraperitoneally injected with 100 μL of SCBT protein solution at a concentration of 1 mg / mL. Two hours after injection, mice in the PBS + HSV-1 group, SCT + HSV-1 group, and SCBT + HSV-1 group were injected with 100 μL of virus suspension containing 1×10 6 PFU of HSV-1 via the tail vein, and the other three groups were injected with an equal volume of PBS. Twelve hours after virus infection, the mice were euthanized with carbon dioxide, and their brains were quickly removed and fixed in 4% paraformaldehyde solution for 24 hours. Subsequently, they were fixed, sectioned, and subjected to immunohistochemistry (IHC) staining experiments.

[0097] 3. Experimental results

[0098] As Figure 8As shown in the figure, a large number of positive staining regions of HSV-1 tegument protein 46 (HSV-1 tegument protein 46 is an important viral protein that antagonizes the host's antiviral immune response, hereinafter referred to as "UL46 protein") can be observed in the brain tissues of mice in the PBS + HSV-1 group, and the average optical density value is relatively high, indicating that HSV-1 replicates abundantly in the mouse brain and expresses UL46 protein. The positive staining region of UL46 protein in the brain tissues of mice in the SCT + HSV-1 group is reduced compared with that in the PBS + HSV-1 group, and the positive staining region of UL46 protein in the brain tissues of mice in the SCBT + HSV-1 group is significantly less than that in the PBS + HSV-1 group and the SCT + HSV-1 group, indicating that the STING protein mutant can significantly inhibit the replication of HSV-1 in the mouse brain and the expression of UL46 protein, further proving that the STING protein mutant can significantly enhance the antiviral ability of mice, improve the function of the STING protein, and effectively reduce the proliferation and diffusion of the virus in vivo.

[0099] Example 9

[0100] 1. Experimental materials

[0101] SPF-grade C57BL / 6 mice at 6 - 8 weeks of age; purified SCBT protein and SCT protein were labeled with the cyanine 5.5 fluorescent dye (Cy5.5) labeling kit (MCE). After the reaction, the unbound Cy5.5 dye was removed by a desalting column, and the concentration and labeling rate of the labeled protein were measured using an ultraviolet spectrophotometer to ensure that the labeling rate reached over 80%. Finally, the concentration of the labeled protein was adjusted to 1 mg / mL and stored in a refrigerator at 4°C for later use. Small animal in vivo imaging system (IVIS Spectrum).

[0102] 2. Experimental methods

[0103] Animal grouping and injection: Twenty-four mice were randomly divided into 3 groups, with 8 mice in each group, namely the PBS group, the SCT + Cy5.5 group, and the SCBT + Cy5.5 group. The weight of each mouse was accurately weighed using an electronic balance, and the injection dose was calculated based on the weight. The PBS group was injected with PBS, the SCT + Cy5.5 group was injected with Cy5.5-labeled SCT protein, and the SCBT + Cy5.5 group was injected with Cy5.5-labeled SCBT protein. Images were captured using a small animal in vivo imaging system 2 hours and 6 hours after injection.

[0104] 3. Experimental results

[0105] As Figure 9As shown, in the in vivo imaging pictures 2 hours after injection, obvious fluorescence signals could be observed in the mice of both the SCT+Cy5.5 group and the SCBT+Cy5.5 group. The fluorescence signals were mainly distributed in the regions of organs such as the liver, spleen, and lungs, indicating that the SCT protein and the SCBT protein could rapidly enter the blood circulation within a short time after injection and accumulate in these major immune and metabolic organs. In the in vivo imaging pictures 6 hours after injection, relatively strong fluorescence signals could still be detected in the mice of both the SCT+Cy5.5 group and the SCBT+Cy5.5 group, and the distribution range of the fluorescence signals was similar to that at 2 hours, but the overall fluorescence intensity decreased slightly. The above results comprehensively showed that the SCT protein and the SCBT protein could be well distributed in mice and could still be detected in the major organs 6 hours after injection, providing important basic data for further studying their functions and action mechanisms in vivo and also providing strong support for their potential clinical applications from the perspectives of in vivo distribution and metabolism.

[0106] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made on the basis of the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

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

1.

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

3. The application according to claim 2, characterized in that, The activator of the stimulator of interferon genes further comprises 2'3'-cyclic guanosine monophosphate.

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

5. Use of the stimulator of interferon genes mutant according to claim 1 in the preparation of a product for preventing or treating herpes simplex virus type 1, characterized in that, The product for preventing or treating herpes simplex virus type 1 comprises a fusion protein, and the fusion protein contains the stimulator of interferon genes mutant according to claim 1 and a transmembrane polypeptide which are connected.

6. The application according to claim 5, characterized in that, The amino acid sequence of the transmembrane polypeptide is as shown in SEQ IDNO.

2.

7. The application according to claim 5, wherein The stimulator of interferon genes mutant and the transmembrane polypeptide are connected through a linker peptide, and the amino acid sequence of the linker peptide is as shown in SEQ ID NO.

3.

8. The application according to claim 5, wherein The amino acid sequence of the fusion protein is: VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLF(BprY)RTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEVTVGSLKTSAVPSTSTMSQEPELLISGMEKPLPLRTDFSGGGGSYGRKKRRQRRR, wherein, (BprY) represents O-(3-bromopropyl)-L-tyrosine.

9. A drug for preventing or treating viral infections, characterized in that, The drug for preventing or treating viral infection comprises the stimulator of interferon genes mutant according to claim 1.

10. The prophylactic or therapeutic agent for viral infection according to claim 9, wherein The virus includes herpes simplex virus type 1.

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