Application of IL-37b gene in preparation of medicine for preventing and treating diseases caused by HSV infection

Abstract

The invention provides an application of an IL-37b gene in preparation of a medicine for preventing and treating diseases caused by HSV infection. In animal model experiments, overexpression of the IL-37b delta1-45 gene can significantly prevent HSK recurrence, relieve corneal functions of a recurrent mouse model, relieve corneal tissue inflammation infiltration, relieve corneal stromal layer fibrosis level and significantly reduce viral load in mouse tears, which prompts that IL-37b delta1-45 is expected to become a new method for preventing HSK recurrence. Wide application prospects are realized.

Description

Field of the Invention

[0001] This application belongs to the fields of infectious disease treatment and gene therapy. Specifically, this application provides the use of the IL-37b gene in the preparation of a medicament for preventing and treating diseases caused by HSV infection. Background Art

[0002] Herpes simplex virus (HSV) belongs to the alpha subfamily of the Herpesviridae family of humans, and is an enveloped spherical virus. It is divided into Herpes simplex virus typeⅠ(HSV-1) and Herpes simplex virus typeⅡ(HSV-2). Both HSV-I and HSV-II are pathogenic viruses, which can cause herpetic stomatitis, herpes encephalitis, herpetic keratitis, genital herpes or neonatal encephalitis. So far, there is no drug to cure them, nor is there a vaccine to prevent infection.

[0003] Humans are the only natural hosts of this virus. In the natural state, human infection with HSV-1 is very common. It is estimated that more than 1 / 3 of the world's population has suffered from recurrent herpetic stomatitis, and 30% - 90% of the surveyed subjects have anti-herpes simplex virus antibodies in their sera, indicating that they have had or are having herpes simplex virus infection. Herpes simplex of oral cavity is an infectious disease mainly characterized by herpes in the acute oral mucosa and perioral skin caused by HSV infection. Traditional Chinese medicine calls it "heat sore", which is the most common viral infection in oral clinics. Among them, primary herpetic stomatitis is the most common oral lesion caused by herpes simplex virus type 1, which may manifest as a relatively severe gingivostomatitis - acute herpetic gingivostomatitis. In addition, recurrent herpetic stomatitis occurs after the healing of primary herpes infection. Regardless of the degree of the lesion, 30% - 50% of the cases may have recurrent lesions. Generally, the site of recurrent infection is on or near the lips, so it is also called recurrent labial herpes.

[0004] Herpes simplex keratitis (HSK) is an infectious corneal disease caused by HSV-1, which can cause blindness in severe cases. According to statistics, 50%-80% of the world's population are carriers of HSV-1, and 1.5 million new patients with keratitis caused by HSV infection are added each year, of which 40,000 patients suffer from visual impairment or blindness. Humans are the only natural host of the HSV-1 virus, and infection with HSV-1 is very common. Most people are carriers, and once infected with HSV-1, the virus will lurk in neurons for life, and when the body's immunity is low, it may be attacked by HSV-1 and cause inflammation. Among them, HSV-1 infects the cornea and causes symptoms of viral keratitis. HSK caused by HSV-1 is the main cause of corneal scarring and corneal opacity leading to blindness in the world.

[0005] Nowadays, the first choice of drugs for the treatment of HSK in clinical practice is acyclovir (ACV), ganciclovir and other similar broad-spectrum antiviral drugs. In addition, glucocorticoids, interferon, cyclosporine, etc. are used for adjuvant treatment; the drugs currently under development are mainly new dosage forms such as liposomes and micelles of acyclovir (Research Progress in Drug Treatment of Herpes Simplex Virus Matrix Keratitis, Acta Ophthalmologica Sinica, 2022, Vol. 37, No. 8). When mild patients use antiviral drugs for the first time, the antiviral effect is very good, but it can only suppress the replication of HSV-1 in the diseased area for a short time, and cannot eliminate the virus. Moreover, there is no countermeasure for the virus lurking in the nerves, resulting in recurrence of the disease. In addition, long-term use of broad-spectrum antiviral drugs by patients is prone to the side effect of drug resistance, which affects the subsequent treatment effect of the disease. Severe HSK patients need corneal transplantation to restore their vision, but the corneal donor resources are scarce and cannot meet the transplant needs of all patients. In addition, there is also the possibility of disease recurrence after corneal transplantation. Therefore, it is urgent to find new therapeutic drugs and new therapeutic targets to solve the above problems.

[0006] IL-37 is a newly discovered cytokine in 2000. It has 5 isoforms (IL-37a-e). It is known that IL-37b protein has a negative immune regulatory effect and plays an important role in controlling a variety of inflammatory diseases. Previous studies have shown that IL-37b protein can alleviate CVB3-induced myocarditis; in addition, further studies have shown that IL-37b protein has an inhibitory effect on influenza virus replication. However, there are currently no reports on the IL-37b gene inhibiting HSV-1 or HSV-2 at home and abroad, nor are there any reports on the use of IL-37b gene in the preparation of drugs for preventing and / or treating diseases infected with recurrent herpes simplex virus. Summary of the invention

[0007] In view of the above problems existing in the current treatment of HSV-infected diseases, and in order to discover and expand new uses of the IL-37b gene and provide new options for the treatment of HSV-infected diseases, on the one hand, the present invention provides the use of AAV9-delivered IL-37b gene in the preparation of a drug for treating diseases caused by HSV. To solve the above problems, the technical solutions adopted in the present invention are as follows:

[0008] On the one hand, the present application provides the application of the IL-37b gene in the preparation of a drug for preventing and treating diseases caused by HSV infection.

[0009] On the other hand, the present application provides the application of a reagent for increasing the expression of the IL-37b gene in the preparation of a drug for preventing and treating HSV-infected diseases.

[0010] Further, the reagent for increasing the expression of the IL-37b gene is a vector overexpressing the IL-37b gene.

[0011] Further, the vector is an adenovirus vector or an adeno-associated virus vector.

[0012] Further, the vector is an AAV9 vector.

[0013] Further, the coding sequence of the IL-37b gene is the protein of SEQ ID NO.6.

[0014] Further, the IL-37b gene sequence is as shown in SEQ ID NO.5.

[0015] The IL-37b gene in the present application can be selected from known and undiscovered IL-37b genes and their fragments, and these gene sequences can be subjected to necessary codon optimization and sequence truncation according to the requirements of the vector and the host. Conventional tags for detection or purification, such as FLAG tags, His tags, etc., can be routinely added or subtracted from the gene and protein sequences in the present application, and those skilled in the art can also add parts such as signal peptides and initiation methionine according to the needs of expression.

[0016] Further, the HSV-infected disease is gingivostomatitis, keratitis, encephalitis, reproductive system infection, neonatal herpes or cervical cancer caused by HSV infection.

[0017] Further, the HSV-infected disease is keratitis.

[0018] Further, the HSV-infected disease is keratitis caused by recurrent HSV infection.

[0019] Further, the drug is an injection dosage form.

[0020] On the other hand, the present application provides a drug for preventing or treating HSV infection diseases, and the drug includes an AAV9 vector overexpressing the IL-37b gene.

[0021] Further, the AAV9 vector overexpressing the IL-37b gene in the drug is the only active ingredient.

[0022] Further, the drug is an injection.

[0023] On the other hand, the present invention provides a method for non-therapeutically inhibiting HSV proliferation or inhibiting HSV infection, and the method includes administering an AAV9 vector overexpressing the IL-37b gene.

[0024] Further, the application object of the drug or method is a mouse or a human.

[0025] The drug in the present application can be in dosage forms known and feasible in the art such as oral, injection, topical administration, etc. The excipients / diluents therein include, but are not limited to, solvents, cosolvents, suspending agents, dispersing agents, acidity regulators, isotonic agents, fillers, binders, coating agents, lubricants, capsule shells, flavoring agents, sweetening agents, etc., which can be routinely selected by those skilled in the art according to pharmaceutical common sense.

[0026] The non-therapeutic methods in the present invention include, but are not limited to, methods for scientific research and methods for inhibiting non-pathogenic latent infections.

[0027] The application of the IL-37bΔ1-45 gene provided by the present invention in the preparation of drugs for preventing and treating recurrent HSK, that is, the treatment regimen of delivering the IL-37bΔ1-45 gene with AAV9 is used for the treatment of HSK and HSK recurrence. In the embodiment of the present invention, 30 days before the establishment of the recurrent HSK mouse model, the mice were given subconjunctival injection of AAV9-IL-37bΔ1-45 adeno-associated virus. Overexpression of IL-37bΔ1-45 can significantly prevent the corneal function of the recurrent HSK mouse model, reduce the inflammatory infiltration of the corneal tissue, reduce the fibrosis of the corneal stromal layer and significantly reduce the virus load in the tears of the mice. The experimental results suggest that IL-37bΔ1-45 is expected to become a new method for preventing HSK recurrence. The present invention confirms through experiments that overexpression of AAV9-IL-37bΔ1-45 can improve the corneal pathological state of HSK by directly hindering the virus load in the corneal tissue and the tears of the mice, so as to achieve a better therapeutic effect and has good application prospects. Description of the Drawings

[0028] Figure 1 It is the plasmid map of GV629;

[0029] Figure 2 It is the experimental flow chart of the present invention;

[0030] Figure 3 Fluorescence detection map showing successful overexpression of the IL-37b gene in corneal tissue;

[0031] Figure 4 Statistical result map showing that the weight loss of the recurrent HSK model mice was reversed after overexpressing the IL-37b gene in corneal tissue;

[0032] Figure 5 Statistical result map showing that the disease score aggravation of the recurrent HSK model mice was reversed after overexpressing the IL-37b gene in corneal tissue;

[0033] Figure 6 HE staining map of the corneal tissue sections taken from the recurrent HSK model mice after overexpressing the IL-37b gene in corneal tissue;

[0034] Figure 7 Result map of fluorescein sodium detection showing that the corneal injury of the recurrent HSK model mice improved significantly after overexpressing the IL-37b gene in corneal tissue;

[0035] Figure 8 Result map of the HSV-1 virus load level in the tears of the recurrent HSK model mice after overexpressing the IL-37b gene in corneal tissue. Detailed implementation manners

[0036] Example 1: Construction of a corneal tissue-specific expression AAV vector for the mouse Il-37b gene

[0037] First, the AAV-293 cell line was provided by Shanghai GeneChem Co., Ltd. The AAV-293 cell line is used to produce limited-related recombinant viruses. The AAV-293 cell line is derived from the commonly used HEK293 cell line and can produce higher-titer viruses. The AAV packaging process is as follows: First, the foreign gene (Il-37b) is cloned into the adeno-associated virus vector, and then the recombinant plasmid carrying the foreign gene is co-transfected with the helper plasmid AAV Helper Vector and the pAAV-rep / cap Vector into the packaging cell AAV-293 cells. A large number of recombinant viruses will be produced in the cells 72 hours after transfection. The culture medium supernatant and cell precipitate are harvested. The virus in the culture medium supernatant is precipitated with PEG8000, and the cell precipitate is lysed to collect the virus. The viruses obtained from the cell precipitate and supernatant are combined. Finally, the collected viruses are purified and concentrated, and are reserved after titer and purity detection.

[0038] I. Preparation of the overexpressing adeno-associated virus clone

[0039] 1. Information on the target gene and the tool vector

[0040] Name of the target gene: Il-37b (AAH20637.1(46-218aa)-3×flag);

[0041] Construction of adeno-associated virus: AAV9-Val46-Asp218. The name of the vector used is GV629. The GV629 vector plasmid contains AAV-pTIE-EGFP-MCS-SV40 polyA, digested with NheI / XhoI, all purchased from GeneChem Co., Ltd. in Shanghai. This company was commissioned to construct the plasmid and package the adeno-associated virus AAV9-Val46-Asp218. The order of viral elements: TIEp-EGFP-MCS-SV40 PolyA, cloning sites: NheI / XhoI. In addition, TIE(165-1458) is a mouse vascular endothelial cell-specific promoter that can promote the expression of human Il-37bΔ1-45 gene in mouse vascular endothelial cells. EGFP is a fluorescent protein that can be used to observe the in vivo infection efficiency of AAV9. The multiple cloning site (MCS) is a short DNA sequence containing up to 20 restriction enzyme cleavage sites that can serve as the insertion site for foreign genes, including the NheI and XhoI sites used in this study. SV40 polyA plays the role of a transcription termination signal. The vector map is as Figure 1 shown. AAV9-vector is the empty vector of GV629. AAV9-IL-37b is the GV629 vector packaged with the human Il-37bΔ1-45 gene (Accession#AAH20637.1), which is from Val46 to Asp218 (AAH20637.1(46-218aa)-3×Flag). The following primers were used to amplify the Il-37b gene: Forward primer: 5'-ACGAGCTGTACAAGGCTAGCATGGTGCACACTAGCCCTAAG-3', Reverse primer: 5'-GCTATCATATGTTACTCGAGTCATTTGTCGTCATCATCCTTATAG-3'. The vector and the human Il-37b gene sequence were digested with the restriction enzymes NheI and Xhol, and then the cloning was completed by the In-Fusion recombination method. The successful construction of the recombinant vector was confirmed by DNA sequencing.

[0042] 2. Vector digestion

[0043] Prepare the digestion system: 42 μL of ddH2O, 5 μL of 10x CutSmart Buffer, 2 μL of purified plasmid DNA (1 μg / μL), 1 μL of NheI restriction endonuclease (10 U / μL). Gently pipette and mix the digestion system, centrifuge and place it at 37°C for 3 h or overnight. Perform agarose gel electrophoresis on the vector digestion product and recover the target band.

[0044] 3. Obtaining the target gene fragment

[0045] (1) Design and synthesize Il-37b gene primers as shown in Table 1 below:

[0046] Table 1 Primers

[0047]

[0048] Primer description: Contains exchange pairing bases, restriction sites, and partial sequence at the 5' end of the target gene for PCR fishing of the target gene.

[0049] (2) PCR amplification of target gene fragment:

[0050] The first step is to clone the foreign gene into a suitable viral vector: Il-37b is cloned into a vector containing ITR / MCS. The inverted terminal repeat (ITR) sequence in these vectors provides all the cis-acting software necessary for AAV replication and packaging. According to the PrimeSTAR HSDNA polymerase instruction manual, the reaction system is prepared and placed in a PCR instrument for reaction. After the reaction is completed, the PCR product is recovered by electrophoresis.

[0051] 4. Exchange PCR products with vectors

[0052] Use the homologous recombination kit to prepare the following reaction system in an ice water bath: ddH2O 3.5μL, 5×CE II Buffer 2μL, digested vector DNA 2.52μL, purified PCR product 1μL, ExnaseTMII 1μL. After preparation, use a pipette to gently blow and mix, centrifuge briefly to avoid bubbles. React at 37℃ for 30min, then cool in an ice water bath for 5min and transform immediately.

[0053] 5. Conversion

[0054] Add 10 μL of the exchange reaction product to 100 μL of competent cells, flick the tube wall several times to mix, and place on ice for 30 minutes. Heat shock at 42°C for 90 seconds, and incubate in an ice water bath for 2 minutes. Add 500 μL of antibiotic-free LB medium and place on a shaker at 37°C for 1 hour. Take an appropriate amount of bacterial solution and evenly spread it on a plate containing the corresponding antibiotics, and invert and culture it in a constant temperature incubator for 12-16 hours.

[0055] 6. Colony PCR identification

[0056] Prepare the identification reaction system: ddH2O 9μL, 2×Taq Plus Master Mix 10μL, upstream and downstream primers 0.5μL (10μM) each. Vortex to mix, centrifuge briefly. Pick a single colony into 20μL identification system, pipette to mix, and place in PCR instrument for reaction.

[0057] Table 2 Primers for PCR identification

[0058]

[0059] Inoculate the identified positive clone transformants into an appropriate amount of LB liquid medium containing the corresponding antibiotics, culture at 37 °C for 12 - 16 h, and take an appropriate amount of the bacterial solution for sequencing. Compare and analyze the sequencing results with the target gene sequence. The sequencing and splicing results are shown below. The sequencing and splicing results show that: the sequencing results are completely consistent with the target sequence. The construction of the Il-37b (AAH20637.1 (46 - 218aa)-3×flag) gene sequence has been optimized for murine codons. The Il-37bΔ1-45 gene and protein sequences are as follows:

[0060]

[0061] II. Plasmid transfection and AAV virus harvest

[0062] 1. 24 h before transfection, digest AAV-293 cells in the logarithmic growth phase with trypsin, adjust the cell density to about 5×10 6 cells / 15 mL with a medium containing 10% serum, and re-inoculate into a 10 cm cell culture dish, and culture in an incubator at 37 °C and 5% CO2. When the cell density reaches 70% - 80% after 24 h, it can be used for transfection;

[0063] 2. Replace with a medium containing 2% serum 2 h before transfection;

[0064] 3. Co-transfect the recombinant expression plasmid with pHelper (carrying genes from adenovirus) and pAAV-RC (carrying AAV replication and capsid genes) into AAV-293 cells (providing trans-acting factors required for AAV replication and packaging). Add the prepared DNA solutions (5 μg of GV629-Il-37b recombinant vector plasmid, 5 μg of pHelper vector plasmid, 5 μg of AAV-RC plasmid) to a sterilized centrifuge tube, mix evenly with the corresponding volume of GeneChem transfection reagent, adjust the total volume to 1 mL, and incubate at room temperature for 15 min;

[0065] 4. Slowly add the mixture dropwise to the culture medium of AAV-293 cells, mix well, and culture in a cell incubator at 37 °C and 5% CO2;

[0066] 5. After culturing for 6 h, discard the medium containing the transfection mixture, add 10 mL of PBS solution to wash once, gently shake the culture dish to wash the remaining transfection mixture, and then discard it;

[0067] 6. Slowly add 10 mL of cell culture medium containing 5% serum, and continue to culture in an incubator at 37°C with 5% CO2 for 48 to 72 h. The recombinant AAV is assembled in the packaging cells.

[0068] III. Concentration and Purification of AAV9 Virus

[0069] 1. Collect AAV virus particles from the infected AAV-293 cells. Generally, AAV particles will be enriched in the packaging cells. Therefore, collecting the cells and then lysing them to release the AAV particles into the supernatant can recover most of the AAV particles. So, according to the cell state, collect the supernatant and cells of AAV-293 cells 72 h after transfection (counting from 0 h when transfection starts).

[0070] 2. Centrifuge at 3000 g for 5 min at 4°C to separate the cells and the supernatant.

[0071] 3. Add the GeneChem AAV virus concentration kit to the supernatant to obtain the virus in the supernatant. Resuspend the cell pellet with the resuspension solution, and repeatedly freeze-thaw 4 times in liquid nitrogen at 37°C, then centrifuge. After that, combine the two parts of the virus.

[0072] 4. Balance the samples respectively, and put the ultracentrifuge tubes with the virus supernatant into the Beckman ultracentrifuge one by one. Set the centrifugation parameters as 63000 rpm, the centrifugation time as 2 h, and control the centrifugation temperature at 18°C.

[0073] 5. Aspirate the separation layer where the virus is located. Put it into an ultrafiltration column for ultrafiltration until it becomes a colorless or light pink clear liquid.

[0074] 6. Sterilize the obtained virus solution through a 0.22 μm filter membrane and aliquot it as required.

[0075] 7. Prepare the samples for detection.

[0076] AAV Virus Titer Detection Method: Detect the AAV virus titer by QPCR, and the result obtained is the number of virus particles packaged. The principle is to perform real-time quantitative PCR with specific primers for the adeno-associated virus genome. Within the linear range of the quantitative curve, the ratio of the Ct value of the virus to the Ct value of the plasmid with known copy number is the initial copy number of the virus genome.

[0077] AAV Virus Purity Detection Method: It can be detected by a sensitive silver staining method. The AAV capsid protein contains 3 subunits, VP1 (82 kDa), VP2 (72 kDa), and VP3 (62 kDa), and the proportion is VP1:VP2:VP3 = 1:1:10. Therefore, a highly pure AAV shows only three bands in SDS-PAGE analysis.

[0078] Example 2: Mouse HSK recurrence model and evaluation indicators

[0079] First, a mouse HSK recurrence model was constructed. An HSV-1 acute corneal infection mouse model was established after modification according to a previously published study (Luo, Z., et al., Inhibitory effects of baicalein against herpes simplex virus type 1. Acta Pharm Sin B, 2020. 10(12): p. 2323-2338.):

[0080] After the mice were anesthetized, the right cornea was scratched into a "#" shape with a 33-gauge needle under a dissecting microscope. Then, 10 μL of the HSV-1 F strain (1.2×10 6 PFU) stock solution was dropped into the scratched eyeball. The HSV-1 strain F was a gift from Dr. Hu Kai, Department of Ophthalmology, Nanjing Gulou Hospital. In the recurrent HSK model, 6-week-old male BALB / c mice were mainly inoculated with 1×10 5 PFU of the HSV-1 F strain in the right eye, which was consistent with the acute HSV-1 corneal infection model. Subsequently, the mice were treated with 1 mL of human serum (Sigma, Temecula, CA, USA) with anti-HSV reactivity at 1 day post-infection (1 dpi) with HSV-1. The 50% virus neutralization effective dose was 1:800 to protect the mice from HSK infection during the initial infection. Then, the mice that survived the acute infection were maintained for 35 dpi, and the right eyes of the mice were continuously exposed to ultraviolet radiation B (UV-B) light at 302 nm for 3 minutes. Starting 5 hours after HSV-1 infection of the right eye of the mice, levofloxacin eye drops were used once a day to control bacterial infection. Corneal images were taken using a stereomicroscope to observe the corneal morphology. The condition of the injured cornea was photographed under a microscope with cobalt blue light with a wavelength of 460 nanometers. The severity of the disease was measured by the HSK disease score and the fluorescein sodium staining score.

[0081] The HSK disease score (0 - 5 points) was performed blindly according to the previously reported criteria (Luo, Z., et al., Inhibitory effects of baicalein against herpes simplex virus type 1. Acta Pharm Sin B, 2020.10(12): p.2323 - 2338.), as shown in Table 3. According to previous studies (Marsh, P. and S.C. Pflugfelder, Topical nonpreserved methylprednisolone therapy for keratoconjunctivitis sicca in syndrome. Ophthalmology, 1999.106(4): p.811 - 6.), the fluorescein staining score was graded from 0 (none) to 12 (most severe), as shown in Table 4.

[0082] Table 3 HSK disease score

[0083]

[0084] Table 4 Sodium fluorescein staining score

[0085]

[0086] Example 3: AAV9 - IL - 37bΔ1 - 45 gene therapy can alleviate corneal injury in recurrent HSK mice

[0087] The mouse experimental procedure is as Figure 2 shown.

[0088] First, a mouse model of recurrent HSK was constructed. Then, the recurrent HSK mice were divided into two different groups: two groups of mice were treated with AAV9 - IL - 37bΔ1 - 45 and AAV9 virus without any gene (AAV9 - Vector), respectively. At 7 days post - infection (7 dpi) of the mouse eyes with HSV - 1, the AAV9 virus carrying the Il - 37b gene (AAV9 - IL - 37bΔ1 - 45) was injected subconjunctivally into the eyes of the experimental group mice, and the AAV9 virus without any gene (AAV9 - Vector) was injected subconjunctivally into the eyes of the control group mice. The amount of virus injected was 1.0×10 11 v.g. In the AAV9 - IL - 37bΔ1 - 45 treatment model of recurrent HSK mice, 1.0×10 11Mice with HSV-1 corneal infection were infected with AAV9-Vector or AAV9-IL-37bΔ1-45 of v.g. At 5 dpi, 10 μL volume of AAV9-IL-37bΔ1-45 was injected subconjunctivally for 3 weeks. Then at 35 dpi, their right eyes were exposed to ultraviolet-B to further induce the recurrence of HSK.

[0089] Figure 3 It was shown that: On the 21st day after injection of AAV9-IL-37bΔ1-45, corneal tissues of control group mice and experimental group mice were taken, and the expression of IL-37b in corneal tissues was detected by immunofluorescence staining. As Figure 3 shown, the control group only showed DAPI fluorescence staining of cell nuclei, while the experimental group showed red FLAG fluorescence labeling in the whole corneal tissue, indicating that exogenous IL-37b was successfully expressed in corneal tissues; Figure 4 It was shown that: Body weights were recorded at 1 dpi, 3 dpi, 5 dpi, 35 dpi, 38 dpi and 40 dpi respectively (n = 5). The statistical results showed that after injection of AAV9-IL-37bΔ1-45, the weight loss of mice with recurrent HSK could be reversed; Figure 5 It was shown that: Disease scores of mice with recurrent HSK were evaluated at 1 dpi, 5 dpi, 35 dpi, 38 dpi and 40 dpi. The results showed that after injection of AAV9-IL-37bΔ1-45, the ocular surface disease state of mice with recurrent HSK could be alleviated; Figure 6 It was shown that: At 5 dpi, 35 dpi, 38 dpi and 40 dpi, corneal tissues were stained with sodium fluorescein to show corneal damage. The results showed that after injection of AAV9-IL-37bΔ1-45, the corneal damage of mice with recurrent HSK could be alleviated. Representative images of corneal morphology were taken under a stereomicroscope. At the same time, sodium fluorescein staining scores were analyzed (n = 5); Figure 7 It was shown that: Representative H&E images of corneal tissues of recurrent HSK mice after treatment with AAV9-IL-37bΔ1-45 at 5 dpi, 35 dpi, 38 dpi and 40 dpi. The results showed that after injection of AAV9-IL-37bΔ1-45, the inflammatory level and pathological state of corneal tissues of mice with recurrent HSK could be alleviated; Figure 8 It was shown that: The virus titer of HSV-1F in mouse tears was determined by the virus plaque assay. The results showed that after injection of AAV9-IL-37bΔ1-45, the virus load in the tears of mice with recurrent HSK could be reduced.

[0090] The above results indicate that AAV9-IL-37bΔ1-45 can improve the corneal pathological conditions of mice with recurrent HSK by reducing the virus load content in mouse tears, thereby achieving a better effect of preventing the recurrence of HSK. The present invention has good application prospects.

Claims

1. Application of IL-37b gene in the preparation of drugs for preventing and treating diseases caused by HSV infection.

2. Use of reagents that increase IL-37b gene expression in the preparation of drugs for preventing and treating HSV infection diseases.

3. The use according to claim 2, wherein the agent for increasing the expression of the IL-37b gene is a vector for overexpressing the IL-37b gene.

4. The use according to claim 3, wherein the vector is an adenovirus vector or an adeno-associated virus vector.

5. The use according to claim 4, wherein the vector is an AAV9 vector.

6. The use according to claim 2, wherein the IL-37b gene coding sequence is a protein of SEQ ID NO.

6.

7. The use according to claim 6, wherein the IL-37b gene sequence is shown as SEQ ID NO.

5.

8. The use according to claim 2, wherein the HSV infection disease is gingivostomatitis, keratitis, encephalitis, reproductive system infection, neonatal herpes or cervical cancer caused by HSV infection.

9. The use according to claim 8, wherein the HSV infection disease is keratitis.

10. A drug for treating HSV infection, characterized in that: The drug includes an AAV9 vector that overexpresses the IL-37b gene.

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