Application of extracellular matrix protein 1 in preparation of medicine for treating HBV infection

Abstract

The invention relates to application of an extracellular matrix protein 1 in preparation of a medicine for treating HBV infection, and belongs to the technical field of biological medicine. The invention provides application of an extracellular matrix protein 1 and / or an extracellular matrix protein 1 accelerant in preparation of drugs for preventing and / or treating HBV infection and related diseases caused by HBV infection. Experiments show that the intrahepatic overexpression extracellular matrix protein 1 can significantly reduce the level of HBsAg and HBeAg in an HBV mouse body and promote the generation of HBsAb in the HBV mouse body; meanwhile, the levels of HBsAg and HBeAg in the HBV mouse can also be remarkably reduced by utilizing the expression of the extracellular matrix protein 1 in the hepatic stellate cells of the HBV mouse and the exogenous supplement of the extracellular matrix protein 1 to the HBV mouse through the adeno-associated virus. Therefore, the extracellular matrix protein 1 can effectively promote the removal of HBV, and provides higher possibility for functional healing of HBV infection.

Description

Technical Field

[0001] This invention relates to the application of extracellular matrix protein 1 in the preparation of drugs for treating HBV infection, and belongs to the field of biomedical technology. Background Technology

[0002] Hepatitis B virus (HBV) is a hepatotropic DNA virus composed of an envelope and nucleocapsid, primarily affecting the human liver. Chronic HBV infection refers to the persistence of HBV surface antigen for 6 months or longer after acute HBV infection. Chronic HBV infection leads to hepatitis, and with long-term progression, can result in liver fibrosis, cirrhosis, and even end-stage liver diseases such as liver cancer. Therefore, timely intervention is necessary to control the progression of chronic HBV infection.

[0003] Persistent negative hepatitis B surface antigen (HBsAg), with or without anti-HBs, HBV DNA below the minimum detection limit, and normal liver biochemical indicators are the ideal therapeutic endpoint for chronic hepatitis B virus infection, namely clinical cure or functional cure. In patients with chronic hepatitis B virus infection, the annual incidence of spontaneous HBsAg serological clearance (defined as two serological HBsAg clearances at least 6 months apart, and still undetectable at the last follow-up) is approximately 0.12–2.38%. Currently, nucleoside (acid) antiviral drugs (NAs) or interferon are commonly used to inhibit hepatitis B virus replication, thereby treating chronic hepatitis B virus infection. However, regardless of whether it is monotherapy or combination therapy with NAs and interferon, the HBsAg clearance rate in patients with chronic hepatitis B virus infection is still less than 15%. Therefore, there is an urgent need to find HBV infection treatment drugs that can effectively improve the HBsAg clearance rate to effectively control the development of chronic hepatitis B virus infection.

[0004] Extracellular matrix protein 1 (ECM1) is an important member of the non-collagenous protein family in the extracellular matrix, playing a crucial role in maintaining hepatic homeostasis. Previous studies have shown that ECM1 delays the progression of liver fibrosis by inhibiting the activation of hepatic stellate cells and can also serve as a serological marker for predicting liver fibrosis. However, the specific mechanism of action of ECM1 in chronic hepatitis B virus infection and its potential role in the functional cure of chronic hepatitis B virus infection remain unclear. Summary of the Invention

[0005] To address the above problems, the present invention provides the application of extracellular matrix protein 1 and / or extracellular matrix protein 1 promoters in the preparation of pharmaceuticals, wherein the extracellular matrix protein 1 promoters include substances capable of increasing the expression level of extracellular matrix protein 1 and / or extracellular matrix protein 1 encoding genes; The drug has at least one of the following uses: (a) Prevention and / or treatment of hepatitis B virus infection; and / or, (b) Prevention and / or treatment of diseases related to hepatitis B virus infection.

[0006] In one embodiment of the present invention, the hepatitis B virus infection is chronic hepatitis B virus infection; the related diseases caused by the hepatitis B virus infection are related diseases caused by chronic hepatitis B virus infection.

[0007] In one embodiment of the present invention, the prevention and / or treatment of hepatitis B virus infection includes promoting the clearance of hepatitis B virus and / or promoting the production of hepatitis B virus antibodies.

[0008] In one embodiment of the present invention, promoting hepatitis B virus clearance includes promoting the clearance of hepatitis B surface antigen (HBsAg) and / or promoting the clearance of hepatitis B e antigen (HBeAg); promoting hepatitis B virus antibody production includes promoting the production of hepatitis B surface antibody (anti-HBs, HBsAb).

[0009] In one embodiment of the present invention, the related diseases caused by hepatitis B virus infection include hepatitis, liver failure, liver fibrosis, cirrhosis and / or liver cancer.

[0010] In one embodiment of the present invention, the drug comprises extracellular matrix protein 1 and pharmaceutically acceptable excipients; or, the drug comprises an extracellular matrix protein 1 promoter and pharmaceutically acceptable excipients; or, the drug comprises extracellular matrix protein, an extracellular matrix protein 1 promoter, and pharmaceutically acceptable excipients.

[0011] In one embodiment of the present invention, the pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and / or release inhibitors.

[0012] The present invention also provides a medicament comprising extracellular matrix protein 1 and / or an extracellular matrix protein 1 promoter; the extracellular matrix protein 1 promoter includes a substance capable of increasing the expression level of extracellular matrix protein 1 and / or the gene encoding extracellular matrix protein 1; the medicament has at least one of the following uses: (a) Prevention and / or treatment of hepatitis B virus infection; and / or, (b) Prevention and / or treatment of diseases related to hepatitis B virus infection.

[0013] In one embodiment of the present invention, the hepatitis B virus infection is chronic hepatitis B virus infection; the related diseases caused by the hepatitis B virus infection are related diseases caused by chronic hepatitis B virus infection.

[0014] In one embodiment of the present invention, the prevention and / or treatment of hepatitis B virus infection includes promoting the clearance of hepatitis B virus and / or promoting the production of hepatitis B virus antibodies.

[0015] In one embodiment of the present invention, promoting hepatitis B virus clearance includes promoting the clearance of hepatitis B surface antigen (HBsAg) and / or promoting the clearance of hepatitis B e antigen (HBeAg); promoting hepatitis B virus antibody production includes promoting the production of hepatitis B surface antibody (anti-HBs, HBsAb).

[0016] In one embodiment of the present invention, the related diseases caused by hepatitis B virus infection include hepatitis, liver failure, liver fibrosis, cirrhosis and / or liver cancer.

[0017] In one embodiment of the present invention, the drug comprises extracellular matrix protein 1 and pharmaceutically acceptable excipients; or, the drug comprises an extracellular matrix protein 1 promoter and pharmaceutically acceptable excipients; or, the drug comprises extracellular matrix protein, an extracellular matrix protein 1 promoter, and pharmaceutically acceptable excipients.

[0018] In one embodiment of the present invention, the pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and / or release inhibitors.

[0019] The present invention also provides a molecular marker for predicting functional cure of hepatitis B virus infection, said molecular marker including extracellular matrix protein 1.

[0020] In one embodiment of the present invention, the molecular marker is the serum extracellular matrix protein 1 expression level.

[0021] This invention also provides the application of reagents for detecting the above-mentioned molecular markers in test samples in the preparation of products for predicting functional cure of hepatitis B virus infection.

[0022] In one embodiment of the present invention, the product includes a test kit.

[0023] The present invention also provides a product for predicting functional cure of hepatitis B virus infection, the product comprising a reagent for detecting the above-mentioned molecular markers in a sample to be tested.

[0024] In one embodiment of the present invention, the product includes a test kit.

[0025] The technical solution of this invention has the following advantages: This invention provides the application of extracellular matrix protein 1 (EMP1) and / or EMP1 promoters in the preparation of drugs for the prevention and / or treatment of hepatitis B virus (HBV) infection and / or related diseases caused by HBV infection. Animal experiments show that intrahepatic overexpression of EMP1 significantly reduces the levels of HBsAg and HBeAg in HBV mice and significantly promotes HBsAb production in HBV mice. Conversely, hepatic-specific knockdown of EMP1 significantly delays viral clearance in HBV mice and inhibits antibody production. Furthermore, animal experiments show that restoring EMP1 expression in hepatic stellate cells of HBV mice using adeno-associated virus (AAV) and exogenous supplementation of EMP1 in HBV mice also significantly reduces the levels of HBsAg and HBeAg in HBV mice. Therefore, EMP1 and / or EMP1 promoters can effectively promote HBV clearance, providing a higher possibility for functional cure of HBV infection. Therefore, extracellular matrix protein 1 and / or extracellular matrix protein 1 promoters are highly promising for the preparation of drugs for the prevention and / or treatment of hepatitis B virus infection, and / or for the prevention and / or treatment of related diseases caused by hepatitis B virus infection. Attached Figure Description

[0026] Figure 1 : A schematic diagram of constructing an HBV mouse model by injecting pAAV-HBV1.2 plasmid into the tail vein under high pressure, while simultaneously injecting ECM1 or an empty vector plasmid.

[0027] Figure 2 Serum HBsAg levels in different mice (injected with ECM1 or empty vector plasmid).

[0028] Figure 3 Serum HBeAg levels in different mice (injected with ECM1 or empty vector plasmid).

[0029] Figure 4 Serum HBsAb levels in different mice (injected with ECM1 or empty vector plasmid).

[0030] Figure 5 : Utilizing hepatic stellate cells to specifically knock down ECM1 (Lrat cre ECM1 fl / + ) and control (Lrat) cre ECM1 + / + (A schematic diagram of the HBV model constructed in mice.)

[0031] Figure 6 Different mice (Lrat) cre ECM1 fl / + Or Lrat cre ECM1+ / + Serum HBsAg level.

[0032] Figure 7 Different mice (Lrat) cre ECM1 fl / + Or Lrat cre ECM1 + / + Immunohistochemical staining results of HBcAg in the liver.

[0033] Figure 8 Different mice (Lrat) cre ECM1 fl / + Or Lrat cre ECM1 + / + Serum HBsAb levels.

[0034] Figure 9 :Lrat cre ECM1 fl / + Or Lrat cre ECM1 + / + The number of mice with serum HBsAb levels above 0.5 mIU / mL (green) or below 0.5 mIU / mL (gray).

[0035] Figure 10 : A schematic diagram of constructing an HBV mouse model by overexpressing ECM1 with recombinant adeno-associated virus in mice with hepatic stellate cell-specific ECM1 knockdown.

[0036] Figure 11 Serum HBsAg levels in different mice (overexpressing ECM1 or control).

[0037] Figure 12 Serum HBeAg levels in different mice (overexpressing ECM1 or control).

[0038] Figure 13 : A schematic diagram of constructing an HBV mouse model by in vivo and exogenous supplementation of recombinant ECM1 protein in mice with specific knockdown of ECM1 in hepatic stellate cells.

[0039] Figure 14 Serum HBsAg levels in different mice (exogenously supplemented with recombinant ECM1 protein or control).

[0040] Figure 15 Serum HBeAg levels in different mice (exogenously supplemented with recombinant ECM1 protein or control). Detailed Implementation

[0041] The following experimental examples are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0042] For experiments in the following examples where specific experimental steps or conditions are not specified, the procedures and conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0043] Experimental Example 1: Effects of ECM1 intervention on HBV mice The experimental procedure is as follows: 1. Experimental Methods 1.1 HBV mouse model construction and dynamic blood collection (1) Select 6-8 week old C57BL / 6 mice (purchased from Guangdong Provincial Medical Animal Experiment Center), weigh and record the weight of each mouse before modeling; (2) The pAAV-HBV 1.2 plasmid was diluted with physiological saline at a volume of 10% of the body weight of each mouse (see the literature "Li-Rung Huang, Hui-Lin Wu, Pei-Jer Chen, et al., Animmunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection[J], Proc Natl Acad Sci USA 2006.103(47) 17862-7." for the pAAV-HBV 1.2 plasmid carrying the HBV virus genome sequence) to obtain the pAAV-HBV 1.2 working solution. HBV modeling was performed by injecting 6 μg of plasmid into each mouse via high-pressure tail vein. (3) At 1, 4, 7, 10 and 14 days after modeling, blood was collected from the orbital vein of HBV mice. The specific procedure was as follows: after anesthetizing HBV mice with isoflurane, blood was collected from the orbital vein using a capillary lancet. 100 μL of peripheral blood was collected and left to stand upright for 2 hours; centrifuged at 6000 rpm at room temperature for 10 min, and the supernatant was transferred to a new 1.5 mL EP tube; the above centrifugation was repeated and the supernatant was collected; the separated serum was aliquoted into EP tubes, labeled and stored in a -20℃ refrigerator (see experimental procedure). Figure 1 ).

[0044] 1.2. Constructing a mouse model of intrahepatic overexpression using ECM1 plasmid. Each mouse was injected with 10 μg of ECM1 plasmid or empty vector plasmid (both ECM1 and empty vector plasmids were constructed by Guangzhou Dahong Biotechnology Co., Ltd. The ECM1 plasmid was obtained by inserting the ECM1 coding gene into the pcDNA3.1 plasmid; the sequence number of the ECM1 coding gene is NM_007899.5; the empty vector plasmid is the pcDNA3.1 plasmid). The ECM1 plasmid or empty vector plasmid was added to the pAAV-HBV1.2 plasmid working solution prepared according to method 1.1 above, and high-pressure tail vein injection and dynamic orbital vein blood collection were performed (experimental procedure see...). Figure 1 ).

[0045] 1.3 Constructing hepatic stellate cell-specific ECM1 knockdown mice using CRISPR / Cas9 technology Using C57BL / 6J mice as the editing target, gene editing was performed using CRISPR / Cas9 technology. Exon 2-8 of the Ecm1-202 transcript (Transcript ID: ENSMUST00000117507.10) were selected as the flux region to construct C57BL / 6J- Ecm1 em1 (flox) Smoc Mice, also known as Lrat cre Ecm1 + / + Mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.). Lrat cre Ecm1 + / + Mice were crossed with Lrat-Cre mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) that expressed Cre recombinase driven by the lecithin retinyltransferase (Lrat) promoter to produce hepatic stellate cell-specific Ecm1 gene knockout mice, namely Lrat mice. cre ECM1 fl / + Mice. Lrat cre Ecm1 + / + Mice and Lrat cre ECM1 fl / + HBV mouse models were constructed using the method described in 1.1 above, and dynamic orbital vein blood collection was performed (experimental procedure see below). Figure 5 ).

[0046] 1.4. Constructing an intrahepatic ECM1 overexpression model using recombinant adeno-associated virus. (1) Propagate a sufficient number of Lrat cre ECM1 fl / + Mice were housed in an SPF-grade animal facility, and their condition and reproductive status were observed daily. (2) When the mice reach 4 weeks of age, inject 100 μL (containing 2×10) via the tail vein. 11The corresponding recombinant adeno-associated virus (AAV6-ECM1 / AAV6-vector) (both AAV6-ECM1 and AAV6-vector were purchased from Shanghai Heyuan Biotechnology Co., Ltd. AAV6-ECM1 was prepared by inserting the ECM1 coding gene into the empty vector GL3009 pAAV-TBG-GdGreen-WPRE, transfecting it into 293T cells, and collecting the virus from the culture supernatant after cell culture; AAV6-vector was prepared by transfecting the empty vector GL3009 pAAV-TBG-GdGreen-WPRE into 293T cells, and collecting the virus from the culture supernatant after cell culture). (3) When the mice reach 6 weeks of age, construct the HBV mouse model according to the method in 1.1 above and perform dynamic orbital vein blood collection (see experimental procedure). Figure 10 ).

[0047] 1.5. Exogenous supplementation of HBV mouse model with recombinant ECM1 protein. (1) Propagate a sufficient number of Lrat cre ECM1 fl / + Mice were housed in an SPF-grade animal facility, and their condition and reproductive status were observed daily. (2) When the mice grow to 6-8 weeks of age, HBV mouse model is constructed according to the method in 1.1 above. One day after modeling, HBV mice are injected into the tail vein with recombinant ECM1 protein (the amino acid sequence of recombinant ECM1 protein is SEQ ID NO.1, purchased from Shanghai Sangon Biotech Co., Ltd., diluted with 200 μL of physiological saline as a solvent for tail vein injection) at a dose of 5 mg / kg / time. The injection is repeated every three days. Control mice are injected into the tail vein with the same dose of physiological saline every day. (3) During the administration period, dynamic orbital vein blood collection was performed on the HBV mouse model with exogenously supplemented ECM1 as described in 1.1 above (experimental procedure is as follows). Figure 13 ).

[0048] The preparation method of the ECM1 recombinant protein is as follows: A gene fragment encoding the recombinant ECM1 protein, with a nucleotide sequence as shown in SEQ ID NO.1, was synthesized; the gene fragment encoding the recombinant ECM1 protein and the pET-22b(+) plasmid were linearized and ligated to obtain a recombinant plasmid; the recombinant plasmid was transformed into Escherichia coli (E. coli). Escherichia coliBL21(DE3) was used to obtain the transformation product. The transformation product was spread on LB solid medium containing 30 μg / mL kanamycin and incubated upside down in a 37℃ incubator for 12 h. Positive clones were screened to obtain single clones of recombinant Escherichia coli. Single clones of recombinant Escherichia coli were picked and inoculated into 10 mL of LB liquid medium containing 30 μg / mL ampicillin and cultured with shaking at 37℃ and 200 rpm until OD. 600 After adding IPTG to a final concentration of 1 mM, the mixture was induced and cultured at 37℃ and 200 rpm for 4 h to obtain the fermentation broth.

[0049] The fermentation broth was centrifuged, and the supernatant and cell pellet were analyzed by SDS-PAGE and Western blotting, respectively. Solubility analysis showed that the target protein (approximately 62 kDa) was mainly present in the cell pellet as inclusion bodies. Subsequently, the culture was expanded to OD400 using the same method in 4 L of LB broth containing 30 µg / mL ampicillin. 600 =0.8, add IPTG to a final concentration of 1 mM, and induce culture at 37℃ and 200 rpm for 4 h. Collect the cell pellet by centrifugation of the fermentation broth. Resuspend the cell pellet in PBS buffer (pH 7.4) containing 0.1% (v / v) Triton X-100, sonicate and centrifuge, and collect the supernatant crude protein for purification.

[0050] Purification was performed using a two-step chromatography method: ① Nickel column affinity chromatography: The crude protein was first incubated with Ni-NTA resin equilibrated with PBS buffer for 1 hour, and the unbound eluent was collected. The target protein was then eluted with PBS buffer containing 500 mM imidazole. SDS-PAGE confirmed that the target band was concentrated in the elution fraction, yielding the affinity-purified product. ② Gel filtration chromatography: The affinity-purified product was further purified by gel filtration chromatography using a Superdex 200 column. The target protein was then eluted with PBS buffer containing 8 M urea, and the target peak fraction was collected to remove impurities. SDS-PAGE confirmed that the target band was concentrated in the elution fraction, yielding the purified protein.

[0051] The purified protein was gradually dialyzed into PBS buffer to remove urea, yielding a refolded product. The refolded product was then concentrated by ultrafiltration, sterilized by a 0.22 μm filter membrane, and finally tested for endotoxin levels, which showed a level of <1 EU / μg (meeting the control standard). The final product was ECM1 recombinant protein with a purity >90%, which was aliquoted and stored at -80℃.

[0052] 1.6 Detection of HBV serum virological markers in mice 1.6.1 HBsAg Detection A hepatitis B virus surface antigen diagnostic kit (purchased from Beijing Wantai) was used to perform semi-quantitative detection of HBsAg in mouse serum based on the principle of the double-antibody sandwich enzyme-linked immunosorbent assay. The HBsAg level in the positive control was detected using the Architect electrochemical immunoluminescence analyzer system, and the positive control was serially diluted to plot a standard curve. The specific procedure is as follows: (1) Sample addition: After diluting the mouse serum sample to an appropriate concentration, add 100 μL each of the test sample and standard diluent to the corresponding well according to the experimental sample layout, and shake to mix. (2) Incubation: After sealing with the sealing film, incubate at 37℃ for 60 min; (3) Add enzyme: After incubation, add 50 μL of enzyme-labeled reagent (HBsAb-HRP) to each well and shake to mix; (4) Incubation: After sealing with the sealing film, incubate at 37°C for 30 min; (5) Preparation of washing solution; dilute the concentrated washing solution with deionized water to 1×; (6) Washing: Tear off the sealing film, discard the liquid in the well, spin dry, wash 5 times with washing solution, soak for 2 min each time, 200 μL / well, and finally dry. (7) Color development: Add 50 μL of color developer solution A and solution B to each well, shake to mix, and place at 37℃ in the dark for 15 min to develop color; (8) Termination: Add 50 μL of termination solution to each well; (9) Reading the plate: After cleaning the bottom of the plate, read the plate within 5 minutes. When reading the plate, set the dual wavelength 450 nm / 630 nm to detect the OD value of each well and use the blank well for calibration.

[0053] 1.6.2 HBeAg Detection A hepatitis B virus e antigen detection kit (purchased from Beijing Wantai) was used to semi-quantitatively detect HBeAg in mouse serum based on the double-antibody sandwich method. The HBeAg level in the positive control was detected using the Architect electrochemical immunoassay analyzer system, and the positive control was serially diluted to plot a standard curve. The specific procedure is as follows: (1) Sample addition: After diluting the mouse serum sample to an appropriate concentration, add 50 μL each of the test sample and standard diluent to the corresponding well according to the experimental sample layout, and shake to mix. (2) Add enzyme: After incubation, add 50 μL of enzyme-labeled reagent (HBeAb-HRP) to each well and shake to mix; (3) Incubation: After sealing with the sealing film, incubate at 37°C for 30 min; (4) Preparation of washing solution; dilute the concentrated washing solution with deionized water to 1×; (5) Washing: Tear off the sealing film, discard the liquid in the well, spin dry, wash 5 times with washing solution, soak for 2 min each time, 200 μL / well, and finally dry. (6) Color development: Add 50 μL of color developer solution A and solution B to each well, shake to mix, and place at 37℃ in the dark for 15 min to develop color; (7) Termination: Add 50 μL of termination solution to each well; (8) Reading the plate: After cleaning the bottom of the plate, read the plate within 5 minutes. When reading the plate, set the dual wavelength 450 nm / 630 nm to detect the OD value of each well and use the blank well for calibration.

[0054] 1.6.3 HBsAb Detection A hepatitis B surface antibody detection kit (Beijing Wantai) was used to semi-quantitatively detect HBsAb levels in mouse serum based on the principle of a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA). The HBsAb levels in the positive control were then quantitatively detected using a Roche cobas e601 electrochemiluminescence immunoassay. A standard curve was plotted by serially diluting the positive control. The specific procedure is as follows: (1) Sample addition: After diluting the mouse serum sample to an appropriate concentration, add 50 μL each of the test sample and standard diluent to the corresponding well according to the experimental sample layout, and shake to mix. (2) Add enzyme: After incubation, add 50 μL of enzyme-labeled reagent (HBsAg-HRP) to each well and shake to mix. (3) Incubation: After sealing with the sealing film, incubate at 37°C for 30 min; (4) Preparation of washing solution; dilute the concentrated washing solution with deionized water to 1×; (5) Washing: Tear off the sealing film, discard the liquid in the well, spin dry, wash 5 times with washing solution, soak for 2 min each time, 200 μL / well, and finally dry. (6) Color development: Add 50 μL of color developer solution A and solution B to each well, shake to mix, and place at 37℃ in the dark for 15 min to develop color; (7) Termination: Add 50 μL of termination solution to each well; (8) Reading the plate: After cleaning the bottom of the plate, read the plate within 5 minutes. When reading the plate, set the dual wavelength 450 nm / 630 nm to detect the OD value of each well and use the blank well for calibration.

[0055] 1.6.4 Immunohistochemical HBcAg staining The specific process is as follows: (1) Sectioning: Take the embedded paraffin tissue, trim it, and cut a 4 μm section of the tissue onto a slide that is designed to prevent detachment; (2) Baking the slide: Preheat the oven to 62°C, place the glass slide in it and bake for 2 hours to allow the paraffin to melt completely; (3) Dewaxing and hydration: In a fume hood, the sections were immersed in xylene I and II for 10 min each; anhydrous ethanol I and II for 2 min each; 95% ethanol, 80% ethanol and 75% ethanol for 2 min each, and gradually hydrated; rinsed with distilled water for 5 min. (4) Blocking endogenous peroxidase: Immerse the slices in 3% (w / v, g / 100mL) hydrogen peroxide solution at room temperature (25℃) for 15 min to inactivate endogenous peroxidase; wash with PBS buffer for 2 min × 3 times; (5) Incubation of primary antibody: Draw a circle 3 mm away from the tissue with an immunohistochemical pen; drop 100 μL of hepatitis B virus core antigen (HBcAg) working solution (purchased from Zhongshan Jinqiao) onto the tissue surface, cover the tissue section, and incubate at 37°C for 60 minutes; wash with PBS buffer for 2 min × 3 times. (6) Incubation with secondary antibody: Add enzyme-labeled goat anti-rabbit / mouse IgG polymer (purchased from Gene Technology, model GK600711) to the tissue surface, cover the tissue section, and incubate at 37℃ for 20 min; wash with PBS buffer for 2 min × 3 times; (7) DAB staining: Prepare the staining solution by mixing C1 and C2 solutions of the DAB staining kit (purchased from Gene Technology, model GK600711) at a ratio of 1:50 (note to avoid light). Add 50 μL of staining agent to each slide and start timing. Observe under a normal optical microscope. When specific staining appears and the degree of staining is moderate, place the slide in tap water to stop the staining reaction. The remaining slides are stained at the same time. (8) Hematoxylin staining: After rinsing with tap water, add hematoxylin staining solution to the tissue section and incubate for 8 min. Observe the degree of staining under a microscope. Stop the staining with tap water and rinse with tap water for 5 min. Differentiate with alcohol containing 1% (v / v) hydrochloric acid for 23 s. Rinse with tap water for 2 min × 2 times. Reverse blue with warm water for 5 min. (9) Dehydration and clearing: Immerse the slices in 75% (v / v) ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol and xylene in turn for 1 min each to gradually dehydrate and clear the slices; (10) Mounting: Place the slide in a fume hood and air dry naturally; use neutral resin to mount the slide, add an appropriate amount of neutral resin to the tissue, cover with a coverslip, examine under a microscope to prevent air bubbles, and store at room temperature.

[0056] 2. Experimental Results The serum HBsAg, HBeAg, and HBsAb levels in different mice and the immunohistochemical staining results of HBcAg in the liver of different mice are shown in the figure. Figures 2-4 , Figures 6-9 , Figures 11-12 and Figures 14-15 .Depend on Figures 2-4 , Figures 6-9 , Figures 11-12 and Figures 14-15 It was found that compared with the high-pressure tail vein injection control plasmid group, injection of ECM1 plasmid significantly reduced serum HBsAg and HBeAg levels and significantly increased HBsAb levels in mice overexpressing ECM1 in the liver; using Lrat cre and ECM1 fl / fl Mice with hepatic stellate cell-specific ECM1 knockdown, bred through crossbreeding, showed significantly increased serum and hepatic HBsAg levels and significantly decreased serum HBsAb levels compared to wild-type mice. Injecting adeno-associated virus (AAV) into these ECM1-specific knockdown mice to replenish ECM1 expression in hepatic stellate cells significantly reduced serum HBsAg and HBeAg levels compared to mice injected with control AAV. Furthermore, injecting recombinant ECM1 protein into these mice significantly reduced serum HBsAg and HBeAg levels compared to mice injected with saline. These results suggest that overexpression of intrahepatic ECM1 or exogenous ECM1 supplementation can significantly promote HBV virus clearance and corresponding antibody production. This ECM1-based therapeutic strategy offers a higher possibility for functional cure of HBV infection.

[0057] Obviously, the above experimental examples are merely illustrative and not intended to limit the implementation methods. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementation methods. However, obvious variations or modifications derived therefrom remain within the scope of this invention.

Claims

1. The use of extracellular matrix protein 1 and / or extracellular matrix protein 1 promoters in the preparation of pharmaceuticals, characterized in that, The promoters of extracellular matrix protein 1 include substances capable of increasing the expression levels of extracellular matrix protein 1 and / or the genes encoding extracellular matrix protein 1; The drug has at least one of the following uses: (a) Prevention and / or treatment of hepatitis B virus infection; and / or, (b) Prevention and / or treatment of diseases related to hepatitis B virus infection.

2. The application as described in claim 1, characterized in that, The prevention and / or treatment of hepatitis B virus infection includes promoting hepatitis B virus clearance and / or promoting the production of hepatitis B virus antibodies.

3. The application as described in claim 2, characterized in that, The promotion of hepatitis B virus clearance includes promoting the clearance of hepatitis B virus surface antigen and / or promoting the clearance of hepatitis B virus e antigen; the promotion of hepatitis B virus antibody production includes promoting the production of hepatitis B virus surface antibody.

4. The application as described in any one of claims 1 to 3, characterized in that, The diseases caused by hepatitis B virus infection include hepatitis, liver failure, liver fibrosis, cirrhosis and / or liver cancer.

5. The application as described in any one of claims 1 to 3, characterized in that, The drug comprises extracellular matrix protein 1 and pharmaceutically acceptable excipients; or, the drug comprises an extracellular matrix protein 1 promoter and pharmaceutically acceptable excipients; or, the drug comprises extracellular matrix protein 1, an extracellular matrix protein 1 promoter, and pharmaceutically acceptable excipients.

6. The application as described in claim 5, characterized in that, Pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and / or release inhibitors.

7. A drug, characterized in that, The drug comprises extracellular matrix protein 1 and / or an extracellular matrix protein 1 promoter; the extracellular matrix protein 1 promoter includes substances capable of increasing the expression level of extracellular matrix protein 1 and / or the gene encoding extracellular matrix protein 1; the drug has at least one of the following uses: (a) Prevention and / or treatment of hepatitis B virus infection; and / or, (b) Prevention and / or treatment of diseases related to hepatitis B virus infection.

8. The drug as described in claim 7, characterized in that, The prevention and / or treatment of hepatitis B virus infection includes promoting hepatitis B virus clearance and / or promoting the production of hepatitis B virus antibodies.

9. The drug as described in claim 8, characterized in that, The promotion of hepatitis B virus clearance includes promoting the clearance of hepatitis B virus surface antigen and / or promoting the clearance of hepatitis B virus e antigen; the promotion of hepatitis B virus antibody production includes promoting the production of hepatitis B virus surface antibody.

10. The medicament according to any one of claims 7 to 9, characterized in that, The diseases caused by hepatitis B virus infection include hepatitis, liver failure, liver fibrosis, cirrhosis and / or liver cancer.

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