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

Abstract

The application relates to application of extracellular matrix protein 1 in preparation of a medicine for treating HBV infection, and belongs to the technical field of biological medicine. The application provides application of extracellular matrix protein 1 and / or a promoter of extracellular matrix protein 1 in preparation of a medicine for preventing and / or treating HBV infection and diseases caused by the HBV infection. Experiments show that overexpression of the extracellular matrix protein 1 in the liver can significantly reduce the levels of HBsAg and HBeAg in HBV mice, and promote the production of HBsAb in the HBV mice; meanwhile, the expression of the extracellular matrix protein 1 in the liver stellate cells of the HBV mice is supplemented by using an adenovirus, and the extracellular matrix protein 1 is supplemented from outside, which can also significantly reduce the levels of HBsAg and HBeAg in the HBV mice. It can be seen that the extracellular matrix protein 1 can effectively promote the clearance of the HBV, and provide higher possibility for functional cure of the HBV infection.

Description

TECHNICAL FIELD

[0001] The application relates to application of extracellular matrix protein 1 in preparation of a medicine for treating HBV infection and belongs to the technical field of biological medicine. BACKGROUND

[0002] Hepatitis B virus (HBV) is a DNA virus that mainly invades the liver of a human body and is composed of an envelope and a nucleocapsid. Chronic hepatitis B virus infection refers to the persistence of hepatitis B virus surface antigen for 6 months or more after acute HBV infection. Chronic hepatitis B virus infection can lead to hepatitis, and long-term development can lead to liver fibrosis, liver cirrhosis, and even liver cancer and other end-stage liver diseases. Therefore, it is necessary to intervene in chronic hepatitis B virus infection in a timely manner to control its development.

[0003] Hepatitis B surface antigen (HBsAg) persistence negative, with or without anti-HBs, HBV DNA below the lower limit of detection, liver biochemistry normal, is the ideal treatment endpoint of chronic hepatitis B virus infection, that is, clinical cure or functional cure. In patients with chronic hepatitis B virus infection, the annual incidence of spontaneous HBsAg seroclearance (defined as the clearance of HBsAg in serum twice at least 6 months apart, and still undetectable in the last follow-up) is about 0.12-2.38%. At present, nucleos(t)ide analogues (NAs drugs) or interferon are usually used to inhibit hepatitis B virus replication, and then to treat chronic hepatitis B virus infection. However, regardless of single treatment or combined treatment of NAs drugs and interferon, the HBsAg clearance rate of patients with chronic hepatitis B virus infection is still less than 15%. Therefore, it is urgent to find an HBV infection treatment drug 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-collagen protein family in the extracellular matrix, which plays a key role in maintaining the homeostasis of the liver internal environment. Previous studies have shown that extracellular matrix protein 1 can delay the progression of liver fibrosis by inhibiting the activation of hepatic stellate cells, and can also be used as a serological marker for predicting liver fibrosis. However, at present, the specific mechanism of action of extracellular matrix protein 1 in chronic hepatitis B virus infection and its potential role in functional cure of chronic hepatitis B virus infection are still unclear. SUMMARY

[0005] To solve the above problems, the present application provides the use of extracellular matrix protein 1 and / or a promoter of extracellular matrix protein 1 in the preparation of a medicament, wherein the promoter of extracellular matrix protein 1 comprises a substance capable of increasing the expression level of extracellular matrix protein 1 and / or a gene encoding extracellular matrix protein 1;

[0006] The medicament has at least one of the following uses:

[0007] (a) preventing and / or treating hepatitis B virus infection; and / or,

[0008] (b) preventing and / or treating diseases related to hepatitis B virus infection.

[0009] In an embodiment of the present application, the hepatitis B virus infection is chronic hepatitis B virus infection; and the diseases related to hepatitis B virus infection are diseases related to chronic hepatitis B virus infection.

[0010] In an embodiment of the present application, the prevention and / or treatment of hepatitis B virus infection comprises promoting hepatitis B virus clearance and / or promoting hepatitis B virus antibody production.

[0011] In an embodiment of the present application, the promotion of hepatitis B virus clearance comprises promoting the clearance of hepatitis B surface antigen (HBsAg) and / or promoting the clearance of hepatitis B e antigen (HBeAg); and the promotion of hepatitis B virus antibody production comprises promoting the production of hepatitis B surface antibody (anti-HBs, HBsAb).

[0012] In an embodiment of the present application, the diseases related to hepatitis B virus infection comprise hepatitis, liver failure, liver fibrosis, liver cirrhosis and / or liver cancer.

[0013] In an embodiment of the present application, the components of the medicament comprise extracellular matrix protein 1 and a pharmaceutically acceptable excipient; or the components of the medicament comprise a promoter of extracellular matrix protein 1 and a pharmaceutically acceptable excipient; or the components of the medicament comprise extracellular matrix protein, a promoter of extracellular matrix protein 1 and a pharmaceutically acceptable excipient.

[0014] In an embodiment of the present application, the pharmaceutically acceptable excipient includes solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, aromatics, anti-caking agents, integrating agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filter aids, and / or release retarders.

[0015] The present application also provides a medicament, wherein the components of the medicament comprise extracellular matrix protein 1 and / or a promoter of extracellular matrix protein 1; the promoter of extracellular matrix protein 1 comprises a substance capable of increasing the expression level of extracellular matrix protein 1 and / or a gene encoding extracellular matrix protein 1; and the medicament has at least one of the following uses:

[0016] (a) preventing and / or treating hepatitis B virus infection; and / or,

[0017] (b) preventing and / or treating a disease associated with hepatitis B virus infection.

[0018] In an embodiment of the present application, the hepatitis B virus infection is chronic hepatitis B virus infection; and the disease associated with hepatitis B virus infection is a disease associated with chronic hepatitis B virus infection.

[0019] In an embodiment of the present application, the preventing and / or treating hepatitis B virus infection comprises promoting hepatitis B virus clearance and / or promoting hepatitis B virus antibody production.

[0020] In an embodiment of the present application, the promoting hepatitis B virus clearance comprises promoting clearance of hepatitis B surface antigen (HBsAg) and / or promoting clearance of hepatitis B e antigen (HBeAg); and the promoting hepatitis B virus antibody production comprises promoting production of hepatitis B surface antibody (anti-HBs, HBsAb).

[0021] In an embodiment of the present application, the disease associated with hepatitis B virus infection comprises hepatitis, liver failure, liver fibrosis, liver cirrhosis, and / or liver cancer.

[0022] In an embodiment of the present application, the components of the drug comprise extracellular matrix protein 1 and pharmaceutically acceptable excipients; or, the components of the drug comprise a promoter of extracellular matrix protein 1 and pharmaceutically acceptable excipients; or, the components of the drug comprise extracellular matrix protein, a promoter of extracellular matrix protein 1 and pharmaceutically acceptable excipients.

[0023] In an embodiment of the present application, the pharmaceutically acceptable excipients include solvents, propellants, solubilizers, co-solvents, emulsifiers, coloring agents, binding agents, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filtration aids and / or release retardants.

[0024] The present application also provides a molecular marker for predicting functional cure of hepatitis B virus infection, which comprises extracellular matrix protein 1.

[0025] In an embodiment of the present application, the molecular marker is serum extracellular matrix protein 1 expression level.

[0026] The present application also provides use of a reagent for detecting the above-mentioned molecular marker in a sample to be tested in the preparation of a product for predicting functional cure of hepatitis B virus infection.

[0027] In an embodiment of the present application, the product comprises a detection kit.

[0028] The present application also provides a product for predicting functional cure of hepatitis B virus infection, which comprises a reagent for detecting the above-mentioned molecular marker in a sample to be tested.

[0029] In an embodiment of the present application, the product comprises a detection kit.

[0030] The technical solution of the present application has the following advantages:

[0031] The application provides application of extracellular matrix protein 1 and / or a promoter of extracellular matrix protein 1 in preparation of a medicine for preventing and / or treating hepatitis B virus infection and / or preventing and / or treating a disease caused by hepatitis B virus infection. Animal experiments show that overexpression of extracellular matrix protein 1 in the liver can significantly reduce the levels of HBsAg and HBeAg in HBV mice, and significantly promote the production of HBsAb in HBV mice; meanwhile, specific knockdown of extracellular matrix protein 1 in the liver can significantly delay the clearance of viruses in HBV mice and inhibit the production of antibodies in HBV mice; at the same time, animal experiments show that the expression of extracellular matrix protein 1 in the liver stellate cells of HBV mice is supplemented by using an adeno-associated virus, and the extracellular matrix protein 1 is supplemented from outside, which can also significantly reduce the levels of HBsAg and HBeAg in HBV mice. It can be seen that the extracellular matrix protein 1 and / or the promoter of extracellular matrix protein 1 can effectively promote the clearance of HBV, and provide a higher possibility for functional cure of HBV infection. Therefore, the extracellular matrix protein 1 and / or the promoter of extracellular matrix protein 1 has a wide application prospect in preparation of a medicine for preventing and / or treating hepatitis B virus infection and / or preventing and / or treating a disease caused by hepatitis B virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 : Schematic diagram of constructing an HBV mouse model by injecting pAAV-HBV1.2 plasmid into the tail vein of a mouse under high pressure, and injecting ECM1 or empty plasmid.

[0033] Figure 2 : Serum HBsAg levels of different mice (injected with ECM1 or empty plasmid).

[0034] Figure 3 : Serum HBeAg levels of different mice (injected with ECM1 or empty plasmid).

[0035] Figure 4 : Serum HBsAb levels of different mice (injected with ECM1 or empty plasmid).

[0036] Figure 5 : Schematic diagram of constructing an HBV model by using liver stellate cell-specific knockdown of ECM1 (Lrat cre ECM1 fl / + ) and control (Lrat cre ECM1 + / + ) mice.

[0037] Figure 6 : Different mice (Lrat cre ECM1 fl / + or Lrat cre ECM1+ / + Serum HBsAg level.

[0038] Figure 7 Serum HBsAg level in different mice (Lrat cre ECM1 fl / + Lrat cre ECM1 + / + Intraliver HBcAg immunohistochemical staining results.

[0039] Figure 8 Serum HBsAb level in different mice (Lrat cre ECM1 fl / + Lrat cre ECM1 + / + Serum HBsAb level in different mice (Lrat

[0040] Figure 9 Serum HBsAb level in different mice (Lrat cre ECM1 fl / + Lrat cre ECM1 + / + Number of mice with serum HBsAb level higher than 0.5 mIU / mL (green) or lower than 0.5 mIU / mL (gray) in mice.

[0041] Figure 10 Schematic diagram of constructing HBV mouse model by overexpressing ECM1 using recombinant adeno-associated virus in Lrat

[0042] Figure 11 Serum HBsAg level in different mice (overexpressing ECM1 or control).

[0043] Figure 12 Serum HBeAg level in different mice (overexpressing ECM1 or control).

[0044] Figure 13 Schematic diagram of constructing HBV mouse model by exogenous supplement of ECM1 recombinant protein in Lrat

[0045] Figure 14 Serum HBsAg level in different mice (exogenous supplement of ECM1 recombinant protein or control).

[0046] Figure 15 Serum HBeAg level in different mice (exogenous supplement of ECM1 recombinant protein or control). DETAILED DESCRIPTION

[0047] The following experimental examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application. Any person under the inspiration of the present application or the combination of the present application with other prior art features will fall within the scope of protection of the present application.

[0048] In the following experimental examples, the specific experimental steps or conditions are not specified, and can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0049] Experimental Example 1: Effect of ECM1 intervention on HBV mice

[0050] The experimental process is as follows:

[0051] 1. Experimental method

[0052] 1.1. HBV mouse model construction and dynamic blood sampling

[0053] (1) Select 6-8 week old C57BL / 6 mice (purchased from Guangdong Medical Animal Experimental Center), and weigh and record each mouse before modeling;

[0054] (2) Dilute the pAAV-HBV 1.2 plasmid (pAAV-HBV 1.2 plasmid, see the literature "Li-Rung Huang, Hui-Lin Wu, Pei-Jer Chen, et al., An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection[J], Proc Natl Acad Sci U S A 2006.103(47) 17862-7.") with physiological saline according to the volume of 10% of the weight of each mouse, to obtain pAAV-HBV 1.2 working solution, and each mouse is injected with 6 μg plasmid through high-pressure tail vein to model HBV;

[0055] (3) On 1, 4, 7, 10, 14 days after modeling, the HBV mice obtained by modeling were taken blood from the orbital vein, and the specific operation was as follows: after the HBV mice were anesthetized with isoflurane, the orbital vein blood was collected by using a capillary blood collection needle, 100 μL of peripheral blood was collected, and the blood was vertically placed for 2 hours; 6000 rpm room temperature centrifugation for 10 min, the supernatant was transferred to a new 1.5 mL EP tube; repeat the above centrifugation and absorb the supernatant; the separated serum was divided into EP tubes, numbered and labeled, and stored in a -20°C refrigerator (see Figure 1 ).

[0056] 1.2, Constructing intrahepatic overexpression mouse model by using ECM1 plasmid

[0057] According to the dose of 10 μg ECM1 plasmid or empty plasmid (ECM1 plasmid or empty plasmid is constructed by Guangzhou Dahong Biological Technology Co., Ltd., wherein the ECM1 plasmid is obtained by inserting the ECM1 coding gene into the pcDNA3.1 plasmid, and the sequence number of the ECM1 coding gene is NM_007899.5, and the empty plasmid is the pcDNA3.1 plasmid) injected into each mouse, the ECM1 plasmid or the empty plasmid is added into the pAAV-HBV1.2 plasmid working solution prepared in the method of 1.1 above, and high-pressure tail vein injection and dynamic orbital vein blood collection are performed (see Figure 1 ).

[0058] 1.3, Constructing hepatostellate cell-specific knockdown ECM1 mouse by using CRISPR / Cas9 technology

[0059] C57BL / 6J mice were used as editing objects, and CRISPR / Cas9 technology was used for gene editing, exon 2-8 of Ecm1-202 transcript (Transcript ID: ENSMUST00000117507.10) was selected as the flox region, and C57BL / 6J- Ecm1 em1 (flox) Smoc mouse, i.e. Lrat cre Ecm1 + / + mouse (purchased from Shanghai Southern Model Organisms Technology Co., Ltd.). The Lrat cre Ecm1 + / + mouse was crossed with the Lrat-Cre mouse (purchased from Shanghai Southern Model Organisms Technology Co., Ltd.) expressing Cre recombinase driven by lecithin retinol acyltransferase (Lrat) promoter to produce hepatostellate cell-specific Ecm1 gene knockout mouse, i.e. Lrat cre ECM1 fl / + mouse. The Lrat cre Ecm1 + / + mouse and the Lratcre ECM1 fl / + Mice were constructed according to the method of 1.1 above and dynamic orbital venous blood sampling was performed (see experimental procedure Figure 5 ).

[0060] 1.4, Constructing an intrahepatic overexpression ECM1 model using recombinant adeno-associated virus

[0061] (1) Propagate a sufficient number of Lrat cre ECM1 fl / + Mice were raised in SPF level animal room, and the state and reproductive situation of mice were observed daily;

[0062] (2) When the mice grew to 4 weeks old, 100 μL (containing 2 x 10 11 v.g virus) of the corresponding recombinant adeno-associated virus AAV6-ECM1 / AAV6-vector (AAV6-ECM1 and AAV6-vector were purchased from Shanghai Heyuan Biotechnology Co., Ltd., wherein the preparation method of AAV6-ECM1 was as follows: after inserting the ECM1 coding gene into GL3009 pAAV-TBG-GdGreen-WPRE empty vector, the vector was transfected into 293T cells, and the virus was collected from the culture supernatant after culturing the cells; the preparation method of AAV6-vector was as follows: GL3009 pAAV-TBG-GdGreen-WPRE empty vector was transfected into 293T cells, and the virus was collected from the culture supernatant after culturing the cells) was injected through the tail vein;

[0063] (3) When the mice grew to 6 weeks old, HBV mouse models were constructed according to the method of 1.1 above and dynamic orbital venous blood sampling was performed (see experimental procedure Figure 10 ).

[0064] 1.5, Exogenous supplementation of ECM1 to HBV mouse models using ECM1 recombinant protein

[0065] (1) Propagate a sufficient number of Lrat cre ECM1 fl / + Mice were raised in SPF level animal room, and the state and reproductive situation of mice were observed daily;

[0066] (2) When the mice grew to 6-8 weeks old, HBV mouse models were constructed according to the method of 1.1 above, and 1 day after modeling, the mice were injected with ECM1 recombinant protein (the amino acid sequence of ECM1 recombinant protein was SEQ ID NO. 1, purchased from Shanghai Sunway Biotech Co., Ltd.) at a dose of 5 mg / kg / time through the tail vein, and the injection was performed once every three days; the control mice were injected with the same dose of normal saline through the tail vein every day;

[0067] (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 ).

[0068] The preparation method of the ECM1 recombinant protein is as follows:

[0069] 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 coli BL21(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.

[0070] 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.

[0071] The purification was performed by two-step chromatography: 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 effluent was collected, and the target protein was eluted with PBS buffer containing 500 mM imidazole. SDS-PAGE verified that the target band was concentrated in the elution fraction, and the affinity purified product was obtained. 2. Gel filtration chromatography: the affinity purified product was further purified by gel filtration chromatography on a Superdex 200 column, and the target protein was eluted with PBS buffer containing 8 M urea, and the target peak fraction was collected to remove impurities. SDS-PAGE verified that the target band was concentrated in the elution fraction, and the purified protein was obtained.

[0072] The purified protein was replaced by stepwise dialysis into PBS buffer to remove urea, and the refolding product was obtained; the refolding product was first ultrafiltrated and concentrated, then sterilized through a 0.22 μm filter membrane, and finally endotoxin detection showed a level <1 EU / μg (in line with the control standard), and finally a purity >90% of the ECM1 recombinant protein was obtained, which was stored at -80°C.

[0073] 1.6, Detection of HBV serological indicators in mouse serum

[0074] 1.6.1, HBsAg detection

[0075] Using a hepatitis B virus surface antigen diagnostic kit (purchased from Beijing Wantai), the mouse serum HBsAg was semi-quantitatively detected according to the principle of double antibody sandwich enzyme-linked immunosorbent assay, the level of HBsAg in the positive control of the kit was detected by electrochemical immunochemiluminescence analyzer Architect system, and the standard curve was drawn by diluting the positive control by multiples. The specific process is as follows:

[0076] (1) Sample addition: after diluting the mouse serum sample to the appropriate concentration, according to the experimental sample layout, add 100 μL of the sample to be tested and the standard diluent to the corresponding wells, and shake well;

[0077] (2) Incubation: after sealing the plate with a sealing film, incubate at 37°C for 60 min;

[0078] (3) Enzyme addition: after incubation, add 50 μL of enzyme-labeled reagent (HBsAb-HRP) to each well, shake well;

[0079] (4) Incubation: after sealing the plate with a sealing film, incubate at 37°C for 30 min;

[0080] (5) Wash solution preparation: dilute the concentrated wash solution with deionized water to 1×;

[0081] (6) Washing: tear off the sealing film, discard the liquid in the wells, shake dry, wash with 200 μL / well of wash solution for 5 times, each time for 2 min, and dry at the end of the last time.

[0082] (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;

[0083] (8) Termination: Add 50 μL of termination solution to each well;

[0084] (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.

[0085] 1.6.2 HBeAg Detection

[0086] 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:

[0087] (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.

[0088] (2) Add enzyme: After incubation, add 50 μL of enzyme-labeled reagent (HBeAb-HRP) to each well and shake to mix;

[0089] (3) Incubation: After sealing with the sealing film, incubate at 37°C for 30 min;

[0090] (4) Preparation of washing solution; dilute the concentrated washing solution with deionized water to 1×;

[0091] (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.

[0092] (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;

[0093] (7) Termination: Add 50 μL of termination solution to each well;

[0094] (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.

[0095] 1.6.3 HBsAb Detection

[0096] The HBsAb in mouse serum was semi-quantified by using the HBsAb detection kit (Beijing Wantai) according to the principle of double antigen sandwich enzyme-linked immunosorbent assay. The HBsAb level of the positive control in the kit was quantitatively detected by Roche cobas e601 electrochemiluminescence method, and the standard curve was drawn by diluting the positive control by ratio. The specific process is as follows:

[0097] (1) Sample addition: after the mouse serum sample was diluted to an appropriate concentration, the test sample and standard diluent were added to the corresponding wells according to the experimental sample layout, 50 μL each, and shaken to mix;

[0098] (2) Enzyme addition: after incubation, 50 μL of enzyme-labeled reagent (HBsAg-HRP) was added to each well, and shaken to mix;

[0099] (3) Incubation: after the plate was sealed with a sealing film, it was incubated at 37°C for 30 min;

[0100] (4) Wash solution preparation: the concentrated wash solution was diluted to 1× with deionized water;

[0101] (5) Washing: the sealing film was torn off, the liquid in the wells was discarded, and the wells were spun dry. The wash solution was washed 5 times, each time for 2 min, 200 μL / well. The last time was spun dry;

[0102] (6) Color development: 50 μL of color developing agent A and B was added to each well, shaken to mix, and incubated at 37°C for 15 min in the dark;

[0103] (7) Termination: 50 μL of termination solution was added to each well;

[0104] (8) Plate reading: the plate bottom was wiped clean, and the plate was read within 5 min. When reading the plate, the OD value of each well was detected at a double wavelength of 450 nm / 630 nm, and the blank well was used for correction.

[0105] 1.6.4, HBcAg staining by immunohistochemistry

[0106] The specific process is as follows:

[0107] (1) Sectioning: the embedded paraffin tissue was trimmed, and the tissue after cutting to 4 μm was placed on a non-falling glass slide;

[0108] (2) Baking: the oven was preheated to 62°C, and the glass slide was placed in the oven for 2 h to completely dissolve the paraffin;

[0109] (3) De-waxing and hydration: in a fume hood, the section was sequentially soaked in dimethylbenzene 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 distilled water for 5 min;

[0110] (4) Blocking endogenous peroxidase: The sections were immersed in 3% (w / v, g / 100 mL) hydrogen peroxide solution at room temperature (25°C) for 15 min to inactivate endogenous peroxidase; PBS buffer was washed for 2 min x 3 times;

[0111] (5) Incubation of primary antibody: The immunohistochemical pen was used to draw a circle at a distance of 3 mm from the tissue periphery; 100 μL of hepatitis B virus core antigen (HBcAg) working solution (purchased from Zhijia Jinqiao) was added to the tissue surface to cover the section tissue, and incubation was performed at 37°C for 60 min; PBS buffer was washed for 2 min x 3 times;

[0112] (6) Incubation of secondary antibody: Enzyme-labeled goat anti-rabbit / mouse IgG polymer (purchased from Genetech, model GK600711) was added to the tissue surface to cover the section tissue, and incubation was performed at 37°C for 20 min; PBS buffer was washed for 2 min x 3 times;

[0113] (7) DAB color development: C1 and C2 liquids of the DAB color development kit (purchased from Genetech, model GK600711) were prepared into a color development liquid at a ratio of 1:50 (note to avoid light), 50 μL of the color development agent was added to each section, and timing was started, and observation was performed under a general optical microscope, and after specific staining was observed and the degree was moderate, the color development reaction was terminated by placing in tap water; the remaining sections were subjected to color development at the same time;

[0114] (8) Hematoxylin nuclear staining: After washing with tap water, hematoxylin staining solution was added to the section tissue for incubation for 8 min, and the degree of tissue staining was observed under a microscope, and the staining was terminated with tap water, and washing was performed with tap water for 5 min; 1% (v / v) hydrochloric acid-containing alcohol was used for differentiation for 23 s, and washing was performed with tap water for 2 min x 2 times; warm water was used for counterstaining for 5 min;

[0115] (9) Dehydration and transparency: The sections were immersed in 75% (v / v) ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene, respectively, for 1 min, and gradual dehydration and transparency were performed;

[0116] (10) Mounting: The glass slide was placed in a fume hood for natural air drying; neutral resin mounting was performed, and an appropriate amount of neutral resin was added to the tissue, and a cover glass was placed thereon, and observation was performed to prevent the generation of bubbles, and room temperature storage was performed.

[0117] 2. Experimental results

[0118] The serum HBsAg, HBeAg, and HBsAb levels of different mice and the results of HBcAg immunohistochemical staining in the liver of different mice are shown in Figure 2~Figure 4 , Figure 6~Figure 9 , Figure 11~Figure 12 and Figure 14~Figure 15 . From Figure 2~Figure 4 ,Figure 6~Figure 9 、 Figure 11~Figure 12 and Figure 14~Figure 15 It is known that, compared with the high-pressure tail vein injection of the control plasmid group, injection of ECM1 plasmid significantly reduces the serum levels of HBsAg and HBeAg in mice overexpressing ECM1 in the liver, and significantly increases the level of HBsAb; using Lrat cre and ECM1 fl / fl The liver stellate cell-specific knockdown ECM1 mice were generated by crossing the mice, and it was found that, compared with wild-type mice, the serum and liver HBsAg levels in ECM1-specific knockdown mice in the liver were significantly increased, and the serum HBsAb level was significantly reduced; injection of adeno-associated virus to replenish ECM1 expression in the liver stellate cells in the liver stellate cell-specific knockdown ECM1 mice significantly reduced the serum levels of HBsAg and HBeAg compared with mice injected with control adeno-associated virus; injection of ECM1 recombinant protein in the liver stellate cell-specific knockdown ECM1 mice significantly reduced the serum levels of HBsAg and HBeAg compared with mice injected with normal saline. The above results suggest that overexpression of ECM1 in the liver or exogenous supplementation of ECM1 can significantly promote the clearance of HBV virus and the production of corresponding antibodies. This ECM1-based treatment strategy provides a higher possibility for the functional cure of HBV infection.

[0119] Obviously, the above experimental examples are only examples for clarity and do not limit the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. The use of extracellular matrix protein 1 and / or extracellular matrix protein 1 promoters in the preparation of medicaments for treating hepatitis B virus infection, characterized in that, The promoter of extracellular matrix protein 1 is a carrier that overexpresses extracellular matrix protein 1; the treatment of hepatitis B virus infection includes promoting the clearance of hepatitis B virus and / or promoting the production of hepatitis B virus antibodies; 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.

2. The application as described in claim 1, 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.

3. The application as described in claim 2, 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.

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