Application of host factor TMEM40 in regulating and controlling hepatitis B virus antigen expression and resisting HBV

Abstract

The invention discloses an application of a host factor TMEM40 in regulation and control of hepatitis B virus antigen expression and HBV resistance. According to the invention, a series of novel host factors for regulating and controlling HBsAg expression are identified by utilizing a protein spectrum, and by screening the host factors, the TMEM40 shows a strong inhibition effect on hepatitis B virus surface antigens in a cell model, and the effect of the TMEM40 gene in inhibiting hepatitis B virus is provided for the first time. Researches show that TMEM40 is expected to be used for developing a novel therapeutic drug for inhibiting hepatitis B virus surface antigen, and a new strategy is provided for HBV functional cure.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine technology, and in particular to the application of host factor TMEM40 in regulating hepatitis B virus antigen expression and anti-HBV. BACKGROUND

[0002] Hepatitis B (referred to as hepatitis B) is an infectious disease caused by hepatitis B virus (HBV), which seriously endangers the health of people all over the world. If hepatitis B is not controlled, it can develop into serious end-stage liver disease such as cirrhosis and hepatocellular carcinoma, endangering the lives of patients.

[0003] The radical cure strategy for chronic viral hepatitis B is to completely eliminate hepatitis B virus in the liver. The current common method is to inhibit the synthesis of HBV DNA by stimulating the production of antiviral enzymes through immunomodulation; or to reduce the production of HBV virus particles by directly inhibiting the reverse transcription activity of HBV DNA polymerase, although these drugs can block the replication of HBV and avoid further damage to liver function in patients, and alleviate the disease, but it is still impossible to inactivate the covalently closed circular DNA (cccDNA) of the original transcription template of HBV. Therefore, it is extremely urgent to find new drug targets and develop new anti-hepatitis B virus drugs.

[0004] During chronic infection, the body's difficulty in eliminating HBV can be caused by the excessive secretion of subviral particles (Subviral particle, SVP, also known as hepatitis B surface antigen (HBsAg or HBs) particles). SVP is considered to be a decoy for the immune system, which can deplete B and T cell responses, thereby contributing to persistent infection. Clinically, the disappearance of HBsAg or the appearance of anti-HBsAg antibodies marks the durable immune control or functional cure of hepatitis B virus infection. Therefore, it is of great significance to achieve this therapeutic goal by developing antiviral drugs targeting HBsAg.

[0005] At present, a variety of candidate molecules targeting HBsAg have been found, including natural products, synthetic compounds, siRNA, NAP, etc. Among them, siRNA and NAP have undergone clinical trials, but due to the short half-life of RNA interference and the easy production of mutant escape; the safety of NAP for liver cirrhosis is not clear, and other drugs targeting HBsAg still need to be developed. It is of great significance to explore the mechanism of drug action on HBsAg for the development of new drugs.

[0006] Host factors are proteins in host cells that participate in the regulation of the transcription and translation of viral genomes after hepatitis B virus infection, thereby affecting viral replication. These host factors can promote viral entry into host cells, assembly and release of progeny virus particles by interacting with viral proteins. The study of the role of host factors in hepatitis B virus infection helps to understand the interaction between virus and host, and provides clues for the development of new antiviral strategies. Since host factors are involved in various stages of the life cycle of hepatitis B virus, the interaction between hepatitis B virus and host factors has been studied more, including identifying the receptor for viral entry into cells, identifying factors that affect cccDNA synthesis, and identifying host factors that affect cccDNA transcription. The focus of these studies is often on the impact on viral replication, that is, the progeny viral DNA, and less on the impact on surface antigens, and there is a lack of systematic screening of host factors related to surface antigen expression.

[0007] TMEM40 (Transmembrane protein 40, transmembrane protein 40) is a 23kDa transmembrane protein mainly involved in intracellular material transport and signal transduction. Recent studies have found that its abnormal expression is related to the progression of tumors such as tongue squamous cell carcinoma. The protein encoded by the TMEM40 gene belongs to the mitochondrial carrier family and is involved in intracellular material transport. SUMMARY

[0008] The purpose of the present application is to solve the above problems, and provide an application of host factor TMEM40 in regulating hepatitis B virus antigen expression and anti-HBV.

[0009] In order to achieve the purpose of the present application, the technical scheme adopted by the present application is:

[0010] The first aspect of the present application provides an application of a host factor or its coding gene in regulating hepatitis B virus replication or the expression level of hepatitis B virus antigen in a host cell, wherein the host factor is TMEM40.

[0011] The hepatitis B virus antigen includes hepatitis B surface antigen HBsAg.

[0012] The host factor inhibits the expression level of hepatitis B virus antigen in the host cell.

[0013] The host factor reduces the expression level of HBV RNA and inhibits HBV DNA replication.

[0014] The second aspect of the present application provides an application of a host factor or its expression promoter in preparing an anti-hepatitis B virus or anti-hepatitis B virus antigen drug, wherein the host factor is TMEM40.

[0015] The expression promoter is a TMEM40 overexpression virus or overexpression plasmid; the hepatitis B virus antigen includes hepatitis B surface antigen HBsAg.

[0016] TMEM40 inhibits HBV by activating JAK-STAT signaling pathway.

[0017] The third aspect of the present application provides application of a host factor or its coding gene as a drug target in screening of drugs against hepatitis B virus or inhibiting hepatitis B virus antigen, wherein the host factor is TMEM40.

[0018] The drug promotes expression of TMEM40, thereby inhibiting expression level of hepatitis B virus antigen in host cells, reducing expression level of HBV RNA, and inhibiting HBV DNA replication; the hepatitis B virus antigen includes hepatitis B surface antigen HBsAg.

[0019] The fourth aspect of the present application provides a product, and an active ingredient of the product is TMEM40 protein; the function of the product is any one of the following:

[0020] (1) inhibiting expression level of hepatitis B virus antigen in host cells; the hepatitis B virus antigen includes hepatitis B surface antigen HBsAg;

[0021] (2) preventing or treating HBV infection;

[0022] (3) inhibiting expression level of HBV RNA;

[0023] (4) inhibiting invasion and replication of HBV.

[0024] Preferably, the product is a drug.

[0025] The beneficial effects of the present application are: by studying the mechanism of compound D4, a series of new host factors regulating HBsAg expression are identified by protein spectrum, by screening these host factors, it is found that TMEM40 shows strong inhibitory effect on hepatitis B virus surface antigen in cell model, which provides the role of TMEM40 gene in inhibiting hepatitis B virus for the first time. The present application finds that compound D4 can inhibit HBsAg secretion by up-regulating TMEM40 expression, overexpression of TMEM40 can dose-dependently inhibit the levels of HBV 3.5 kb and total RNA in two kinds of cells, and can inhibit the level of HBV DNA, and further verification is carried out by knockdown experiment, after knocking down TMEM40, the levels of HBV 3.5 kb and total RNA are increased, which indicates that TMEM40 can play an anti-HBV virus effect by inhibiting HBV RNA; TMEM40 can inhibit HBV by activating JAK-STAT signaling pathway; the Q-PCR results of mouse experiment show that overexpression of TMEM40 can significantly reduce the levels of HBV DNA and HBV RNA in liver tissue, and the Western Blot results show that overexpression of TMEM40 can inhibit HBsAg in mouse liver. The present application shows that TMEM40 is expected to be used for developing a new type of therapeutic drug for inhibiting hepatitis B virus surface antigen, and provides a new strategy for HBV functional cure. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The compound D4 target point screening is shown: (A, B) transcriptional genomic gene analysis statistics of HepG2.2.15 cells after compound D4 acts; (C) transcription factor PCR array result figure shows the differential genes produced after compound D4 acts on HepG2.2.15 cells; (D) luciferase reporter gene assay of 19 up-regulated genes on the influence of HBsAG level in HepG2 cells; (E) ELISA method detects the influence of 19 up-regulated genes on the influence of HBsAG level in HepG2 cells.

[0027] Figure 2Showed whether TMEM40 is the target of the effect of compound D4 on HepG2.2.15 cells: (A) Western blot detected the effect of compound D4 on the RNA and protein levels of TMEM40 in HepG2.2.15 cells; (B) ELISA method detected the effect of overexpression of TMEM40 on the level of HBsAG in HepG2.2.15 cells; (C) Western blot detected the effect of overexpression of TMEM40 on the level of HBsAG in HepG2.2.15 cells; (D) Knockdown of TMEM40 in HepG2.2.15 cells; (E) ELISA method detected the effect of knockdown of TMEM40 on the level of HBsAG in HepG2.2.15 cells; (F) Western blot detected the effect of knockdown of TMEM40 on the level of HBsAG in HepG2.2.15 cells; (G) ELISA method detected the effect of adding compound D4 and knockdown of TMEM40 at the same time on the level of HBsAG in HepG2.2.15 cells; (H) Western blot detected the effect of adding compound D4 and knockdown of TMEM40 at the same time on the level of HBsAG in HepG2.2.15 cells; (I) ELISA method detected the effect of overexpression of TMEM40 on the level of HBV 3.5 kb and total RNA in HepG2.2.15 cells; (J) ELISA method detected the effect of overexpression of TMEM40 on the level of HBV DNA in HepG2.2.15 cells; (K) ELISA method detected the effect of knockdown of TMEM40 on the level of HBsAG in HepG2.2.15 cells; (L) ELISA method detected the effect of knockdown of TMEM40 on the level of HBV DNA in HepG2.2.15 cells.

[0028] Figure 3 Showed the effect of TMEM40 activating JAK-STAT pathway on HBV: (A) ELISA method detected the effect of adding Ruxolitinib and overexpression of TMEM40 at the same time on the level of HBsAG in HepG2.2.15 cells; (B) Western blot detected the effect of adding Ruxolitinib and overexpression of TMEM40 at the same time on the level of HBsAG in HepG2.2.15 cells; (C) Western blot detected the effect of overexpression of TMEM40 on the phosphorylation level of JAK1 and STAT1 in HepG2 cells; (D) Immunofluorescence image of staining IRF9 in HepG2 cells overexpressing TMEM40; (E) Detected the level of translocation of IRF9 to the nucleus in HepG2 cells overexpressing TMEM40; (F) Luciferase reporter gene detected the level of translocation of IRF9 to the nucleus in HepG2 cells overexpressing TMEM40.

[0029] Figure 4 The following data were presented: (A) Effect of TMEM40 overexpression on HBV levels in AML12 cells as detected by ELISA; (B) Effect of TMEM40 overexpression on HBsAG levels in AML12 cells as detected by Western blot; (C) Effect of TMEM40 overexpression on HBsAg levels in mouse liver tissue as detected by ELISA; (D) Effect of TMEM40 overexpression on HBsAg levels in mouse liver tissue as detected by luciferase reporter gene assay; (E) Dissection of mouse liver tissue on day 7; (F) Effect of TMEM40 overexpression on HBV DNA levels in liver tissue as detected by Q-PCR; (G) Effect of TMEM40 overexpression on HBV RNA levels in liver tissue as detected by Q-PCR; (H) Effect of TMEM40 overexpression on HBsAG levels in liver tissue as detected by Western blot. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0032] The main reagent materials in this invention are sourced from:

[0033]

[0034] Plasmids: pCH9 / 3091 plasmid, which contains the HBV1.1 ploid genome driven by the CMV promoter, was stored in our laboratory. pAAV-CMV-EGFP plasmid was also stored in our laboratory. pAAV-Vector, pAAV-TMEM40, and pAAV-HBV1.2 plasmids were constructed by our research team in previous studies.

[0035] Cells: The human liver cancer cell lines HepG2 and HepG2.2.15 were both preserved by our research group.

[0036] Immortalized mouse liver cells AML12: US Type Culture Collection Center.

[0037] Compound D4 and compound D3 are compounds 1-4 and 1-3 in the patent CN2024111747002 (publication number CN 119055636 A) previously filed by our research team.

[0038] Compound D4: 4-amino-5-benzoyl-2-[(3,4-dichlorophenyl)amino]-N-(4-ethoxyphenyl)thiophene-3-carboxamide, CAS No.: 702655-24-1, molecular formula: C 26 H 21 The chemical structural formula of Cl2N3O3S is shown below:

[0039]

[0040] Compound D3: 4-amino-5-benzoyl-2-[(2-ethoxyphenyl)amino]-N-(1,2,3,4-tetrahydronaphthyl-1-yl)thiophene-3-carboxamide, with the molecular formula C 30 H 29 The chemical structural formula of N3O3S is shown below:

[0041]

[0042] C57BL / 6 mice were purchased from the Experimental Animal Center of Chongqing Medical University. The animals were housed in a specific pathogen-free (SPF) clean environment at a temperature maintained at (23±0.5)℃ and a relative humidity maintained at (50±5)%. The animals had free access to food and water during the rearing period. The animal experiments were approved by the university's Animal Experiment Ethics Review Committee.

[0043] The amplification primer sequences used in the embodiments of this invention are shown in Table 1 below:

[0044] Table 1

[0045]

[0046] The siRNA sequences used for gene knockdown in this invention embodiment are shown in Table 2:

[0047] Table 2

[0048]

[0049] The experimental methods involved in this invention are as follows:

[0050] 1. Cell Culture and Transfection Methods: HepG2, HepG2.2.15, HepG2-NTCP, and HepAD38 cell lines were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO2 using standard methods. Plasmids were transfected according to the Lipo8000 protocol. TM Transfection according to the instructions for the transfection reagent.

[0051] 2. HBV virus collection and concentration: Collect 500 mL of HepAD38 cell culture supernatant, centrifuge at 2000 g for 10 min at 4 °C, filter the supernatant through a 0.45 μm bacterial filter into a new sterile collection bottle, add 10 mL of 30% PEG8000 to every 40 mL of virus supernatant, seal the tube with sealing film, and incubate overnight at 4 °C. The next day, centrifuge at 4000 g for 35 min at 4 °C, discard the supernatant, add 400 μL of Lopti-MEM to dissolve the precipitate, gently pipette to mix, and aliquot into EP tubes and store at -80 °C.

[0052] 3. HBV infection of HepG2-NTCP cells: HepG2-NTCP cells were pretreated with pretreatment medium (2% DMSO + 2 μg / mL doxycycline). After 24 h, the pretreatment medium was discarded and replaced with infection medium containing HBV virus (4% PEG8000, 2% DMSO, and 2 μg / mL doxycycline), and the cells were gently inverted to mix. After 24 h of infection, the medium was discarded, the cells were washed three times with PBS, and the cells were re-cultured with the pretreatment medium.

[0053] 4. HBsAg detection: The HBsAg level in the cell culture supernatant was detected according to the instructions of the enzyme-linked immunosorbent assay kit (Kehua).

[0054] 5. Detection of HiBiT luciferase: Detect the fluorescence value in the cell culture supernatant according to the Nano-Glo® HiBiT Lytic Detection System (Promega) instructions.

[0055] 6. CCK-8 assay for cytotoxicity: The effect of the drug on cell viability was detected according to the CCK-8 kit (Li Ji) instructions.

[0056] 7. Western blot analysis: 72 h after cell transfection, wash twice with PBS. Add 50 μL RIPA lysis buffer and protease inhibitor to each well (for 24-well plates), lyse on ice for 30 min, extract total cell protein, and determine protein concentration using the BCA method. Denature an equal volume of total cell protein at 100℃ for 10 min. Separate proteins in 12.5% ​​SDS-PAGE, transfer to a membrane at 200 mA on ice for 2 h, block with skim milk for 1 h, then replace with primary antibody, and incubate overnight at 4℃ on a shaker. The next day, wash three times with TBST for 10 min each time. Then add secondary antibody, incubate on a shaker at room temperature for 1 h, wash three times with TBST, and develop with ECL.

[0057] 8. RNA extraction and real-time quantitative PCR: (Taking 12-well plate cells as an example) Wash cells twice with PBS, add 1 mL of Trizol lysis buffer, lyse at room temperature for 10 min, add 200 μL of chloroform, vortex thoroughly, carefully transfer the supernatant to a new EP tube, add an equal volume of isopropanol, vortex thoroughly, centrifuge at 13000g for 10 min at 4℃, discard the supernatant, add 500 μL of 75% ethanol to wash the precipitate, centrifuge at 13000g for 10 min at 4℃, discard the supernatant and dry the RNA precipitate, dissolve in 20 μL of water, measure the concentration, and use a reverse transcription kit to remove genomic DNA and synthesize cDNA. After the qPCR reaction, export the results and use... The method calculates the relative expression level.

[0058] 9. Construction of overexpression plasmids (taking TMEM40 as an example):

[0059] The human TMEM40 gene sequence involved in plasmid construction is (SEQ ID NO.39).

[0060] Using pCH9 / 3091 plasmid as a template, fragment 1 was amplified with primers F SV40GG2+ R G4S. Fragment 2 was amplified with primers F TMEM40+R TMEM40 using cDNA as a template. Fragments 1 and 2 were then ligated using the Golden Gate cloning method to obtain plasmid TMEM40.

[0061] The PCR reaction system consisted of: 10 ng plasmid as template plasmid, 0.4 μL each of primers F (10 μM) and R (10 μM), 10 μL of 2×PrimeSTAR Max Premix, and sterile ultrapure water to a final volume of 20 μL. Reaction conditions were: 98℃ pre-denaturation for 2 min; 35 cycles of 98℃ for 10 s, 55℃ for 5 s, and 72℃ for 30 s. The amplified fragments were recovered using a gel extraction kit, and their concentration was determined.

[0062] The Golden Gate ligation reaction system consisted of: 1 μL Tango buffer, 1 μL DTT, 1 μL ATP, 0.75 μL BsmBI enzyme, 0.25 μL T7 DNA ligase, and the fragments to be ligated. The amount of each fragment added was calculated based on the Kb*20 / Gel recovery concentration (ng / μL), and sterile ultrapure water was added to bring the total volume to 10 μL. Reaction conditions were: 37℃ for 4 min, 20℃ for 4 min, 20 cycles, followed by 65℃ for 20 min. The Golden Gate ligation product was transformed into TOP10 competent bacteria, plated, and single colonies were picked for culture. The bacterial culture was sequenced for identification. Selected colonies with correct sequencing were extracted to obtain the target overexpression plasmid.

[0063] 10. High-pressure hydrodynamic method for establishing a mouse model: 6-8 week old C57BL / 6J mice weighing 17-22g were randomly divided into Vector and RAB10 groups. A PBS solution containing 15μg plasmid was prepared, with a solution volume of 10% of the mouse's body weight. The mouse tail was fixed using a restraint device, and the tail skin was disinfected with a 75% alcohol swab. The solution was injected into the mouse through the tail vein within 5 seconds.

[0064] 11. Extraction of HBV DNA from mouse serum and liver tissue: Extraction was performed according to the instructions of the Virus Genome Extraction Kit (Magen).

[0065] Example 1: Target Screening and Identification of Compound D4

[0066] 1. Target screening of compound D4

[0067] The aforementioned studies have shown that compound D4 inhibits the production of HBV RNA (see Chinese patent application CN2024111747002, publication number CN119055636A, invention title: Compounds that inhibit HBV transcription and hepatitis B surface antigen and their applications). To further screen its target, we performed transcriptomics analysis and transcription factor PCR array experiments. Compound D4 and a negative control (compound D3, a structural analog with virtually no effect on HBV) were added to HepG2.2.15 cells. After 72 hours of drug treatment, cells were collected, cellular RNA was extracted, and transcriptomics analysis and transcription factor PCR array experiments were performed.

[0068] Transcriptomic analysis showed that when the fold change between the experimental and control groups was greater than 2-fold, 804 genes were upregulated and 1832 genes were downregulated. Figure 1 A). Due to the large number of differentially expressed genes, we increased the fold change factor to select more differentially expressed genes. We then screened for genes based on their expression levels and performed QRT-PCR experiments to verify whether the expression of these differentially expressed genes was consistent with the transcriptomic analysis. A total of 19 differentially expressed genes were identified. Figure 1 B). Transcription factor PCR array is a method for rapid screening of target genes using QRT-PCR experiments. Its 96-well plate contains specific primers for transcription factors and growth factors downstream of cytokine and chemokine signals, providing a total of 90 screenable genes, 4 internal control genes, and 2 blank control wells. Compared to transcriptomics, the number of screenable genes is limited. The results of the transcription factor PCR array experiment showed that, compared to the negative control (compound D3), treatment with compound D4 produced 28 differentially regulated genes, of which 24 were upregulated and 4 were downregulated. Figure 1 C).

[0069] We then used QRT-PCR to verify the results of the transcription factor PCR array experiment and selected 7 genes with significant differences in upregulation (ATF3, EGR1, HEY1, CEBPG, JUN, JUNB, GATA2). Based on the combined results of transcriptomics analysis and transcription factor PCR array experiments, a total of 23 differentially expressed genes were selected. Among them, 19 genes were upregulated: 1-1 (CYP1A1), 1-2 (AREG), 1-3 (ATF3), 1-4 (EGR1), 1-5 (HSPA5), 1-6 (NPRL2), 1-7 (IL23A), 1-8 (RRAD), 1-9 (IQCG), 1-10 (HEY1), 1-11 (CEBPG), 1-12 (ATG4A), 1-13 (JUN), 1-14 (JUNB), 1-15 (GATA2), 1-16 (TMEM40), 1-17 (NPR3), 1-18 (IL-11), and 1-19 (HSPA6). 4 genes were downregulated: 2-1 (PTGDR2), 2-2 (FABP1), 2-3 (APOA5), and 2-4 (AGXT). Subsequently, we constructed overexpression plasmids for these differentially expressed genes and transfected HepG2 cells to verify their effects on HBsAg. The results showed that, compared with the negative control Pac1 group, upregulated genes 1-16 (transmembrane protein 40, TMEM40) significantly inhibited HBsAg. Figure 1 D, 1E).

[0070] 2. Target identification of compound D4

[0071] Therefore, we conducted further research on TMEM40. First, we compared the effects of D4 on TMEM40 with the results of transcriptomics. The results showed that after adding compound D4 to HepG2.2.15 cells, both the RNA and protein levels of TMEM40 were upregulated, consistent with the transcriptomics results. Figure 2 A). Subsequently, TMEM40 was overexpressed in HepG2.2.15 cells, and the results showed that TMEM40 dose-dependently inhibited extracellular and intracellular HBsAg levels ( Figure 2 B, 2C). We also conducted a knockdown experiment, which showed that knocking down TMEM40 increased both extracellular and intracellular HBsAg levels (B, 2C). Figure 2 D, 2E, 2F). Subsequently, while adding compound D4, we knocked down TMEM40, and the results showed that the HBsAg inhibitory effect of compound D4 was weakened (D, 2E, 2F). Figure 2G, 2H). The results in summary indicate that compound D4 inhibits HBsAg by upregulating the expression of TMEM40 protein. We further examined the antiviral effect of TMEM40 on HBV, and the results showed that overexpression of TMEM40 dose-dependently inhibited the levels of HBV 3.5 kb and total RNA in both cell lines, and also inhibited the level of HBV DNA (G, 2H). Figure 2 I, 2J), and further verification was performed by knockdown experiments, which showed that knockdown of TMEM40 increased the levels of HBV 3.5 kb and total RNA (I, 2J). Figure 2 K, 2L). In summary, this indicates that TMEM40 can exert an anti-HBV effect by inhibiting HBV RNA.

[0072] Example 2: The Mechanism of Action of the TMEM40 Gene—TMEM40 Inhibits HBV by Activating the JAK-STAT Pathway

[0073] TMEM40 is a member of the TMEM family and has been reported to be associated with cervical cancer and squamous cell carcinoma of the skin, but there are no studies on its association with hepatitis B or liver cancer. According to literature, TMEM2, a member of the TMEM family, can inhibit hepatitis B virus infection in HepG2 and HepG2.2.15 cells by activating the JAK-STAT signaling pathway. Therefore, we investigated whether TMEM40 exerts its effect through the same pathway. First, we added the JAK1 inhibitor (Ruxolitinib) to HepG2.2.15 cells overexpressing TMEM40. The results showed that the HBsAg inhibitory effect of TMEM40 was weakened. Figure 3 (A and 3B) indicates that TMEM40 may inhibit HBV through the JAK-STAT pathway. Subsequent overexpression of TMEM40 in HepG2 cells and Western blot results showed that TMEM40 overexpression upregulated the phosphorylation levels of JAK1 and STAT1 ( Figure 3 C); Immunofluorescence results showed that overexpression of TMEM40 enhanced the translocation of IRF9 to the nucleus ( Figure 3 This was further confirmed by luciferase reporter gene assays (D, 3E). Figure 3 F). In summary, overexpression of TMEM40 can inhibit HBV by activating the JAK-STAT signaling pathway.

[0074] Example 3: Anti-HBV Effects of TMEM40 in Mice In Vitro and In Vitro - TMEM40 exerted HBV inhibitory effects in mice both in vivo and in vitro.

[0075] Subsequently, we investigated the effects of TMEM40 in mice. First, we examined the inhibitory effect of TMEM40 on HBsAg in α-mouse liver 12 (AML12) cells in vitro. The results showed that overexpression of TMEM40 in AML12 cells inhibited intracellular and extracellular HBsAg levels (…). Figure 4 (A and 4B).

[0076] Next, we verified the in vivo anti-HBV effect of TMEM40. Six- to eight-week-old male C57BL / 6 mice, weighing 17-22 g, were randomly divided into two groups: a TMEM40 group and a Vector group, with six mice in each group. Each mouse was injected via tail vein using a high-pressure hydrodynamic method with 10 μg pAAV-Vector or pAAV-TMEM40 + 5 μg pAAV-HBV1.2, with an injection volume (mL) equal to 10% of the mouse's body weight. Blood samples were collected from the orbital sinus on days 3, 5, and 7 post-injection to measure HBsAg and HBV DNA levels. On day 7, liver tissue sections were collected for pathological examination, including HE staining and HBsAg immunohistochemical staining. HBV DNA and HBV RNA in the liver tissue were also measured. The results showed that TMEM40 significantly reduced HBsAg levels in peripheral blood in the mouse hydrodynamic HBV replication model. Figure 4 C), and can also inhibit HBV DNA replication ( Figure 4 D). The liver tissue obtained from mouse dissection on day 7 showed no significant differences or lesions. Figure 4 E). Q-PCR results showed that overexpression of TMEM40 significantly reduced the levels of HBV DNA and HBV RNA in liver tissue. Figure 4 F and 4G); meanwhile, Western blotting results showed that overexpression of TMEM40 inhibited intrahepatic HBsAg (F and 4G); Figure 4 H).

Claims

1. Use of a host factor or its encoding gene in regulating the replication of hepatitis B virus or the expression level of hepatitis B virus antigen in a host cell, wherein the host factor is TMEM40.

2. Use according to claim 1, characterized in that: The hepatitis B virus antigen comprises hepatitis B surface antigen HBsAg. The host factor inhibits the expression level of hepatitis B virus antigen in a host cell.

3. Use according to claim 1, characterized in that: The host factor reduces the expression level of HBV RNA and inhibits the replication of HBV DNA.

4. Use of a host factor or its expression promoter in preparing a medicine for resisting hepatitis B virus or inhibiting hepatitis B virus antigen, wherein the host factor is TMEM40.

5. Use according to claim 4, characterized in that: The expression promoter is a virus or plasmid overexpressing TMEM40; the hepatitis B virus antigen comprises hepatitis B surface antigen HBsAg.

6. Use according to claim 4, characterized in that: TMEM40 inhibits HBV by activating JAK-STAT signaling pathway.

7. Use of a host factor or its encoding gene as a drug target in screening a medicine for resisting hepatitis B virus or inhibiting hepatitis B virus antigen, wherein the host factor is TMEM40.

8. Use according to claim 7, characterized in that: The medicine promotes the expression of TMEM40, thereby inhibiting the expression level of hepatitis B virus antigen in a host cell, reducing the expression level of HBV RNA and inhibiting the replication of HBV DNA; the hepatitis B virus antigen comprises hepatitis B surface antigen HBsAg.

9. A product, whose active ingredient is TMEM40 protein, and whose function is any one of the following: (1) inhibiting the expression level of hepatitis B virus antigen in a host cell; the hepatitis B virus antigen comprises hepatitis B surface antigen HBsAg; (2) preventing or treating HBV infection; (3) inhibiting the expression level of HBV RNA; (4) inhibiting the invasion and replication of HBV.

10. The product of claim 9, wherein: The product is a medicine.

Images