CN114452374A - Application of ubiquitin-binding protein UBXN7 in preparation of anti-HBV drugs - Google Patents

Abstract

The invention provides application of ubiquitin-binding protein UBXN7 in preparing anti-HBV drugs. According to the invention, researches show that after HBV infects cells, UBXN7 is promoted to be ubiquitinated and degraded through a proteasome pathway, so that UBXN7 is degraded; over-expression of UBXN7 inhibits s and e antigen secretion of HBV, reducing its RNA and encapsidated DNA expression levels; in the aspect of molecular mechanism, the UBXN7 protein inhibits HBV replication by inhibiting NF-kB signal activity; the function and molecular mechanism of UBXN7 in inhibiting HBV replication are respectively confirmed in human primary hepatocytes PHHs and mice. The invention discovers for the first time that the purpose of treating HBV related diseases can be achieved by up-regulating the expression of UBXN7, and therefore, the UBXN7 can be applied to the preparation of anti-HBV related disease drugs.

Description

Application of ubiquitin-binding protein UBXN7 in preparation of anti-HBV drugs

Technical Field

The invention relates to the technical field of molecular biology, in particular to application of ubiquitin-binding protein UBXN7 in preparation of anti-HBV drugs.

Background

Chronic infection with Hepatitis B Virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma, and there are approximately 2.57 billion chronic HBV infected individuals worldwide, however, the pathogenic mechanism of HBV is not fully elucidated. Protein ubiquitination modification can regulate various vital activities in cells, and the most prominent of them is the specific regulation of two major proteolytic systems, the ubiquitin-protease system and autophagy. Ubiquitin is a small protein consisting of 76 amino acids, widely present in eukaryotes and highly conserved, with a molecular weight of about 8.5 kD. Eukaryotic cellular proteins are regulated by post-translational modifications, with ubiquitination being one of the most common post-translational modifications of proteins. In eukaryotic cells, ubiquitin is covalently bound to a target protein through three cascade enzymatic reactions under the action of ATP, and the process is ubiquitination. More and more studies have shown that protein ubiquitination modification plays an important role in the replication and pathogenesis of HBV. The ubiquitin-proteasome system regulates the function, abundance and localization of host and viral proteins in cells, thereby affecting HBV replication; meanwhile, HBV can also promote self-replication or escape from host immune reaction by manipulating the ubiquitin-proteasome system of the host; in addition, HBV can promote self-replication by regulating autophagy. However, the regulatory mechanisms of this process are still poorly understood.

Therefore, there is a need to develop new anti-HBV drugs.

Disclosure of Invention

The invention aims to provide application of ubiquitin-binding protein UBXN7 in preparation of anti-HBV drugs, and finds the regulation and control effect of UBXN7 in the HBV replication process and the relation between UBXN7 and HBV related diseases for the first time, namely the purpose of treating HBV related diseases can be achieved by up-regulating the expression of UBXN 7. According to the function of UBXN7, it can be applied to prepare anti-HBV related disease drugs, and provides important basis for developing new anti-HBV related disease drugs or diagnostic reagents.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of ubiquitin-binding protein UBXN7 in preparing anti-HBV drugs.

Further, the anti-HBV drug is a drug for inhibiting secretion of HBsAg and HBeAg antigens.

Further, the anti-HBV drug is a drug that inhibits HBV replication.

Further, the ubiquitin-binding protein UBXN7 is obtained by expression of an expression vector, and the expression vector of the ubiquitin-binding protein UBXN7 comprises: the expression vector pEF-Flag-UBXN7, the preparation method of the expression vector pEF-Flag-UBXN7 is as follows:

cloning a primer of SEQ ID NO.1-SEQ ID NO.2 from a human genome sequence to obtain an expression gene fragment of UBXN 7;

and carrying out gel electrophoresis on the expression gene fragment of the UBXN7, recovering, carrying out double enzyme digestion on the recovered expression gene fragment and a vector pEF-Flag by EcoRI/XbaI, recovering, connecting and transforming to obtain the UBXN 7.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides an application of ubiquitin-binding protein UBXN7 in preparation of anti-HBV drugs, and researches show that (1) under HBV infection conditions, HBV can interact with UBXN7 protein molecules to promote UBXN7 to be subjected to ubiquitination modification, so that UBXN7 molecules are degraded through a proteasome pathway. (2) Over-expression of UBXN7 could inhibit s and e antigen secretion of HBV, reducing RNA and encapsidated DNA expression levels. (3) UBXN7 reduces HBV replication by inhibiting NF-. kappa.B signaling activity. (4) The function and molecular mechanism of UBXN7 for inhibiting HBV replication are confirmed in human primary hepatocytes PHHs and mice. Therefore, UBXN7 is a new inhibitor of NF-kB signaling pathway, and the deletion of UBXN7 can lead to the enhancement of NF-kB signaling activity and the promotion of HBV replication. HBV promotes the ubiquitination modification of UBXN7 protein molecules, so that the UBXN7 protein molecules are degraded through a proteasome pathway, and the replication of the HBV is promoted. The research results show that the UBXN7 protein molecule can be used as a new potential target for developing anti-HBV drugs.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 shows that HBV inhibits UBXN7 protein levels but does not affect mRNA expression in different cells. A. Stably transfected HBV inhibited UBXN7 protein levels but did not affect UBXN7mRNA levels. HepG2 and hepg2.2.15 cells were plated separately, cellular RNA and protein were extracted, and intracellular UBXN7mRNA (upper) and protein (lower) levels were detected by qPCR and western blot experiments, respectively. B. Transient transfection of HBV in Huh7 cells inhibited UBXN7 protein levels but did not affect UBXN7mRNA levels. pUC18 or phbv1.3 plasmid was transiently transferred to Huh7 cells, cellular RNA and protein were extracted, and intracellular UBXN7mRNA (upper) and protein (lower) levels were detected by qPCR and western blot experiments, respectively. C. The experiment with B was repeated in HepG2 cells. ns, indicates no significant difference.

FIG. 2 shows the inhibition of UBXN7 protein expression level by HBV through proteasome pathway. A. pHBV1.3 plasmid was transiently transferred to Huh7 cells in a concentration gradient, and MG132 and NH were added before harvesting₄Cl and CQ, with DMSO as control; intracellular UBXN7 protein levels were detected by western blot experiments after transfection. B. Experiments with A were performed in HepG2 cells. C. HBV promotes total ubiquitination of intracellular UBXN7 protein. plasmid pEF-Flag-UBXN7, plasmid pUC18/pHBV1.3 and plasmid HA-Ub are transiently transferred into Huh7 cells, a proteasome inhibitor MG132 is added before collection, and the ubiquitination level of UBXN7 protein is detected by immunoprecipitation and Western blot experiment of the post-transfection protein.

FIG. 3 shows that a concentration gradient of UBXN7 inhibits HBV antigen secretion. A. UBXN7 concentration gradients in Huh7 and HepG2 cells transiently transfected with HBV inhibited s and e antigen secretion of HBV. The upper diagram: pHBV1.3 and pEF-Flag-UBXN7 plasmids were transiently transfected into Huh7 cells, and culture supernatants were collected after transfection for ELISA to detect the secretion of s and e antigens of HBV. The following figures: the same experiment was performed in HepG2 cells. B. Knock-down of UBXN7 in Huh7 and HepG2 cells transiently transfected with HBV promotes s and e antigen secretion of HBV. The upper graph is as follows: pHBV1.3 and siNC or siUBXN7 were transiently transfected into Huh7 cells, and culture supernatants were collected after transfection for detection of HBV secretion of s and e antigens by ELISA. The following figures: the same experiment was performed in HepG2 cells. C. UBXN7 concentration gradient in HepG2.2.15 cells of the stably transformed HBV inhibits the secretion of s and e antigens of the HBV, and the knocking-down of UBXN7 promotes the secretion of s and e antigens of the HBV. The upper diagram: the pEF-Flag-UBXN7 plasmid was transiently transferred to HepG2.2.15 cells, and culture supernatant was collected after transfection for ELISA to detect the secretion of s and e antigens of HBV. The following figures: the same experiment was performed with siNC or siUBXN7 transiently transfected into hepg2.2.15 cells. P < 0.05, P < 0.01, P < 0.001.

FIG. 4 shows the inhibition of HBV RNA expression levels by a concentration gradient of UBXN7 in different cell models. A. In Huh7 and HepG2 cells, the UBXN7 concentration gradient inhibited HBV RNA expression levels in HBV transiently transfected cells. The upper diagram: pHBV1.3 and pEF-Flag-UBXN7 plasmids were transiently transfected into Huh7 cells, and HBV RNA levels were measured by qPCR after transfection. The following figures: similar experiments were performed in HepG2 cells. B. Transient HBV transformation in Huh7 and HepG2 cells simultaneously knockdown UBXN7 expression and promote HBV RNA expression level. The upper diagram: pHBV1.3 and siNC or siUBXN7 were transiently transfected into Huh7 cells and qPCR assayed for HBV RNA levels after transfection. The following figures: similar experiments were performed in HepG2 cells. C. The UBXN7 concentration gradient in HepG2.2.15 cells of the stably transformed HBV inhibits the HBV RNA, and the UBXN7 is knocked down to promote the HBV RNA expression level. The upper diagram: the pEF-Flag-UBXN7 plasmid was transiently transferred to HepG2.2.15 cells, and qPCR was performed to detect HBV RNA levels after transfection. The following figures: similar experiments were performed with siNC or siUBXN7 transiently transfected into hepg2.2.15 cells. P < 0.05, P < 0.01, P < 0.001.

FIG. 5 shows UBXN7 inhibition of HBV RNA and encapsidated DNA expression levels. A. Northern blot experiments showed that UBXN7 inhibited HBV RNA expression levels in cells transiently transfected with HBV. pHBV1.3 and pEF-Flag-UBXN7 were transiently transferred to Huh7 cells, and the Northern blot assay examined HBV RNA levels after transfection. B. UBXN7 inhibited HBV encapsidated DNA expression levels in HBV transiently transfected cells. pHBV1.3 and pEF-Flag-UBXN7 were transiently transfected into Huh7 cells, and the expression level of HBV encapsidated DNA was examined by Southern blot analysis after transfection.

FIG. 6 shows that UBXN7 inhibits HBV-induced NF- κ B activity. pRL-TK, pUC18/pHBV1.3 and pEF-Flag/pEF-Flag-UBXN7 were transiently transferred to Huh7 cells, and NF- κ B activity was detected by double fluorescence assay after transfection. P < 0.001.

FIG. 7 shows that UBXN7 inhibits HBV antigen secretion and RNA expression levels in HBV-infected PHHs. A. UBXN7 inhibits s antigen secretion of HBV in human primary hepatocytes infected with HBV. After HBV infects primary human hepatocytes, pEF-Flag-UBXN7 plasmid is transiently transformed, and ELISA is performed after transfection to detect s antigen secretion of HBV. B. UBXN7 inhibited HBV RNA expression levels in HBV infected human primary hepatocytes. After HBV infects primary human hepatocytes, pEF-Flag-UBXN7 plasmid is transiently transformed, and qPCR is carried out after transfection to detect HBV RNA level. P < 0.01, P < 0.001.

FIG. 8 shows that UBXN7 inhibits HBV antigen secretion and RNA expression levels in mice. A. UBXN7 inhibited s and e antigen secretion of HBV in mice. Tail vein high pressure injection pUC18/pHBV1.3, pEF-Flag/pEF-Flag-UBXN7 plasmid into mouse body, raising for a period of time, then blood sampling by eyeball to test the secretion of s and e antigens of HBV in serum by ELISA. B. UBXN7 inhibited HBV RNA expression levels in mice. Injecting pUC18/pHBV1.3, pEF-Flag/pEF-Flag-UBXN7 plasmids into mice under high pressure in tail vein, feeding for a period of time, taking liver tissues to extract RNA, and carrying out qPCR to detect the level of HBV RNA. P < 0.05, P < 0.01, P < 0.001.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

in previous work we found that the host proteome affected by HBV and the ubiquitination modification group are negatively correlated, indicating that proteome expression levels are negatively regulated by ubiquitination. Protein-protein interaction network analysis indicates enrichment of metabolic, ribosomal, secretory, immune system and NF- κ B regulatory-related proteins. In addition, the ubiquitination modification level of proteins such as ubiquitin-binding proteins (e.g., UBXN7-K84/K99) is significantly up-regulated, while the protein level is significantly down-regulated.

In the present invention, we first compared host cell proteins and ubiquitination-modified changes after HBV integration by bioinformatic analysis and looked for ubiquitination-modified proteins of interest. Analyzing the expression level of UBXN7 in HBV stably integrated or transiently transfected liver cancer cell lines by Western blot experiments and qRT-PCR; changes in protein ubiquitination levels were detected by immunoprecipitation experiments. Analyzing the effect of UBXN7 on HBV antigen secretion, RNA and encapsidated DNA expression levels by ELISA, Northern hybridization and Southern hybridization experiments; finally, the influence function of UBXN7 protein on HBV replication and related molecular mechanisms are verified in human primary hepatocytes PHHs and mice.

The following will describe in detail the application of ubiquitin-binding protein UBXN7 in preparing anti-HBV drugs in combination with examples and experimental data.

Mice used in the present invention were purchased from experimental animals ltd, slyke landsda, lake south.

The commercial human primary hepatocytes PHHs used in the present invention were purchased from shanghai reed liver institute.

The human hepatoma cell line HepG2.2.15 used in the present invention was stored in the laboratory and HepG2 and Huh7 were purchased from cell banks of Chinese academy of sciences (Shanghai).

The vector used in the present invention, pEF-Flag, was purchased from addgene, cat # MLCC 12737.

The pHBV1.3 and pUC18 plasmids used in the present invention were both gifted by the laboratory of the Hirschmannide researchers of the Chinese academy of sciences. The pHBV1.3 plasmid is a plasmid based on pUC18 to which 1.3 times of type D HBV genome is ligated. The HA-Ub plasmid was stored in this laboratory.

Antibodies used in the present invention: GAPDH antibody, Flag-Tag antibody and HA-Tag antibody were purchased from Proteintech, China, and UBXN7 antibody was purchased from Novus Biologicals, USA.

Reagents relevant to the immunoblotting experiment used in the present invention: cell lysates and 100 × PMSF were purchased from bi yun sky biotechnology; 100 × phosphatase inhibitors (PhosSTOP) were purchased from MCE; 100 XCocktail and 0.45nm NC transfer films were purchased from Merck Millipore, USA; skim milk was purchased from BD, usa; ECL chemiluminescence color development kit was purchased from Bio-Rad; the BCA protein quantification kit and the protein Marker are purchased from ThermoFisher company.

The dual-luciferase reporter gene detection kit produced by Promega company is used in the invention; plasmid pRL-TK (available in Biyunyan), NF-kB luciferase reporter plasmid were all stored in this laboratory.

Chemicals used in the present invention: chloroquine (CQ), ammonium chloride (NH)₄Cl), MG-132, and the protease inhibitor Cocktail, all available from Sigma-Aldrich, USA.

The reverse transcription kit used in the present invention was purchased from Takara. 2 × Sybr Green Mix and octal tubing were purchased from ThermoFisher, Inc.

The kit for detecting the HBV surface antigen and the e antigen used in the invention is purchased from Shanghai Kowa Bio Inc.

Northern blot and Southern blot experiments related reagents used in the present invention: DIG Northern Starter kit was purchased from Roche and Nylon Membrane was purchased from GE Amersham.

The co-immunoprecipitation experiment used in the invention is related to the reagents: the lysate is western blot and immunoprecipitation cell lysate of Byuntian biotechnology company. Flag-Beads was purchased from sigma (aldrich).

The disposable sterile 1ml and 2ml syringes used in the present invention were purchased from BD corporation, and the RNA protective solution was purchased from Tiangen Biotechnology Ltd.

Example 1: the down regulation of UBXN7 expression caused by HBV infection and the molecular mechanism thereof

HBV inhibits the expression of UBXN7 protein levels in different cells

1. In order to verify the results of previous experiments in different cell lines, total proteins of HepG2.2.15 cells and HepG2 cells stably transfected with HBV genomes were extracted first to perform a western blot experiment, and protein levels of UBXN7 were detected; meanwhile, RNA of two cells is extracted, qPCR experiment is carried out after reverse transcription, and UBXN7mRNA level is detected.

The results of the experiment are shown in FIG. 1A, indicating that: there was no significant difference in UBXN7mRNA levels in HepG2 and HepG2.2.15 cells, but UBXN7 protein levels in HepG2.2.15 cells were significantly lower than in HepG2 cells.

2. In addition, we transiently transfected pUC18 empty vector or phbv1.3 plasmid (present in chinese academy of sciences highlight man researcher laboratories) in Huh7 and HepG2 cells, respectively, and 48 hours after transfection, total protein of Huh7 and HepG2 cells was extracted for western blotting experiments to detect UBXN7 protein level; meanwhile, RNA of two cells is extracted, qPCR experiment is carried out after reverse transcription, and UBXN7mRNA level is detected.

The results are shown in FIGS. 1B and 1C, indicating that: UBXN7mRNA levels were not significantly changed in cells after transient transfection of the phbv1.3 plasmid, but UBXN7 protein levels were significantly reduced.

The above experimental results show that HBV inhibits UBXN7 protein levels in both transient and stable presence, but has no significant effect on UBXN7mRNA levels.

II, HBV (hepatitis B virus) molecular mechanism for inhibiting UBXN7 protein expression

The above results indicate that HBV inhibits UBXN7 protein levels but does not affect UBXN7 gene mRNA expression, suggesting that HBV may promote UBXN7 protein degradation. There are two major proteolytic systems in eukaryotic cells: ubiquitin-protease system and autophagy. Wherein the ubiquitin-proteasome system is a process for ubiquitination labeling of substrate proteins and transfer to proteasome for degradation. Autophagy is a process of degrading proteins by lysosomes. Therefore, it is necessary to further explore the pathways by which HBV promotes the degradation of UBXN 7.

We transiently transformed pUC18 or pHBV1.3 plasmids in Huh7 and HepG2 cells, respectively, and added DMSO (control) and proteasome (proteasome) before harvestingInhibitor MG132, autophagy inhibitor NH₄Cl and CQ (chloroquine), extracting total cell protein, performing Western blotting experiment, and comparing protein levels of UBXN7 in cells after gradient transient transformation of pHBV1.3 plasmid.

The results are shown in fig. 2A and 2B, indicating that: when DMSO and the autophagy inhibitor NH are added₄HBV dose-dependent inhibition of UBXN7 protein levels when Cl, CQ; when proteasome inhibitor MG132 was added, the inhibitory effect of HBV on UBXN7 protein disappeared, indicating that HBV promotes UBXN7 degradation via the proteasome pathway.

Previous ubiquitination modification group results showed that HBV promoted the level of ubiquitination modification of UBXN7, which we further validated in cells. The CDS sequence of UBXN7 gene is shown in SEQ ID NO. 6.

Construction of the expression vector pEF-Flag-UBXN 7: cloning an expressed gene fragment of UBXN7 from a human genome sequence by using primers shown in SEQ ID NO.1(UBXN 7-FP: 5'-CCGGAATTCATGGCTGCCCACGGGGGCTCC-3') and SEQ ID NO.2(UBXN 7-RP: 5'-CTAGTCTAGATTAATTTCTTTCCTGTACAAAGACAGTCT-3');

and carrying out gel electrophoresis on the expression gene fragment of the UBXN7, recovering, carrying out double enzyme digestion on the recovered expression gene fragment and a vector pEF-Flag by EcoRI/XbaI, recovering, connecting and transforming to obtain the UBXN 7.

On the basis of transient transfection of pEF-Flag-UBXN7 and HA-Ub plasmids in Huh7 cells, pUC18 empty vectors or pHBV1.3 plasmids are co-transformed, a proteasome inhibitor MG132 is added before collection, and UBXN7 ubiquitination modification change is detected through immunoprecipitation and a western blot experiment.

The results are shown in fig. 2C, indicating that: infection with HBV significantly contributes to the overall ubiquitination modification level of intracellular UBXN7 protein.

Example 2: over-expression of UBXN7 protein in cells can inhibit replication of HBV

It has been found that a number of UBXN family proteins can inhibit retroviral production, and in view of the fact that there is a reverse transcription process in the HBV life history, we guess whether UBXN7 can inhibit HBV replication.

The small interfering RNA used in the present invention (siUBXN7 and control siNC) was purchased from Yangzhou Ribo Biotech, Inc. in the following sequence:

siUBXN7-1：5’-GCAAGTGTCTCTACTGTCA-3’(SEQ ID NO.3)；

siUBXN7-2：5’-GTTGCGGTATCCAGATGGA-3’(SEQ ID NO.4)；

siUBXN7-3：5’-CCATTGATTTGATGCATAA-3’(SEQ ID NO.5)；

we co-transfected pHBV1.3 and pEF-Flag/pEF-Flag-UBXN7 plasmids or siNC/siUBXN7 in Huh7 and HepG2 cells, and 48 hours later, cell culture supernatants were removed and tested for secretion of s-antigen and e-antigen of HBV by ELISA. The results show that: in cells transiently transfected with HBV, a concentration gradient of UBXN7 inhibited s and e antigen secretion from HBV (fig. 3A), and knock-down of UBXN7 promoted s and e antigen secretion from HBV (fig. 3B).

In addition, we transfected pEF-Flag/pEF-Flag-UBXN7 plasmid or siNC/siUBXN7 in HepG2.2.15 cells stably integrated with HBV genome, and 48 hours later, cell culture supernatant ELISA method was taken to detect the secretion of s antigen and e antigen of HBV. The results show that: the steady transfection of UBXN7 concentration gradient in HBV cells inhibited the s and e antigen secretion of HBV, and the knockdown of UBXN7 promoted the s and e antigen secretion of HBV (fig. 3C).

To further explore the effect of UBXN7 on HBV RNA, we co-transfected pHBV1.3 and pEF-Flag-UBXN7 expression plasmids or siUBXN7 in Huh7 and HepG2 cells, and extracted cellular RNA 48 hours later and performed qPCR experiments to detect HBV RNA. The results show that: transient HBV UBXN7 concentration gradients inhibited HBV RNA levels (fig. 4A), interfering with UBXN7 expression promoted HBV RNA levels (fig. 4B).

Similarly, pEF-Flag-UBXN7 expression vector or siUBXN7 was transfected in HepG2.2.15 cells, and after 48 hours, cellular RNA was extracted for qPCR assay to detect HBV RNA. The results show that: the concentration gradient of UBXN7 in the stably transfected HBV cells suppressed HBV RNA levels, and interfering with UBXN7 expression promoted HBV RNA levels (fig. 4C).

In addition, we also co-transfected pHBV1.3 and pEF-Flag-UBXN7 expression vectors in Huh7 cells, and after 48 hours, extracted cellular RNA and detected HBV RNA changes by Northern blot experiments. The experimental results show that: UBXN7 inhibited HBV RNA levels (fig. 5A).

We further explored the effect of UBXN7 on HBV DNA replication by co-transfecting pHBV1.3 and pEF-Flag-UBXN7 expression vectors in Huh7 cells and detecting changes in HBV encapsidated DNA levels after 96 hours by Southern blot experiments. The experimental results show that: UBXN7 inhibited HBV DNA replication (fig. 5B).

Example 3: molecular mechanism of UBXN7 protein for inhibiting HBV replication

A variety of UBX family proteins have been found to be involved in NF-. kappa.B signaling. Early studies found that UBXN1, UBXN2C and UBXN3A could inhibit TNF- α -induced NF- κ B signaling, but UBXN7 and UBXN3B had no inhibitory effect on TNF- α -induced NF- κ B signaling. Subsequent studies have shown that UBXN1, UBXN9 and UBXN11 inhibit TNF- α -induced NF- κ B signaling to inhibit partial retroviral production, and it is noted that UBXN7 was found to inhibit TNF- α -induced NF- κ B activity in previous screening procedures, but no further study was conducted. Therefore, whether UBXN7 can affect NF-. kappa.B signaling remains controversial. Our previous results show that UBXN7 is able to inhibit transcription and replication of HBV, so we sought to explore whether UBXN7 also inhibits replication of HBV by inhibiting NF- κ B signaling.

To investigate the effect of UBXN7 on NF-. kappa.B signaling, we co-transformed pRL-TK, pUC18/pHBV1.3 and pEF-Flag-UBXN7 expression vectors in Huh7 cells, and lysed the cells after 48 hours for dual luciferase assays. The results are shown in FIG. 6, indicating that: UBXN7 significantly inhibited HBV-induced NF- κ B activity.

Example 4: verification of UBXN7 inhibition of HBV replication in human primary hepatocytes PHHs

For a long time, the most important reason why humans cannot overcome hepatitis B is the lack of good in vitro models. Human primary hepatocytes (PHHs) are ideal models, but due to lack of suitable culture conditions, the isolated primary hepatocytes undergo morphological and gene expression pattern changes in a short time during in vitro culture, lose hepatocyte functions, and cannot be further tested. Liver cancer cells are currently studied in most laboratories, but there is still some difference compared to human primary liver cells. In the past decades, researchers have attempted to change the culture materials, but have not always established a simple, efficient, stable in vitro culture system for human primary hepatocytes. In 2019, 4, the Dang Hongkui research group at Beijing university, et al published in the journal of Science in research papers (Xiang C, Du Y, Meng G, Soon Yi L, Sun S, Song N, Zhang X, Xiao Y, Wang J, Yi Z, Liu Y, Xie B, Wu M, Shu J, Sun D, Jia J, Liang Z, Sun D, Huang Y, Shi Y, Xu J, Lu F, Li C, Xiang K, Yuan Z, Lu S, Deng H.Long-term functional maifangnance of primary human hepatology in science.2019,364(6438):399-402), which first demonstrated the use of small chemical molecule regulatory cell signaling pathways to achieve long-term maintenance of human primary hepatocytes in vitro, and established a model of HBV infection and persistent hepatitis C (HBV infection in vitro). This study offers the possibility of preparing a large number of functionally mature hepatocytes and their use. The liver cells under the culture condition support the high-efficiency infection of HBV, can produce cccDNA for a long time, can be used as an ideal research model of HBV infection related diseases, and brings hope for curing the HBV related diseases. Based on this, the present invention uses commercial human primary hepatocytes PHHs purchased from shanghai reed liver institute as a research model.

In the above experiments we found that UBXN7 inhibited HBV antigen secretion and RNA by inhibiting HBV-induced NF- κ B signaling, but these results were obtained in HBV genome-integrated hepg2.2.15 cells, and we further explored whether UBXN7 could exert the same function in HBV-infected human primary hepatocytes PHHs. PHHs are paved in a 12-well plate, after 24 hours of culture, cells in one well are trypsinized and counted, concentrated HBV virus solution, 1% DMSO and 4% PEG8000 are added according to the proportion of 200copies per cell for auxiliary infection, and the culture medium is replaced with fresh medium after 24 hours of infection. And (3) transfecting a pEF-Flag-UBXN7 expression vector after the infection of the fourth day, collecting the supernatant of the PHHs culture solution infected with HBV on the sixth day, and detecting the s antigen secretion of the HBV by an ELISA method. The results show that: overexpression of UBXN7 significantly inhibited s antigen secretion by HBV (fig. 7A).

We extracted PHHs RNA infected with HBV and performed qPCR experiments to detect HBV RNA level changes. The experimental results show that: overexpression of UBXN7 significantly suppressed HBV RNA expression levels (fig. 7B).

Example 5 verification of UBXN7 inhibition of HBV replication Process in mice

At present, the pathogenic mechanism and interaction between HBV and host are not fully elucidated, and one important reason is the lack of suitable animal model for HBV infection, so if a suitable animal model can be found, we can make intensive research on HBV in vivo. The mouse model is a more common and widely used animal model for HBV infection. The mouse used in the invention is purchased from lake south Slek Jingda laboratory animals GmbH, and the adopted tail vein high pressure injection mouse model can simulate HBV acute infection and is easier to obtain than other mouse models, thus being a better choice.

We found by the above experiments that UBXN7 inhibited HBV antigen secretion, but these results were obtained under conditions of transient cell transfection. A mouse acute HBV infection model is constructed by a tail vein high pressure injection method, and the influence of UBXN7 on the HBV replication process in a mouse is researched. 30 male 10-week-old Balb/C mice were divided into 3 groups of 10 mice each. The corresponding pUC18/pHBV1.3 and pEF-Flag/pEF-Flag-UBXN7 plasmids were mixed well with 2ml of physiological saline, and the solution was injected into the tail vein of the mouse within 10 seconds using a 2ml syringe. After the mice are raised for four days, serum of the mice is collected by an eyeball blood sampling method to carry out ELISA experiment detection on the secretion of s and e antigens of HBV; mouse liver tissues were divided into 2 parts for western blotting and qPCR experiments, respectively. The results show that: after tail vein high pressure injection of pHBV1.3 plasmid, secretion of s and e antigens of HBV can be detected in mouse serum, which indicates that the mouse HBV acute infection model is successfully constructed, and simultaneously, after pEF-Flag-UBXN7 expression vector is added, secretion of s and e antigens of HBV is remarkably reduced (FIG. 8A).

We detected HBV RNA levels in mouse liver tissue by qPCR. The experimental results show that: after the tail vein high pressure injection of pHBV1.3 plasmid, the level of HBV RNA in the liver of mice was significantly increased, and after the pEF-Flag-UBXN7 expression vector was added, the level of HBV RNA was significantly decreased, indicating that UBXN7 significantly inhibited the level of HBV RNA (FIG. 8B).

In summary, our findings show that: UBXN7 is a new inhibitor of NF-kB signal channel, and UBXN7 deletion can lead to the enhancement of NF-kB signal activity and the promotion of HBV replication; HBV promotes the ubiquitination modification of UBXN7 protein molecules, so that the UBXN7 protein molecules are degraded through a proteasome pathway, and the replication of the HBV is promoted. The research results show that the UBXN7 protein molecule can be used as a new potential target for developing anti-HBV drugs. The invention provides the function and potential application of ubiquitin-binding protein UBXN7 in anti-HBV, and provides an important basis for developing new anti-HBV related disease drugs or diagnostic reagents.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

<110> Wuhan university

Application of <120> ubiquitin-binding protein UBXN7 in preparation of anti-HBV drugs

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccggaattca tggctgccca cgggggctcc 30

<210> 2

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctagtctaga ttaatttctt tcctgtacaa agacagtct 39

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcaagtgtct ctactgtca 19

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gttgcggtat ccagatgga 19

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccattgattt gatgcataa 19

<210> 6

<211> 1470

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggctgccc acgggggctc cgcggcgtcc tcggcgctga aggggttaat tcaacagttc 60

accaccatta ccggtgcaag tgaaagtgta ggaaaacata tgcttgaagc gtgcaacaat 120

aatctggaaa tggcagtcac tatgtttttg gatggtggag gaatcgctga agagcccagt 180

accagttcag caagtgtctc tactgtcaga ccacacacag aagaagaagt tcgtgcccca 240

attcctcaaa agcaggaaat actggtggaa ccagaaccat tatttggtgc tcctaaaaga 300

cgacggcctg cacgttcaat ttttgatggt ttccgggatt ttcagactga aactattcgg 360

caagaacaag aattaagaaa tggaggagct atcgataaga aattaactac ccttgcagat 420

ctattccggc cacccattga tttgatgcat aaaggcagct ttgaaacagc caaagagtgt 480

ggccagatgc aaaataagtg gctgatgata aacattcaaa atgttcaaga ctttgcatgt 540

cagtgcctca accgcgatgt gtggagcaac gaagctgtga agaatattat ccgggaacat 600

ttcattttct ggcaggttta tcatgacagt gaggaaggtc agagatacat acagttttat 660

aagttagggg atttccccta tgtttccata ttggacccac ggacaggtca gaagctagta 720

gaatggcacc agttagatgt atcttctttc ttggaccaag tgacgggatt tctgggtgaa 780

catggacaac tggatggact ttctagcagt ccccccaaaa aatgtgcccg ttcagagagc 840

cttatagatg caagtgaaga cagccagcta gaagctgcca tcagagcctc cttacaagaa 900

acacattttg attcaacaca gacaaaacag gatagccgct cagatgaaga atctgaatct 960

gaactttttt ctggcagtga ggagttcata tccgtttgtg gctctgatga agaagaagag 1020

gtagagaatc ttgccaagtc cagaaagtct ccccacaaag atttggggca tagaaaagag 1080

gagaatagaa ggccgctgac tgagccacca gtcagaactg atcctggaac agccacaaac 1140

caccaaggat tgccagctgt ggattcagag atactggaga tgccacctga aaaagcagat 1200

ggagtagtgg aggggataga tgtaaatgga ccaaaagcac agctgatgtt gcggtatcca 1260

gatggaaaaa gggaacagat cactcttcca gagcaagcta aactgctagc tttggtgaag 1320

cacgtgcagt ctaaaggata cccaaatgaa cgttttgaac ttctcaccaa ctttcctcga 1380

aggaaattat ctcatctgga ctatgatatt acattgcaag aggcaggcct ttgtcctcaa 1440

gagactgtct ttgtacagga aagaaattaa 1470

Claims (4)

1. Application of ubiquitin-binding protein UBXN7 in preparing anti-HBV drugs.

2. The use according to claim 1, wherein the anti-HBV agent is an agent that inhibits secretion of HBsAg and HBeAg antigens.

3. The use according to claim 1, wherein the anti-HBV agent is an agent that inhibits HBV replication.

4. The use according to claim 1, wherein the ubiquitin binding protein UBXN7 is expressed by an expression vector, and the expression vector of the ubiquitin binding protein UBXN7 comprises: the expression vector pEF-Flag-UBXN7, the preparation method of the expression vector pEF-Flag-UBXN7 is as follows:

cloning a primer of SEQ ID NO.1-SEQ ID NO.2 from a human genome sequence to obtain an expression gene fragment of UBXN 7;

and carrying out gel electrophoresis on the expression gene fragment of the UBXN7, recovering, carrying out double enzyme digestion on the recovered expression gene fragment and a vector pEF-Flag by EcoRI/XbaI, recovering, connecting and transforming to obtain the UBXN 7.

Images