阅读说明：本技术 乙型肝炎病毒重组cccDNA小鼠模型的建立方法及用途 (Establishment method and application of hepatitis B virus recombinant cccDNA mouse model ) 是由 袁正宏 邬敏 张小楠 陈捷亮 于 2020-05-11 设计创作，主要内容包括：本发明属微生物动物细胞系技术领域,具体涉及一种8型腺相关病毒载体介导建立的乙型肝炎病毒重组cccDNA(rcccDNA)小鼠模型及其建立方法和用途。本发明利用8型腺相关病毒载体,将两端带有loxP位点的HBV单拷贝基因组带入Alb-Cre转基因小鼠体内,建立了AAV-rcccDNA小鼠模型。建立的AAV-rcccDNA小鼠模型具有1)建模成功率高；2)模型小鼠肝内rcccDNA表达水平高、维持时间长,易于用Southern blot方法进行检测；3)模型小鼠血清及肝内病毒抗原表达水平、维持时间长；4)模型小鼠肝内HBV特异性T细胞免疫反应偏弱等特点。本发明建立的小鼠模型适用于筛选和测试HBV cccDNA靶向药物、免疫调节剂以及研究慢乙肝治愈的合适模型中。(The invention belongs to the technical field of microbial animal cell lines, and particularly relates to a hepatitis B virus recombinant cccDNA (rccccDNA) mouse model established by mediation of 8-type adeno-associated virus vectors, and an establishment method and application thereof. The invention utilizes 8 type adeno-associated virus vector to bring HBV single copy genome with loxP sites at two ends into Alb-Cre transgenic mouse body, and establishes AAV-rcccDNA mouse model. The established AAV-rcccDNA mouse model has 1) high modeling success rate; 2) the rcccDNA in the liver of the model mouse has high expression level and long maintenance time, and is easy to detect by a Southern blot method; 3) the expression level and the maintenance time of the serum and the liver virus antigen of the model mouse are long; 4) weak HBV specific T cell immunoreaction in the liver of the model mouse and the like. The mouse model established by the invention is suitable for screening and testing HBV cccDNA targeted drugs, immunomodulators and suitable models for researching chronic hepatitis B cure.)

1.A method for establishing a hepatitis B virus recombinant cccDNA mouse model is characterized in that an 8-type adeno-associated virus vector is utilized to bring HBV single copy genome with loxP sites at two ends into an Alb-Cre transgenic mouse, and the 8-type adeno-associated virus vector is utilized to mediate and construct a hepatitis B virus HBV recombinant covalent, closed and circular DNA (rcccDNA) mouse model, and the method comprises the following steps:

constructing pAAV8-rcccDNA plasmid;

packaging the virus: pAAV8-rcccDNA, pRC and pHelper plasmids were cotransformed into HEK293T cells;

purifying the virus: purifying by chloroform treatment-PEG/NaCl precipitation-chloroform extraction method to obtain concentrated and purified recombinant gland-associated virus liquid, quantifying by quantitative PCR method, and taking pAAV8-rcccDNA plasmid gradient diluted as standard curve;

establishing a model: AAV8-rcccDNA virus injected tail vein in Alb-Cre male mice at the injection dose, a single copy of HBV genome will be released from the viral vector, circularized separately to form rcccDNA.

2. The method of claim 1, wherein the biological properties of the mouse model AAV-rcccDNA are:

1) the rcccDNA in the liver of the model mouse obtained after modeling has high expression level and long maintenance time, and is easy to detect by a Southern blot method within 30 weeks;

2) the expression level of the virology indexes in the serum and the liver of the model mouse is high, and the maintenance time is long;

3) the HBV specific T cell in the liver of the model mouse has weak immunoreaction, no obvious inflammatory reaction and can observe slight fibrosis hyperplasia and metabolism level change.

3. The method of creating according to claim 1, characterized in that the use of the created mouse model AAV-rccccdna for the preparation of HBV cccDNA related biological research model.

4. The method according to claim 1, wherein the mouse model AAV-rccccdna is used for preparing a model for studying HBV cccDNA-related immunological properties.

5. The method according to claim 1, wherein the mouse model AAV-rcccDNA is used for screening anti-HBV drug models.

6. The method according to claim 1, wherein the use of the established mouse model AAV-rcccDNA for the preparation of a model for the evaluation of anti-HBV drugs.

Technical Field

The invention belongs to the technical field of microbial animal cell lines, relates to a hepatitis B virus recombinant cccDNA (rccccDNA) mouse model, and particularly relates to a hepatitis B virus recombinant cccDNA (rccccDNA) mouse model established by mediation of a 8-type adeno-associated virus vector, and an establishment method and application thereof.

Background

The prior art discloses that Hepatitis B caused by Hepatitis B Virus (HBV) infection is a serious infectious disease that seriously harms human health. According to statistics, the risk of patients suffering from Chronic Hepatitis B (CHB) infection is remarkably higher (> 20%) than that of normal people suffering from liver cirrhosis, liver failure and liver cancer. At present, more than 9000 tens of chronic hepatitis B patients exist in China, wherein 2800 tens of patients need to be treated, and although the treatment of the chronic hepatitis B has been advanced to some extent in recent years, a series of problems such as treatment course, drug resistance, relapse after drug withdrawal and the like still remain clinical problems, wherein one important reason is the continuous existence and difficult elimination of a structural molecule 'covalently closed circular DNA (cccDNA)' which is characteristic of HBV.

Research has shown that HBV cccDNA is formed from relaxed circular DNA (rcDNA) of viral genome through a series of steps in the hepatocyte nucleus, wherein rcDNA can be derived from newly infected viral particles or newly synthesized viral core particles during viral replication; HBV cccDNA is transcribed to generate virus mRNA, wherein pregenomic RNA (pgRNA) is a template for translation and reverse transcription replication of virus protein, so that the possibility of completely curing chronic hepatitis B can be realized only by clearing cccDNA in cell nucleus, which is also the target for research and development of new HBV medicines.

There are still many gaps in the understanding of the biological properties of cccDNA formation, functional activity and stability regulation in the current field. Research practices show that the development of HBV animal models is subject to strict species specificity of HBV, and mice are used as the most thoroughly studied mammalian experimental animals with the largest use amount and are not easy to be infected with HBV; even in a human sodium taurocholate cotransporter polypeptide (hNTCP) transgenic mouse model, effective infection cannot be established due to low cccDNA formation efficiency. Most of the HBV mouse models at present rely on the methods of transgene, viral vector transduction or high-pressure tail vein injection for establishment, in which cccDNA is difficult to detect. The human liver chimeric mouse is the only mouse model which effectively supports HBV infection and cccDNA formation, but the high technical challenge in the model establishment process and the lack of adaptive immunity of the model mouse are important factors which limit the universal application of the model. Therefore, establishing a mouse model which is easy to culture and has a relatively stable cccDNA library and normal immunocompetence is an urgent need in the field of hepatitis b research.

In recent years, the development of recombinant cccDNA (rccccdna) mouse models has been fast, rccccdna can effectively support the replication and protein expression of viruses and establish chronic infection, however, in the reported rccccdna mouse model, the level and duration of the rccccdna library in mouse liver cells are still not suitable for cccDNA targeting research, although the rccccdna band can be clearly detected by Southern blot within one week of modeling, the rccccdna level is rapidly reduced afterwards, which limits the application of the rccccdna in researching the related biological mechanism of cccDNA and developing new cccDNA targeting drugs.

Based on the characteristics of high infection rate, no carcinogenic hidden danger, low immunogenicity and the like of adeno-associated virus (AAV), wherein the affinity of type 8 to liver is highest, the invention establishes a novel HBV rcccDNA mouse model by utilizing the mode of introducing type 8 adeno-associated virus vector (AAV8) on the basis of the strategy of generating rcccDNA in Cre/loxP system. The model is expected to become a suitable model for screening and testing cccDNA targeted drugs and researching chronic hepatitis B cure.

Disclosure of Invention

The invention aims to overcome the defects of an HBV rccccDNA mouse model in the prior art, and establishes an rccccDNA mouse model with higher expression level and longer maintenance time, wherein the mouse model can be used for researching related biological characteristics of HBV cccDNA and screening and evaluating anti-HBV drugs, particularly novel drugs directly targeting cccDNA.

Specifically, the invention provides a hepatitis B virus recombinant cccDNA (rcccDNA) mouse model and an establishment method thereof, wherein the mouse model of the Hepatitis B Virus (HBV) recombinant covalent, closed and circular DNA (rcccDNA) is constructed by using an adeno-associated virus (AAV) vector.

More specifically, the invention provides a hepatitis B virus recombinant cccDNA (rccccDNA) mouse model established by the mediation of an 8-type adeno-associated virus vector, and an establishing method and application thereof.

In the invention, an 8-type adeno-associated virus vector is adopted, HBV single copy genome with loxP sites at two ends is brought into an Alb-Cre transgenic mouse body, and an AAV-rcccDNA mouse model is established.

In the invention, the expression level and the maintenance time of the rcccDNA in the liver of the established AAV-rcccDNA mouse model are detected; the results showed that 8-week-old male Alb-Cre mice received 2X 10¹¹After the tail vein injection modeling of the vg recombinant virus, rcccDNA in the liver is easy to detect by a Southern blot method within 30 weeks.

In the invention, the AAV-rcccDNA mouse serum and the liver virology index are detected, and the result shows that the expression of the model mouse serum HBsAg, HBeAg and the liver HBsAg and HBcAg can be maintained for more than one year.

In the invention, the intrahepatic immunology and pathology of the AAV-rcccDNA mouse model are detected, and the result shows that the HBV specific T cell immunoreaction in the liver of the model mouse is weak, no obvious inflammatory reaction exists, but slight fibrosis proliferation and metabolism level change can be observed.

The invention further provides application of the established AAV-rcccDNA mouse model in research on HBVcccDNA related biological characteristics and screening and evaluation of anti-HBV drugs, particularly novel drugs directly targeting cccDNA. In the embodiment of the invention, the antiviral effects of AKSZ0002-B, Bay41-4109 and Entecavir (ETV) are evaluated in an AAV8-rcccDNA model mouse, and the results show that the changes of serum and intrahepatic virus indexes of the model mouse can well reflect different action mechanisms of the three antiviral preparations.

The invention establishes a novel HBV rcccDNA mouse model which becomes a suitable model for screening and testing cccDNA targeted drugs and researching chronic hepatitis B cure.

Drawings

Figure 1 is a graphical representation of the intrahepatic rcccDNA generation of the AAV-rcccDNA mouse model.

Figure 2.AAV-rcccDNA mouse model intrahepatic rcccDNA production assay.

Figure 3 AAV-rcccDNA mouse model serum and intrahepatic viral marker detection.

FIG. 4 detection of HBV-specific T lymphocyte expression and function in liver of AAV-rcccDNA mouse model.

Figure 5 AAV-rcccDNA mouse model for antiviral effect evaluation.

Detailed Description

The following examples are provided to illustrate the present invention, but are not intended to limit the scope of the present invention, nor are the scope of the present invention limited by the following examples.

EXAMPLE 1 establishment of AAV-rcccDNA mouse model

Construction of pAAV8-rcccDNA plasmid: AAV8 vector is obtained by XhoI/BamHI double digestion of pAAV8-CAG-RFP plasmid, HBV single copy genome sequence with loxP sites at two ends is amplified from prcccDNA plasmid, and cloned into pAAV8 vector by recombination method;

packaging the virus: pAAV8-rcccDNA, pRC and pHelper plasmids were cotransformed into HEK293T cells, and PEI was used as a transfection reagent. After 72 hours of transfection, the cells were collected into a 50ml centrifuge tube together with the medium;

purifying the virus: purifying by chloroform treatment-PEG/NaCl precipitation-chloroform extraction method, collecting cells and culture solution, adding 1/10 volume chloroform, placing in a shaking table at 37 ℃ and violently shaking for 1h, adding solid sodium chloride to a final concentration of 1mol/L, shaking for dissolution, centrifuging for 15min at 4 ℃ and 12000r/min, taking out an upper layer water phase, adding PEG8000 to a final concentration of 10% (w/v), shaking for dissolution, placing in an ice bath for 1h, centrifuging for 15min at 11000r/min, discarding supernatant, blowing and eluting precipitates on the bottom and wall of a centrifuge tube by using a proper amount of PBS, subpackaging into 1.5ml centrifuge tubes, defining rAAV as an intermediate product, digesting at room temperature for 30min by benzonase nuclease (50U/ml), adding isometric chloroform for extraction to obtain concentrated and purified adeno-associated virus liquid, quantifying by PCR method, the plasmid pAA V8-rcccDNA is used as a standard curve after being diluted in a gradient way;

establishing a model: AAV8-rcccDNA virus is injected into Alb-Cre male mice by tail vein according to injection dosage, virus is diluted to 200 μ l/mouse by PBS for injection, the liver of Alb-Cre transgenic mice specifically expresses recombinase Cre, so in mouse liver cells, the Cre enzyme recognizes and cuts loxP sites, single copy HBV genome is released from virus vector, and rcccDNA is formed by cyclization alone (shown in figure 1).

Example 2 AAV8-rcccDNA mouse model intrahepatic rcccDNA detection

Establishing a model: male Alb-Cre mice at 8 weeks of age received 2X 10¹¹Death of liver at the corresponding time points after vg AAV8-rcccDNA virus injection;

② extracting DNA: weighing 100mg of mouse liver tissue, placing the mouse liver tissue in 100 mu l of PBS precooled by ice, cutting the mouse liver tissue into small blocks as much as possible by using scissors, grinding the small blocks by using a tissue grinder, and sieving the small blocks by using a cell sieve with the aperture of 100 mu m to obtain liver cell suspension. Extracting by using a Hirt extraction method;

detecting rcccDNA by Southern blot: southern Blot Detection (DIG High Prime DNA Labeling and Detection Starter Kit II manual) was performed using the digoxin probe Labeling and Detection system from Roche; within 30 weeks, a clear signal of rcccDNA could be detected; the property that rcccDNA bands can be linearized by EcoRI endonuclease but insensitive to T5 exonuclease confirms the structure of its supercoiled form (as shown in fig. 2A);

fourthly, quantitative PCR detection of rcccDNA: the primers for specific intron sequences on rcccDNA were used for detection, and rcccDNA specific signals were still detected by quantitative PCR at week 52 after modeling (as shown in fig. 2B), and the half-life of rcccDNA in AAV8-rcccDNA mouse liver was 7.8 weeks (6.113-10.64 weeks) by fitting analysis to the quantitative data.

Example 3 detection of mouse model serum and hepatology index of AAV8-rcccDNA

Establishing a model: male Alb-Cre mice of specified week age receive AAV8-rcccDNA virus injection of specified dose to establish a model;

② serum virus index detection: every two weeks after injectionPerforming primary orbital angular vein plexus blood collection with the blood collection amount of 200 mul anticoagulated whole blood, standing the collected blood at room temperature for 2 hours, centrifuging at 8000rpm at room temperature for 10 minutes to separate serum for HBsAg, HBeAg, alanine aminotransferase ALT and HBV DNA detection, and displaying the result that the serum HBsAg and HBeAg of AAV-rcccDNA mouse model are respectively 10⁴IU/ml and 10³COI levels (as shown in fig. 3A, B) and remained positive throughout the experimental period (51 weeks); serum HBV DNA initial amount is 10⁴IU/ml, then gradually decreased (as shown in figure 3C), AAV8-rcccDNA mouse model serum viral antigen and DNA expression levels correlated with the injected recombinant viral dose, but not much with the mouse's week-old;

③ detecting the virus indexes in the liver: after the mice are dead at corresponding time points and perfused by a heart, organs/tissues such as liver, kidney and the like are separated, a part of each organ/tissue is placed in 4% paraformaldehyde fixing solution for paraffin embedding and slicing, and is used for immunohistochemical staining and the like, the result shows that HBsAg and HBcAg in the liver of the AAV-rcccDNA mice are expressed for a long time (as shown in figure 3D), HBsAg signals are positioned in cytoplasm, HBcAg signals are polymorphic, cells with strong cytoplasmic staining, strong nuclear staining and uniform nuclear staining can be observed, the positive expression areas of HBsAg and HBcAg at the early stage (2 weeks) of modeling are 60.71 +/-6.73% and 17.43 +/-6.27%, and the positive expression areas of HBsAg and HBcAg at the late stage (51 weeks) are 22.83 +/-4.24% and 12.04 +/-1.07%, respectively.

Example 4 detection of HBV-specific T lymphocytes expression and function in the liver of AAV8-rcccDNA mouse model

Establishing a model: male Alb-Cre mice 8 weeks old received 1X 10¹²Modeling by injecting the vg recombinant virus; three additional controls were set: a PBS group; EmptyAAV group: tail vein receiving 1X 10¹²vg of unloaded AAV8 virus injection; pAAV-rcccDNA group, high pressure tail vein received 10 μ g pAAV-rcccDNA plasmid injection;

② hepatitis B specific CD8⁺T lymphocyte number detection: two weeks after modeling mice were sacrificed, hepatic lymphocytes were isolated, surface stained with CD3, CD8, HBsAg or HBcAg specific Tetramer, and flow assayed for hepatitis B specific CD8⁺The proportion of T lymphocytes and the results show that AAV8-rcccDNA modelHBsAg and HBcAg specific CD8 in type mouse liver⁺The proportion of T lymphocytes is significantly lower than that of the plasmid group (env3534.62 ± 1.52% vs20.77 ± 4.98%, p ═ 0.0047; core930.919 ± 0.34% vs 3.91 ± 0.59%, p ═ 0.0017). However, no HBsAg or HBcAg-specific CD8 was evident in the PBS group and AAV No-load virus group mice⁺Expression of T lymphocytes (as shown in fig. 4A);

③ detection of hepatitis B specific CD8+ T lymphocyte function: after stimulating the separated mouse hepatic lymphocytes in two weeks of modeling with HBsAg peptide fragment for 5 hours, detecting the proportion of IFN-gamma and TNF-alpha positive T lymphocytes by intrinsic factor staining, and the result shows that the proportion of the IFN-gamma and TNF-alpha positive T lymphocytes is obviously lower than that of a plasmid group (IFN-gamma 2.27 +/-0.47% vs 5.78 +/-0.99%, p is 0.023, TNF-alpha 0.48 +/-0.06% vs 1.94 +/-0.56%, p is 0.004) after the AAV8-rcccDNA model mouse hepatic lymphocytes are stimulated by the S peptide fragment (as shown in figure 4B); further detection of PD-1 molecules Env 353-specific CD8T in AAV8-rcccDNA model mouse liver⁺The expression level on cells shows that the expression level (2309.33 +/-77.56) of the AAV-rcccDNA group PD-1 is obviously higher than that of the plasmid group (1377 +/-189), which indicates that the HBV specificity CD8T in the liver of the AAV-rcccDNA transgenic mouse⁺Cell function is impaired (as shown in figure 4C).

Example 5 AAV-rcccDNA mouse model for evaluation of antiviral Effect

Establishing a model: male Alb-Cre mice of 8 weeks old were treated with 2X 10¹¹Four weeks after vg AAV8-rcccDNA virus injection modeling, mice were randomly divided into a control group, an HBV expression inhibitor AKSZ0002-B treatment group, a nucleocapsid assembly inhibitor Bay41-4109 treatment group and a reverse transcription inhibitor ETV treatment group, the AKSZ0002-B and Bay41-4109 belong to two types of novel preparations, the two preparations respectively show good effects on indexes such as reduction of HBsAg expression and inhibition of virus replication, but the influence on the cccDNA in the liver is detected, the AKSZ0002-B group is administrated with 4 mg/kg/piece of drug gavage twice a day, the Bay 41-9 group is administrated with 15 mg/kg/piece of drug gavage twice a day, the ETV group is administrated with 0.03 mg/kg/piece once a day, the control group is administrated with 30% PEG400, 0.2% DMSO, 200. mu.l/piece, twice a day, all receiveTest compounds were administered at the indicated dose and frequency for 28 days, day 28, with some mice sacrificed per group and the remaining mice were observed to be off for 49 days;

collecting and detecting samples: blood was collected weekly to monitor serum antigen and viral load, on day 28, a portion of mice were sacrificed per group, livers were removed, and a portion of livers were used for fixation, paraffin embedding, sectioning and HBsAg, Core immunohistochemical staining, and the remainder were stored in RNAlater for Hirt extraction and Southern blot analysis, and the results showed that HBsAg in mouse serum was significantly reduced (1.6 log10 reduction, p 0.002) after one week of administration in the AKEX0007 group, and thereafter maintained at a lower level (as shown in fig. 5A, left); HBeAg levels were reduced by 0.35log10(p <0.001) (as shown in fig. 5A, middle); serum HBV DNA was slightly reduced by 0.1log10 (as shown in fig. 5A, right), compared to ETV group which observed only significant reduction in serum HBV-DNA (1.17 log10 reduction, p 0.007), while serum HBsAg, HBeAg did not change significantly, the serum viral indicators of Bay41-4109 group changed similarly to ETV group (as shown in fig. 5B), there was no significant change in serum viral antigen levels, but viral load was reduced (0.88 log10 reduction, p 0.005); the immunohistochemistry result shows that the liver HBsAg of mice in the AKEX0007 group is obviously reduced and the HBcAg is slightly reduced (the HBsAg 39.32 +/-3.5% vs13.55 +/-5.43%, the HBcAg 13.32 +/-2.69% vs 9.7 +/-1.82%) (as shown in FIG. 5C) compared with the control group of mice; a significant drop in HBcAg (17.68 ± 2% vs 8.89 ± 1.13%) in the liver of Bay41-4109 groups (as shown in fig. 5D); whereas neither virus antigen in the ETV group changed significantly (as shown in fig. 5C); further monitoring of the liver rcccDNA levels of each group of mice showed no significant change in the rcccDNA levels in the livers of each group of mice compared to the control group at the end of treatment (as shown in figures 5E-F) and at the end of follow-up (as shown in figure 5G); consistent with this result, significant rebound of HBV serum markers to baseline levels was observed in the AKEX0007 and ETV groups after discontinuation of dosing (as shown in fig. 5A).

In conclusion, the AAV8-rcccDNA mouse model can well reflect different action mechanisms of the tested three antiviral preparations on the virological indexes in serum and liver.

Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art from this description that certain modifications may be made within the scope of the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.