Construction of a recombinant plasmid and a hepatitis b virus full genome knock-in mouse model

Abstract

The application discloses a recombinant plasmid and a construction of a hepatitis B virus full-genome knock-in mouse model. The application firstly constructs a HBV full-genome 1.3-fold recombinant plasmid, which can be applied to screening of HBV drug-resistant sites, research of HBV drug-resistant mechanism, construction of HBV mouse models and the like. The application further establishes a hepatitis B virus full-genome knock-in mouse model with normal immune capacity. The mouse is mated with AlbCreERT2 to obtain a double-transgenic mouse. By adjusting a tamoxifen administration scheme and an age of the double-transgenic mouse, different stages of a natural history of HBV infection can be simulated. Therefore, the model can be used for basic researches on how an immune system of an acute infection host clears HBV and how chronic infection HBV causes tolerance and exhaustion of the immune system of the host, and can also be used for application researches on development and evaluation of new drugs for curing hepatitis B. In a word, the establishment of the model lays a solid foundation for analyzing a host immune panorama of each period of the natural history of hepatitis B virus infection and developing an immune treatment method for curing hepatitis B.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the construction of a recombinant plasmid and a hepatitis B virus whole genome knock-in mouse model. Background Technology

[0002] Hepatitis B virus (HBV) causes hepatitis B (HB). Although the incidence of new HBV infections has decreased significantly due to ongoing hepatitis B vaccination efforts, a large number of people worldwide still suffer from chronic HBV infection. Chronic HBV infection greatly increases the risk of cirrhosis and liver cancer, making it a significant infectious disease threatening public health. Antiviral therapy, including interferon and nucleoside analogues, has improved the prognosis for patients with chronic hepatitis B, but current treatments cannot effectively cure chronic hepatitis B.

[0003] Most existing in vitro studies related to HBV, such as those exploring HBV replication, transcriptional regulation, and antiviral mechanisms, are conducted by transfecting recombinant HBV DNA plasmids into liver cancer cell lines (such as HepG2 and Huh7 cells). Replicable HBV DNA recombinants must contain at least 1.1 times the HBV genome. Due to the unique structure of the HBV genome, constructing replicable recombinants in vitro is challenging. Literature reports that most HBV in vitro recombinants require multiple complex enzymatic digestion and ligation processes to replace fragments from laboratory standard viral strains. These methods are limited by the high heterogeneity of HBV, thus lacking versatility and making them unsuitable for multi-sample clinical trials.

[0004] HBV is a non-cytopathic virus, and the acute and chronic hepatitis it causes after infection is generally believed to be caused by the host's antiviral immune response. Establishing an animal model that can closely simulate the natural history of HBV infection is crucial for understanding the entire dynamic process of the interaction between the host immune system and HBV.

[0005] Human HBV exhibits high host species and tissue specificity, naturally infecting only humans, chimpanzees, and tree shrews. The use of chimpanzees is limited by their large size, high cost, and ethical constraints. Tree shrew infection with HBV is transient, with low HBV titers, and they are not inbred strains, making them unsuitable as routine laboratory animals. HBV-related viruses such as marmot HBV (WHV) and duck HBV (DHBV) have also been used in HBV-related research, but these models differ significantly from clinical acute and chronic hepatitis B models and are not ideal animal models for HBV.

[0006] Mice are the best choice for constructing HBV models due to their clear genetic background and ease of rearing. In recent years, mouse models used for hepatitis B research have made some progress, including HBV transgenic mice, mouse models of naked HBV DNA injected via high-pressure tail vein, mouse models of HBV genome delivered by adenovirus or adeno-associated virus vectors, and human-mouse chimeric liver models.

[0007] The work of Chisari et al. resulted in the creation of high-throughput HBV transgenic mice by inserting a 1.3-fold HBV genome into the mouse genome. Based on this HBV transgenic mouse model, it was elucidated that HBV does not cause host hepatocyte damage through direct cytopathic effects, but rather that the HBV-induced immune response, while mediating viral clearance, also leads to host hepatocyte damage. Furthermore, the HBV transgenic mice continuously produce high-titer viral particles, making this model important for antiviral drug evaluation. However, the expression of the HBV genome in these transgenic mice cannot be artificially regulated, and HBV replication begins in the suckling stage, leading to immune tolerance to HBV antigens. Therefore, this model is largely unsuitable for studying adaptive immune responses to HBV.

[0008] The high-pressure injection model involves injecting a large volume of liquid containing naked HBV DNA into mice via the tail vein within a short period. This model allows HBV replication and can be partially used to study the host's immune response to HBV. For example, this model has shown that CD4+ T cells, in addition to CD8+ CTLs, also play an important role in HBV clearance. However, high-pressure injection causes liver stress damage, and HBV is cleared rapidly in high-pressure tail vein mouse models, which typically only simulate acute HBV infection and are therefore unsuitable for studying chronic hepatitis B. In recent years, mouse models transduced with HBV genome via adenovirus vectors have been reported. In these models, HBV can persist for more than 3 months, mimicking the chronic HBV infection state to some extent. However, for studying the host's immune response to HBV, the immune response induced by the adenovirus vector itself cannot be ignored.

[0009] Chimeric liver models are based on three aspects: destruction of the mouse's own hepatocytes, absence of immune rejection, and transplantation of human hepatocytes. Destruction of the mouse's own hepatocytes can be achieved through methods such as uPA transfection or fah knockout; inducing immunodeficiency in mice through recombinase knockout can avoid transplant rejection. The HBV virus particles generated by this model can continue to infect hepatocytes and can form cccDNA. Therefore, it is suitable for evaluating antiviral drugs targeting cccDNA. However, obtaining human hepatocytes is difficult and costly, and this model is usually immunodeficient, which is not conducive to husbandry or studying the host's antiviral immune process.

[0010] Therefore, in order to more accurately analyze the host immune landscape at each stage of the natural history of hepatitis B virus infection and develop immunotherapies to cure chronic hepatitis B, it is crucial to construct a mouse model that can closely simulate the natural history of HBV infection. Summary of the Invention

[0011] This invention provides a 1.3-ploid recombinant plasmid of the HBV whole genome and a HBV whole genome knock-in mouse model with normal immune capacity, which can be used for research on the innate and adaptive immune response mechanisms of antiviral therapy in acute and chronic hepatitis B, as well as for application research such as the development and evaluation of drugs and vaccines that can cure hepatitis B.

[0012] To achieve this objective, the present invention provides the following technical solution:

[0013] In a first aspect, the present invention provides a 1.3-ploid recombinant plasmid of the whole HBV genome, the nucleotide sequence of which is SEQ ID NO.10.

[0014] Because most existing HBV recombinant strains are formed through a complex multi-step enzyme digestion and ligation process involving fragment replacement of laboratory standard viral strains, these methods are limited by the high heterogeneity of HBV, resulting in poor versatility and making them unsuitable for multi-sample clinical trials. In contrast, the recombinant plasmid provided in this application possesses a 1.3-ploidy of the entire HBV genome, enabling it to replicate in vitro and be used for screening HBV drug resistance sites, studying HBV drug resistance mechanisms, and constructing HBV mouse models.

[0015] In a second aspect, the present invention provides the application of the HBV whole genome 1.3-ploid recombinant plasmid described herein in screening HBV drug resistance sites, studying HBV drug resistance mechanisms, and constructing HBV mouse models.

[0016] A third aspect of the present invention provides a method for preparing the HBV whole genome 1.3-ploid recombinant plasmid described herein, comprising the following steps:

[0017] S1. An LSL expression cassette is introduced between the nt202 and nt203 sites in the HBV genome. The LSL expression cassette contains three termination signals: BGH polyA, SV40 polyA, and TK polyA. The nucleotide sequence of the LSL expression cassette is terminated by LoxP at both ends.

[0018] S2. An HDV genomicribozyme-miRNA122 target-HDV antigenomic ribozyme sequence is further introduced between the first LoxP and the termination signal site of the LSL expression cassette, wherein the miRNA122 target is in 5 repeats;

[0019] S3. Add 5' introns and 3' introns from the pCI-neo vector to both sides of the LSL expression cassette.

[0020] Preferably, the nucleotide sequence of the 5' intron of the pCI-neo vector is SEQ ID NO.1.

[0021] Preferably, the nucleotide sequence of the 3' intron of the pCI-neo vector is SEQ ID NO.2.

[0022] Preferably, the nucleotide sequence of the LoxP is SEQ ID NO.3.

[0023] Preferably, the nucleotide sequence of the miRNA122 target is SEQ ID NO.4.

[0024] Preferably, the nucleotide sequence of the HDV genomic ribozyme is SEQ ID NO.5.

[0025] Preferably, the nucleotide sequence of the HDV antigenomic ribozyme is SEQ ID NO.6.

[0026] Preferably, the nucleotide sequence of the BGH polyA is SEQ ID NO.7.

[0027] Preferably, the nucleotide sequence of the SV40 polyA is SEQ ID NO.8.

[0028] Preferably, the TK polyA nucleotide sequence is SEQ ID NO.9.

[0029] In this invention, the HBV genome is modified and edited by inserting “5'intron-LoxP-HDVgenomic ribozyme-miRNA122 target-HDV antigenomic ribozyme-BGH polyA-SV40polyA-TK polyA-LoxP-3'intron” between nt202 and nt203. The HDV genomic ribozyme and reverse genomic ribozyme (HDV genomic ribozyme, HDV antigenomic ribozyme) have site-specific RNA self-cleavage activity, effectively avoiding the expression of HBcAg and HBeAg caused by immature HBV mRNA induced by fentamoxifen.

[0030] In this invention, the miRNA122 target can guide the silencing complex (RISC) to degrade mRNA or inhibit its translation by pairing with the bases of the target gene mRNA, effectively avoiding the expression of HBcAg and HBeAg caused by immature HBV mRNA induced by fentamoxifen.

[0031] In this invention, the introduction of the intron (5'intron, 3'intron) causes the "5'intron-LoxP-3'intron" in the precursor HBV mRNA to be cleaved, thus allowing the mature HBV mRNA to be seamlessly recombined.

[0032] A fourth aspect of the present invention provides a method for constructing a hepatitis B virus whole genome knock-in mouse model with normal immune function, comprising the following steps:

[0033] R1. Obtain the 1.3-ploid recombinant plasmid of the HBV whole genome as described in claim 1;

[0034] R2. The 1.3-ploid recombinant plasmid of the whole HBV genome was knocked into mice by homologous recombination.

[0035] R3. The mice obtained in step R2 were bred with AlbCreERT2 mice to obtain double transgenic mice.

[0036] Preferably, step R1 includes the method for preparing the HBV whole genome 1.3-ploid recombinant plasmid provided by the present invention.

[0037] A preferred method for preparing a 1.3-ploid recombinant plasmid of the HBV whole genome includes the following steps:

[0038] S1. An LSL expression cassette is introduced between the nt202 and nt203 sites in the HBV genome. The LSL expression cassette contains three termination signals: BGH polyA, SV40 polyA, and TK polyA. The nucleotide sequence of the LSL expression cassette is terminated by LoxP at both ends.

[0039] S2. An HDV genomicribozyme-miRNA122 target-HDV antigenomic ribozyme sequence is further introduced between the first LoxP and the termination signal site of the LSL expression cassette, wherein the miRNA122 target is in 5 repeats;

[0040] S3. Add 5' introns and 3' introns from the pCI-neo vector to both sides of the LSL expression cassette.

[0041] Preferably, the nucleotide sequence of the 5' intron of the pCI-neo vector is SEQ ID NO.1.

[0042] Preferably, the nucleotide sequence of the 3' intron of the pCI-neo vector is SEQ ID NO.2.

[0043] Preferably, the nucleotide sequence of the LoxP is SEQ ID NO.3.

[0044] Preferably, the nucleotide sequence of the miRNA122 target is SEQ ID NO.4.

[0045] Preferably, the nucleotide sequence of the HDV genomic ribozyme is SEQ ID NO.5.

[0046] Preferably, the nucleotide sequence of the HDV antigenomic ribozyme is SEQ ID NO.6.

[0047] Preferably, the nucleotide sequence of the BGH polyA is SEQ ID NO.7.

[0048] Preferably, the nucleotide sequence of the SV40 polyA is SEQ ID NO.8.

[0049] Preferably, the TK polyA nucleotide sequence is SEQ ID NO.9.

[0050] A fifth aspect of the present invention provides a method for constructing a mouse HBV infection model, comprising the following steps: applying an HBV gene expression inducer to a double transgenic mouse model obtained by the method of the present invention.

[0051] Preferably, the HBV gene expression inducer includes tamoxifen.

[0052] Preferably, a central immune tolerance model of HBV can be established by injecting double transgenic mice in the infant stage with HBV gene expression inducers once or multiple times in a short period of time.

[0053] Preferably, an acute HBV infection model can be established by single or multiple injections of an HBV gene expression inducer into adult double-transgenic mice over a short period of time. More preferably, the HBV gene expression inducer is tamoxifen injection, 175 mg / kg intraperitoneally, once every 48 hours, for a total of 3 times.

[0054] Preferably, adult double-transgenic mice are continuously injected with an HBV gene expression inducer to initially establish an acute infection, and then establish a chronic HBV infection model as the immune system is exhausted. More preferably, the HBV gene expression inducer is tamoxifen injection, with an initial dose of 175 mg / kg, once every 48 hours for a total of 3 times; and a maintenance dose of 120 mg / kg, once every 6 days.

[0055] Preferably, the infant mice are mice aged 10 days to 4 weeks.

[0056] Preferably, the adult mice are 6-8 weeks old mice.

[0057] In this invention, establishing acute and chronic HBV infection models can be used for research on host antiviral immune mechanisms. It can also provide a new platform for the development and evaluation of novel drugs to cure hepatitis B.

[0058] Compared with the prior art, the beneficial effects and significant progress of the present invention are as follows:

[0059] 1. This invention provides a 1.3-ploid recombinant plasmid of the entire HBV genome, which is strictly regulated by Cre and can be used to screen HBV drug resistance sites, study HBV drug resistance mechanisms, and construct HBV mouse models.

[0060] 2. This invention establishes a hepatitis B virus whole-genome knock-in mouse model with normal immune function. This mouse is related to AlbCreER. T2 Double transgenic mice were obtained through crossbreeding. By adjusting the tamoxifen dosing regimen and the age of the tested double transgenic mice, different stages of the natural history of HBV infection can be simulated. Therefore, this model can be used for basic research on how the host immune system clears HBV during acute infection and how chronic HBV infection leads to host immune system tolerance and exhaustion. It can also be used for applied research in the development and evaluation of novel drugs to cure hepatitis B. In summary, the establishment of this model lays a solid foundation for understanding the host immune landscape at each stage of the natural history of hepatitis B virus infection in order to develop immunotherapies to cure hepatitis B. Attached Figure Description

[0061] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0062] Obviously, the accompanying drawings described below are only some of the drawings of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort, but these other drawings are also within the scope of the drawings required for the embodiments of the present invention.

[0063] Figure 1A is a schematic diagram of the 1.3-ploid recombinant plasmid of the whole HBV genome in Example 1 of the present invention;

[0064] Figure 1 B is a schematic diagram of gRNA-mediated insertion of the HBV genome into the host genome in Embodiment 1 of the present invention;

[0065] Figure 2 A is a schematic diagram of Southern Blot identification of enzyme cleavage sites in Example 3 of the present invention;

[0066] Figure 2 B represents the insertion of the Southern Blot-confirmed knock-in segment in Embodiment 3 of this invention;

[0067] Figure 2 C is a schematic diagram of the PCR identification primer target region in Example 3 of the present invention;

[0068] Figure 2 D is a graph showing the PCR identification results in Example 3 of this invention;

[0069] Figure 3 A represents the 4-week and 8-week-old HBV genome single transgenic mice and HBV genome and AlbCreERT2 double transgenic mice in Example 4 of the present invention, whose serum HBsAg and HBeAg were detected by ELISA.

[0070] Figure 3 B represents the 10-day-old (left) and 6-week-old (right) double transgenic mice in Example 4 of this invention. The serum levels of HBsA and HBeAg were detected by ELISA after tamoxifen induction.

[0071] Figure 4 This is a schematic diagram illustrating how the HBV whole genome knock-in mice in Example 4 of the present invention simulate different stages of the natural history of HBV infection by adjusting the tamoxifen dosing regimen and the age of the test mice. Detailed Implementation

[0072] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0073] Obviously, all the embodiments described are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0074] What needs to be understood is:

[0075] Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0076] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments.

[0077] The technical solution of the present invention will now be described in detail with reference to specific embodiments.

[0078] Example 1: HBV Genome Modification

[0079] According to such Figure 1 The method shown in A modifies the HBV genome.

[0080] 1.1 First, a Loxp-Stop-Loxp (LSL) expression cassette is introduced between the nt202 and nt203 sites in the HBV genome. The LSL expression cassette contains three termination signals (BGH polyA, SV40 polyA, and TK polyA sequences as shown below), with LoxPs at both ends (LoxP sequences as shown below). Because the LSL expression cassette already contains complete HBV core antigen and e antigen (HBcAg, HBeAg) open reading frames upstream, incomplete HBV transcripts will lead to Cre-independent HBcAg and HBeAg expression.

[0081] 1.2. The sequence “HDV genomicribozyme-miRNA122 target-HDV antigenomic ribozyme” was further introduced between the first LoxP and Stop sites of the LSL expression cassette. This allowed the incomplete HBV transcript to be separated from the three termination signals, BGHpolyA, SV40 polyA, and TK polyA, and degraded by tissue cells to limit the expression of non-Cre-dependent HBcAg and HBeAg. Because the wild-type hepatitis D virus ribozyme (HDV genomic ribozyme, HDV antigenomicribozyme, sequence below) has site-specific RNA self-cleavage activity, its active core domain is generally considered to be 85 nt. However, some studies have shown that its flanking sequences may affect its catalytic activity. Therefore, this study used a 126 nt genomic ribozyme sequence and a 129 nt reverse genomic ribozyme sequence. The microRNA122 (miRNA122) molecule is extremely abundant in liver tissue. It can guide the silencing complex (RISC) to degrade mRNA or inhibit its translation by forming complementary pairing with target gene mRNA. Therefore, in this study, five repeating miRNA122 target gene sequences totaling 35 nt were placed between two ribozyme sequences.

[0082] 1.3. Add 5' intron and 3' intron from the pCI-neo vector (Promega) to both sides of the LSL expression cassette (5' intron, 3' intron sequence below). Through the posttranscriptional splicing effect brought about by the intron, the final mature HBV mRNA can be seamlessly recombined.

[0083] In summary, this invention modifies and edits the HBV genome by inserting “5'intron–LoxP-HDVgenomic ribozyme-miRNA122 target-HDV antigenomic ribozyme-BGH polyA-SV40polyA-TK polyA-LoxP-3'intron” between nt202 and nt203. The nucleotide sequence of the modified HBV whole genome 1.3-ploid recombinant plasmid is SEQ ID NO.10. The above sequence is shown in Table 1 below.

[0084] Table 1

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] Example 2: Construction of a whole-genome knock-in mouse model of hepatitis B virus

[0091] 2.1. The pre-modified HBV whole genome 1.3-ploid recombinant plasmid (SEQ ID NO.10) from Example 1 was microinjected into mouse zygotes. Using the CRISPR-Cas9 method, the HBV genome was retrogradely inserted into the first intron located at the rosa26 site on chromosome 6 via gRNA guidance. The specific operational principle is described in [link to detailed procedure]. Figure 1 B. To construct the targeting vector, a BAC clone from the C57BL / 6 library was used as a template, and homologous arms were generated by PCR. Cas9 mRNA and gRNA were co-injected with the targeting vector into fertilized eggs (gRNA sequences are SEQ ID NO.11 and SEQ ID NO.12) to generate gene knock-in mice. All of the above procedures were performed by Guangzhou Cyagen Biotech Co., Ltd.

[0092] Table 2

[0093] SEQ ID NO.11 gRNA1 5'-GGCAGGCTTAAAGGCTAACCTGG-3' SEQ ID NO.12 gRNA 2 5'-CTCCAGTCTTTCTAGAAGATGGG-3'

[0094] Example 3: Gene-level validation in mice

[0095] This example validates the hepatitis B virus whole-genome knock-in mice with normal immune function prepared in Example 2. The insertion of the target sequence was validated at the gene level in the constructed hepatitis B virus whole-genome knock-in mice using Southern Blot and PCR methods.

[0096] Experimental groups: normal wild-type mice (WT) and hepatitis B virus whole genome knock-in mice (MT) with normal immune capacity prepared in Example 2.

[0097] Experimental methods:

[0098] Southern Blot Method and Results

[0099] 3.1 Preparation of DNA for testing: Genomic DNA was obtained from WT and MT mice. Cells were lysed using appropriate chemical reagents, and most proteins and RNA were digested using proteases and RNases; proteins were removed using organic reagent (phenol / chloroform) extraction.

[0100] Prepare Southern blot probes. Take the WT group mouse DNA obtained in step 3.1 and perform PCR amplification using the following Southern Blot primers to obtain 5' and 3' hybridization fragments that can form complementary base pairs with the DNA sample to be tested. Clone these fragments into the pUC19 vector (containing the T7 in vitro transcription promoter), and perform in vitro transcription and digoxigenin labeling using the Roche in vitro transcription probe preparation kit (Cat. No. 12039672910). Aliquot the probes and store them at -80℃ for later use.

[0101] The primer sequences for Southern Blot are as follows:

[0102] Primers for 5'Probe:

[0103] 5'Probe forward primer(SEQ ID NO.13):5'-AAACGTGGAGTAGGCAATACCCAGG-3'

[0104] 5'Probe reverse primer(SEQ ID NO.14):5'-AAAGAAGGGTCACCTCAGTCTCCCT-3'

[0105] Primers for 3'Probe:

[0106] 3'Probe forward primer(SEQ ID NO.15):5'-TTCTGGGCAGGCTTAAAGGCTAAC-3'

[0107] 3'Probe reverse primer(SEQ ID NO.16):5'-AGGAGCGGGAGAAATGGATATGAAG-3'

[0108] 3.2. Shearing with Bsu36I restriction endonuclease. Take the sample DNA solution obtained in step 3.1 and add it to a 0.5 ml Eppendorf tube. Add 2 μl of 10× restriction endonuclease buffer, 6–10 U of the corresponding restriction endonuclease, and sterile double-distilled water to a total volume of 20 μl. Incubate at 37°C for 2 hours, then heat at 65°C for 5 minutes or terminate the reaction with an appropriate amount of 0.5 mol / L EDTANa2.

[0109] The results are as follows Figure 2 As shown in Figure A, WT mice can obtain a fragment of 4.48KB, while MT mice can obtain a fragment of 6.52kb.

[0110] 3.3 Separation of DNA samples by agarose gel electrophoresis. Take 2 μl of the DNA digest obtained in step 3.2 and add 10 μl of sample buffer (containing bromophenol blue indicator and glycerol), and perform horizontal electrophoresis on a 0.8% agarose gel (containing 0.5 μg / ml ethidium bromide) at a voltage <5V / cm for about 2 hours.

[0111] After alkaline denaturation, the DNA samples separated by electrophoresis were transferred to a solid support (nylon membrane). The agarose gel after electrophoresis was denatured in alkaline denaturing solution (0.5M NaOH, 1.5M NaCl) for 45 min, rinsed three times with double-distilled water, and treated with neutralization solution (1M Tris-HCl, 1.5M NaCl, pH 7.4) for 45 min. The transfer system was then assembled using the descending capillary transfer method, and the membrane was allowed to stand for 8 hours.

[0112] DNA immobilized on the membrane was annealed and hybridized with a digoxigenin-labeled probe. The nylon membrane containing the sample DNA obtained above was baked at 80°C for 2 hours and then placed in a hybridization tube. 5 ml of hybridization solution was added and pre-hybridized at 42°C for 30 minutes. Then, the prepared Southern blot probe was added (denatured at 65°C for 10 minutes before adding the probe and immediately placed on ice for 5 minutes). Hybridization was carried out for about 8 hours.

[0113] The membrane was incubated with alkaline phosphatase-labeled digoxigenin antibody. The hybridization solution in the hybridization tube was discarded, and the membrane was washed with low-strength wash buffer at 25°C for 5 minutes. This was repeated once. Then, the membrane was washed with high-strength wash buffer at 68°C for 15 minutes. This was repeated once. Blocking buffer was added and the membrane was blocked at 25°C for 1 hour. Finally, the membrane was incubated with digoxigenin antibody at 25°C for 30 minutes.

[0114] Add alkaline phosphatase substrate for color development. Discard the antibody incubation buffer in the hybridization tube, add washing buffer and wash at 25°C for 15 minutes, repeat once. Then add detection buffer and wash for 5 minutes, repeat once. Remove the membrane and place it on parchment paper, add chemiluminescence solution for development and detection.

[0115] The results are as follows Figure 2 As shown in the left figure (B), WT represents the WT mouse group, and 9 and 13 represent the MT mouse group. MT mice can obtain two fragments, 6.52kb and 4.48kb, while WT mice only have a 4.48kb fragment.

[0116] 3.4. Using the same method as steps 3.2 and 3.3, perform Southern Blot experiments with another restriction endonuclease, EcoNI.

[0117] The results are as follows Figure 2 As shown in Figure B on the right, WT represents the WT mouse group, and 9 and 13 represent the MT mouse group. MT mice can obtain two fragments, 8.99kb and 3.80kb, while WT mice only have a 3.80kb fragment.

[0118] PCR method

[0119] 3.5. Prepare the DNA to be tested. The genomic DNA of WT group mice and MT group mice was obtained by the method in step 3.1.

[0120] 3.6 Design two pairs of specific PCR primers, Primes1 and Primes2, such as... Figure 2 As shown in C, both Primes1 and Primes2 primer pairs contain fragments that pair with the inserted sequence and the non-inserted sequence, respectively.

[0121] 3.7. The DNA obtained in step 3.5 was amplified by PCR using the two primer pairs Primes1 and Primes2 designed in step 3.6.

[0122] The results are as follows Figure 2 As shown in Figure D, WT represents the WT mouse group, 9 and 13 represent the MT mouse group, and Water represents the blank control. MT mice were amplified using these two primer pairs to obtain fragments of 5.2 kb (Primes1) and 5 kb (Primes2), respectively. No products were amplified in the WT and Water groups.

[0123] However, during routine breeding, the genotype of this mouse strain is identified using Primes3 and Primes4. WT mice can be amplified using Primes3 to produce a 453bp fragment. Homozygous MT mice can be amplified using Primes4 to produce a 272bp fragment. Heterozygous MT mice can be amplified using both Primes3 and Primes4.

[0124] The above experiments demonstrate that the present invention successfully guided the Cas9 enzyme to insert the modified target HBV genome sequence into the rosa26 site of C57BL / 6 mice via gRNA.

[0125] Table 3

[0126]

[0127]

[0128] Example 4 Antigen Level Validation in Mice

[0129] This embodiment validates the MT mice constructed in Example 2 at the antigen level using the ELISA method.

[0130] 4.1 Experimental Mice: The MT mice constructed in Example 2 were in the LoxP-HBV group. The MT mice constructed in Example 2 were compared with AlbCreER... T2 Mice were bred to obtain double transgenic mice containing the HBV genome and AlbCreERT2, namely LoxP-HBV-AlbCreERT2. T2 Group.

[0131] 4.2. Four-week-old juvenile and eight-week-old adult HBV genome single transgenic mice (LoxP-HBV group) and HBV genome and AlbCreERT2 double transgenic mice (LoxP-HBV-AlbCreERT2 group) that were not induced by tamoxifen were used. T2 Blood was drawn from the orbital cavity of the group.

[0132] 4.3 ELISA detection of HBsAg and HBeAg in serum.

[0133] The results are as follows Figure 3 As shown in Figure A, there was no leakage expression of HBsAg and HBeAg without tamoxifen induction.

[0134] 4.4 Ten-day-old suckling mice and six-week-old adult mice were intraperitoneally injected with tamoxifen dissolved in corn oil at a dose of 175 mg / kg (single injection for suckling mice; once every 48 hours for adult mice, for a total of three injections). Blood samples were collected from the orbital rim at a series of time points after the injection (5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, and 9 weeks for suckling mice; and 0 days, 2 days, 3 days, 4 days, 6 days, 9 days, 12 days, 15 days, and 18 days for adult mice).

[0135] 4.5. ELISA detection of HBsAg and HBeAg in serum.

[0136] The results are as follows Figure 3 As shown in B, after tamoxifen induction, CreERT2 translocates into the nucleus and exerts recombinase activity. The sequence “5'LoxP-HDV genomic ribozyme-miRNA122 target-HDV antigenomic ribozyme-BGH polyA-SV40 polyA-TK polyA-LoxP” is cleaved, leaving one LoxP sequence. The “5'intron-LoxP-3'intron” sequence in the precursor HBV mRNA is then cleaved by posttranscriptional splicing, thus allowing HBV-related antigens to be induced and regulated.

[0137] ELISA analysis showed that a single dose of tamoxifen induced chronic HBV infection in double-transgenic neonatal mice, with serum HBsAg reaching a peak (approximately 540 IU / ml) at 6 weeks of age, while serum HBeAg levels fluctuated around 10 PEIU / ml, and both HBsAg and HBeAg persisted for more than 9 weeks. A short-term dose of tamoxifen induced acute HBV infection in double-transgenic adult mice, with serum HBsAg reaching a peak (approximately 160 IU / ml) 3 days after injection and persisting for about 2 weeks; serum HBeAg reached a peak (approximately 5 PEIU / ml) 9 days after injection and persisted for more than 18 days. Therefore, this invention successfully obtained 1.3-fold HBV genome knock-in mice. Double-transgenic mice bred from these mice with AlbCreERT2 do not exhibit antigen expression induced by non-tamoxifen and can be induced by tamoxifen to simulate acute and chronic HBV infection.

[0138] In addition, such as Figure 4 As shown, by adjusting the tamoxifen dosing regimen and the age of the test mice, different stages of the natural history of HBV infection can be simulated. Therefore, the hepatitis B virus whole-genome knock-in mice we designed and constructed can serve as an important tool for studying viral dynamics and host antiviral immune mechanisms in acute and chronic HBV infection, and can also provide a new and effective platform for developing and evaluating novel drugs to cure hepatitis B.

[0139] In summary, this invention designed a 1.3-fold HBV genome knock-in mouse, which was then bred with AlbCreERT2 to obtain a double transgenic mouse. In this model mouse, HBV gene expression is tamoxifen-dependent. Therefore, compared with conventional HBV transgenic mice, the hepatitis B mouse model designed in this invention does not exhibit natural immune tolerance to HBV antigen. In this invention, the 1.3-fold HBV genome was modified and edited by introducing functional elements such as the miRNA122 target and HDV ribozyme, effectively avoiding the expression of HBcAg and HBeAg caused by immature HBV mRNA induced by tamoxifen.

[0140] In the description process of the above instruction manual:

[0141] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0142] Finally, it should be noted that:

[0143] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0144] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A 1.3-ploid recombinant plasmid of the entire HBV genome, characterized in that, The nucleotide sequence of the recombinant plasmid is SEQ ID NO.

10.

2. The application of the HBV whole genome 1.3-ploid recombinant plasmid as described in claim 1 in screening HBV drug resistance sites, studying HBV drug resistance mechanisms, and constructing HBV mouse models.

3. The method for preparing the HBV whole genome 1.3-ploid recombinant plasmid according to claim 1, characterized in that, Includes the following steps: S1. An LSL expression cassette is introduced between the nt202 and nt203 sites in the HBV genome. The LSL expression cassette contains three termination signals: BGHpolyA, SV40polyA, and TKpolyA. The nucleotide sequence of the LSL expression cassette is terminated by LoxP at both ends. S2. An HDVgenomicribozyme-miRNA122 target-HDV antigenomicribozyme sequence is introduced between the first LoxP and the termination signal site of the LSL expression cassette, wherein the miRNA122 target is in 5 repeats. S3. Add 5' introns and 3' introns from the pCI-neo vector to both sides of the LSL expression cassette.

4. The method as described in claim 3, characterized in that, The nucleotide sequence of the 5' intron of the pCI-neo vector is SEQ ID NO.

1.

5. The method as described in claim 3, characterized in that, The nucleotide sequence of the 3' intron of the pCI-neo vector is SEQ ID NO.

2.

6. The method as described in claim 3, characterized in that, The nucleotide sequence of the LoxP is SEQ ID NO.

3.

7. The method as described in claim 3, characterized in that, The nucleotide sequence of the miRNA122 target is SEQ ID NO.

4.

8. The method as described in claim 3, characterized in that, The nucleotide sequence of the HDV genomicribozyme is SEQ ID NO.

5.

9. The method as described in claim 3, characterized in that, The nucleotide sequence of the HDV antigenomicribozyme is SEQ ID NO.

6.

10. The method as described in claim 3, characterized in that, The nucleotide sequence of BGHpolyA is SEQ ID NO.

7.

11. The method as described in claim 3, characterized in that, The nucleotide sequence of SV40polyA is SEQ ID NO.

8.

12. The method as described in claim 3, characterized in that, The TKpolyA nucleotide sequence is SEQ ID NO.

9.

13. A method for constructing a hepatitis B virus whole-genome knock-in mouse model with normal immune function, characterized in that, Includes the following steps: R1. Obtain the 1.3-ploid recombinant plasmid of the HBV whole genome as described in claim 1; R2. The 1.3-ploid recombinant plasmid of the whole HBV genome was knocked into mice by homologous recombination. R3. The mice obtained in step R2 were bred with AlbCreERT2 mice to obtain double transgenic mice.

14. The method as described in claim 13, characterized in that, Step R1 includes the method described in any one of claims 3-9.

15. A method for constructing a mouse HBV infection model, characterized in that, The method includes the following steps: applying an HBV gene expression inducer, wherein the HBV gene expression inducer is tamoxifen, to a double transgenic mouse model obtained by the method of claim 13 or 14.

16. The method as described in claim 15, characterized in that, A central immune tolerance model of HBV can be established by injecting double transgenic mice during infancy with a single or multiple injections of HBV gene expression inducers over a short period of time.

17. The method as described in claim 15, characterized in that, An acute HBV infection model can be established by injecting adult double-transgenic mice with HBV gene expression inducers once or multiple times over a short period of time.

18. The method as described in claim 15, characterized in that, Adult double-transgenic mice can be continuously injected with HBV gene expression inducers to first establish an acute infection model, and then establish a chronic HBV infection model as the immune system is exhausted.

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