Application of donkey-derived biological components in analyzing human HBV or its satellite virus infection

Abstract

The present invention discloses the use of donkey-derived biological components in analyzing human HBV or its satellite virus infection. Through genetic phylogenetic tree analysis, the present invention screened and identified donkey sodium ion / taurocholic acid co-transporting peptides that are similar to human ones. Furthermore, experimental confirmation confirmed that the donkey sodium ion / taurocholic acid co-transporting peptides can support human HBV and / or its satellite virus infection, and that primary donkey hepatocytes can support human HBV and / or its satellite virus infection. This invention enables the construction of an economical and efficient animal model for HBV infection, which is of great significance for antiviral research and drug development against HBV and / or its satellite viruses.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of donkey-derived biological components in analyzing human HBV or its satellite virus infection. Background Art

[0002] Hepatitis B virus (HBV) is a worldwide epidemic pathogen that causes diseases such as cirrhosis and liver cancer, posing a significant threat to human health and life. Therefore, there is an urgent need for basic and translational research aimed at curing hepatitis B. Hepatitis D virus (HDV) is a satellite virus of HBV, utilizing the HBV envelope protein for infection and the production and release of progeny viruses, thus sharing the same early mechanisms of HBV infection of hepatocytes. Hepatitis D is a severe viral liver disease caused by HDV infection in hepatitis B carriers or by co-infection with HBV and HDV. Compared to HBV infection alone, co-infection with HBV and HDV significantly develops into a chronic infection and significantly accelerates the progression of liver disease to terminal, severe conditions such as cirrhosis and liver cancer. 70-80% of patients develop cirrhosis within 5-10 years, and the incidence of liver cancer is 2-3 times that of HBV infection alone. Approximately half of patients die from related liver diseases within 10 years of diagnosis.

[0003] HBV has a very narrow host infection spectrum, primarily infecting humans and gorillas, with low efficiency infecting tree shrews, but unable to infect experimental animals such as mice, rats, pigs, dogs, and non-human primates. Currently, commonly used alternative in vivo HBV research models include hydrodynamic injection into the mouse tail vein, Adv / AAV-mediated in vivo expression of HBV in mice or monkeys, and HBV transgenic mouse models. However, these models can only simulate HBV replication in vivo and suffer from issues such as short viral persistence and immune tolerance. They cannot represent de novo viral infection or simulate long-term chronic HBV infection and host-virus interactions. In recent years, human liver chimeric mouse models have been gradually developed and applied. By transplanting primary human hepatocytes into immunodeficient recipient mice, a mouse model that supports HBV infection has been constructed. However, the limited availability and extremely high cost of primary human hepatocytes have severely restricted the construction and widespread application of human liver chimeric mice. Therefore, there is an urgent need to develop a cost-effective and efficient animal model of HBV infection. Summary of the Invention

[0004] To address at least some of the problems in the prior art, the present invention provides a method for analyzing human HBV or its satellite virus infection using a donkey-derived biological component. Specifically, the present invention includes the following:

[0005] The first aspect of the present invention provides a use of a donkey-derived biological component in constructing a human HBV and / or its satellite virus infection model, wherein the donkey-derived biological component includes a donkey sodium ion / taurocholic acid co-transporter peptide or its gene, and / or donkey hepatocytes.

[0006] In certain embodiments, according to the use of the present invention, the human HBV and / or its satellite virus include human hepatitis B virus and / or human hepatitis D virus.

[0007] In certain embodiments, according to the use of the present invention, the sources of the donkey hepatocytes include primary hepatocytes, immortalized donkey liver cell lines, donkey hepatocytes obtained by induced differentiation, genetically engineered hepatocytes or liver organoids.

[0008] In certain embodiments, according to the use of the present invention, the infection model comprises a molecular model, a cell model or an animal model.

[0009] A second aspect of the present invention provides a method for constructing an organism for simulating infection with human HBV and / or its satellite virus, comprising the following steps:

[0010] (1) providing an organism comprising a donkey sodium ion / taurocholic acid co-transporting peptide or a gene thereof or being capable of producing a donkey sodium ion / taurocholic acid co-transporting peptide under desired conditions; and

[0011] (2) contacting human HBV and / or its satellite virus with the organism.

[0012] The third aspect of the present invention provides an organism for simulating human HBV and / or its satellite virus infection, wherein the organism contains a donkey sodium ion / taurocholic acid co-transporting peptide or its gene or is capable of producing a donkey sodium ion / taurocholic acid co-transporting peptide under required conditions, wherein the donkey sodium ion / taurocholic acid co-transporting peptide or its gene is inherent to the organism or is artificially introduced into the organism from an exogenous source.

[0013] In a fourth aspect, the present invention provides a method for constructing an animal model of human HBV and / or its satellite virus infection, comprising the step of introducing donkey hepatocytes into a heterologous host animal to form a chimeric liver or a complete liver.

[0014] In a fifth aspect, the present invention provides a method for screening drugs for treating HBV and / or its satellite virus infection, which comprises the step of using the organism or animal model of the present invention.

[0015] In a sixth aspect, the present invention provides a method for screening a drug for treating HBV and / or its satellite virus infection according to the present invention, comprising the following steps:

[0016] (I) measuring the amount of HBV and / or its satellite virus in the organism or animal model to obtain a first measurement value;

[0017] (II) contacting the test drug with the organism or animal model;

[0018] (III) measuring the amount of HBV and / or its satellite virus in the organism or animal model after exposure to the test drug to obtain a second measurement value;

[0019] (IV) comparing the first measurement value and the second measurement value; when the second measurement value is less than the first measurement value, screening the drug to be tested as a candidate drug capable of treating HBV and / or its satellite virus infection; and when the second measurement value is greater than or equal to the first measurement value, screening the drug to be tested as a drug that is useless for treating HBV or its satellite virus infection.

[0020] The seventh aspect of the present invention provides the use of an organism or animal model in antiviral research or drug development, wherein the virus includes human HBV and / or its satellite virus, the organism is the organism described in the third aspect of the present invention, and the animal model is an animal model obtained by the method according to the fourth aspect of the present invention.

[0021] The present invention screened for a donkey sodium / taurocholate cotransporting peptide (NTCP) close to human origin through genetic phylogenetic tree analysis. Furthermore, experiments confirmed that donkey NTCP can support infection with human HBV and / or its satellite viruses, and that primary donkey hepatocytes can support infection with human HBV and / or its satellite viruses. This invention enables the construction of an economical and efficient animal model for HBV infection, which is of great significance for antiviral research and drug development against HBV and / or its satellite viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of genetic phylogenetic tree analysis of NTCP amino acid sequences from different species are shown.

[0023] Figure 2 The results of the comparison of NTCP amino acid sequences from different species are shown.

[0024] Figure 3 Donkey NTCP was shown to be able to support HBV binding.

[0025] Figure 4 Donkey NTCP was shown to be able to support infection with the HBV satellite virus HDV.

[0026] Figure 5 It shows that donkey NTCP can support HBV virus infection, where A is the HBV infection and detection process; B is the detection result of the virological marker HBeAg in the cell infection supernatant; C is the detection result of HBV cccDNA in the infected cells.

[0027] Figure 6It shows that donkey primary hepatocytes can support HBV infection, wherein A shows that donkey primary hepatocytes can adhere to the wall and culture after isolation and show good epithelial cell characteristics; B shows that donkey primary hepatocytes support HBV infection. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0031] Uses of donkey-derived bioingredients

[0032] One aspect of the present invention provides the use of donkey-derived biological components in establishing a human HBV and / or its satellite virus infection model, wherein the donkey-derived biological components include a donkey sodium / taurocholate co-transporting peptide or its gene, and / or donkey hepatocytes. HBV satellite viruses include hepatitis delta virus (HDV), which utilizes the HBV envelope for progeny replication and release, resulting in HDV infection of hepatocytes similar to HBV.

[0033] In the present invention, the donkey-derived biological components should be understood in a broad sense, including donkey liver cells, donkey tissues, donkey organs of natural origin, or artificially synthesized recombinant proteins containing donkey sodium ion / taurocholic acid co-transporting peptides, organoids containing donkey sodium ion / taurocholic acid co-transporting peptides, nucleic acid molecules encoding donkey sodium ion / taurocholic acid co-transporting peptides, vector molecules containing the nucleic acid molecules, or host cells containing the vector molecules.

[0034] In the present invention, the human HBV and / or its satellite virus include human hepatitis B virus and / or human hepatitis D virus. In a preferred embodiment, the HBV and / or its satellite virus is human hepatitis B virus. In another preferred embodiment, the HBV and / or its satellite virus is human hepatitis B virus and human hepatitis D virus.

[0035] In the present invention, the sources of the donkey hepatocytes include, but are not limited to, primary hepatocytes, immortalized donkey liver cell lines, donkey hepatocytes derived by induced differentiation, genetically engineered hepatocytes, or liver organoids. The induced differentiated donkey hepatocytes may be differentiated products of totipotent stem cells, pluripotent stem cells, unipotent stem cells, or iPS cells.

[0036] In the present invention, the infection model includes, but is not limited to, a molecular model, a cell model, or an animal model. Examples of cells include, but are not limited to, PHH, HepG2, Huh7, and HepaRG cells. Examples of animals include, but are not limited to, rats, mice, rabbits, pigs, dogs, donkeys, cattle, sheep, and monkeys. In a preferred embodiment, the infection model is a HepG2 cell model. In another preferred embodiment, the infection model is a mouse model.

[0037] Method for constructing an organism for simulating human HBV and / or its satellite virus infection

[0038] One aspect of the present invention provides a method for constructing an organism for simulating infection by human HBV and / or its satellite virus.

[0039] In a preferred embodiment, the method for constructing an organism for simulating human HBV and / or its satellite virus infection comprises the following steps:

[0040] (1) providing an organism comprising a donkey sodium ion / taurocholic acid co-transporting peptide or a gene thereof or being capable of producing a donkey sodium ion / taurocholic acid co-transporting peptide under desired conditions; and

[0041] (2) contacting human HBV and / or its satellite virus with the organism.

[0042] In the present invention, examples of the organism include, but are not limited to, cells (such as, but not limited to, naturally derived donkey hepatocytes, donkey hepatocytes obtained by induced differentiation, genetically engineered hepatocytes, etc.), cell populations (such as, but not limited to, donkey hepatocyte populations), tissues (such as, but not limited to, donkey liver tissue), organoids (such as, but not limited to, donkey liver organoids), and organs (such as, but not limited to, donkey liver organs).

[0043] In the present invention, "capable of producing the donkey sodium ion / taurocholic acid co-transporting peptide under desired conditions" means that the organism already contains the donkey sodium ion / taurocholic acid co-transporting peptide before contacting the organism with human HBV and / or its satellite virus. For example, the organism contains the donkey sodium ion / taurocholic acid co-transporting peptide by containing a nucleic acid molecule encoding the donkey sodium ion / taurocholic acid co-transporting peptide, a vector molecule containing the nucleic acid molecule, or a host cell containing the vector molecule.

[0044] organism

[0045] One aspect of the present invention provides an organism for simulating human HBV and / or its satellite virus infection, wherein the organism contains a donkey sodium ion / taurocholic acid co-transporting peptide or its gene or is capable of producing a donkey sodium ion / taurocholic acid co-transporting peptide under desired conditions, wherein the donkey sodium ion / taurocholic acid co-transporting peptide or its gene is inherent to the organism or is artificially introduced into the organism from an exogenous source.

[0046] In the present invention, the term "inherent in an organism" refers to an organism naturally containing the donkey sodium ion / taurocholic acid co-transporter peptide or its gene. Examples of such organisms are described above and will not be repeated here. The term "artificial means" is not particularly limited, as long as the donkey sodium ion / taurocholic acid co-transporter peptide or its gene can be introduced into the organism and achieve the corresponding function. Examples include, but are not limited to, direct protein delivery, gene injection, liposome transfection, introduction into biological vectors, and gene editing.

[0047] Method for constructing an animal model of human HBV and / or its satellite virus infection

[0048] One aspect of the present invention provides a method for constructing an animal model of human HBV or its satellite virus infection.

[0049] In a preferred embodiment, the method of the present invention for constructing an animal model for infection with human HBV and / or its satellite viruses comprises the step of introducing donkey hepatocytes into a heterologous host animal to form a chimeric or intact liver. The host animal is not particularly limited, and examples include, but are not limited to, rats, mice, rabbits, pigs, dogs, donkeys, cattle, sheep, monkeys, and the like. In a specific embodiment, the method for constructing an animal model for infection with human HBV and / or its satellite viruses comprises the step of introducing donkey hepatocytes into a mouse to form a chimeric liver.

[0050] Method for screening drugs for treating HBV and / or its satellite virus infection

[0051] In one aspect, the present invention provides a method for screening a drug for treating HBV and / or its satellite virus infection, comprising using the organism or animal model described herein. The organism or animal model is as described above and will not be further described here.

[0052] In a preferred embodiment, the method for screening drugs for treating HBV and / or its satellite virus infection comprises the following steps:

[0053] (I) measuring the amount of HBV and / or its satellite virus in the organism or animal model to obtain a first measurement value;

[0054] (II) contacting the test drug with the organism or animal model;

[0055] (III) measuring the amount of HBV and / or its satellite virus in the organism or animal model after exposure to the test drug to obtain a second measurement value;

[0056] (IV) comparing the first measurement value and the second measurement value; when the second measurement value is less than the first measurement value, screening the drug to be tested as a candidate drug capable of treating HBV and / or its satellite virus infection; and when the second measurement value is greater than or equal to the first measurement value, screening the drug to be tested as a drug that is useless for treating HBV and / or its satellite virus infection.

[0057] Use of organisms or animal models in antiviral research or drug development

[0058] One aspect of the present invention provides the use of the organisms or animal models described herein in antiviral research or drug development, wherein the virus comprises human HBV or its satellite virus. Examples of such antiviral research include, but are not limited to, studies of viral structure and function, viral gene editing, viral-host interaction mechanisms, and host immune responses. Drug development includes target identification and validation, in vitro experiments, and the like.

[0059] Example

[0060] It is shown below that donkey-derived biological components are capable of supporting infection with HBV and / or its satellite viruses.

[0061] 1. Donkey NTCP is similar to human NTCP

[0062] 1.1 Experimental Methods

[0063] In this example, a genetic phylogenetic tree analysis was performed on the NTCP amino acid sequence, including humans, Old World monkeys, New World monkeys, donkeys, tree shrews, pigs, cattle, sheep, rabbits, mice, marmots, and squirrels. The NTCP sequences of these species were compared in detail, and amino acids 84-87 and 157-165, which are known to be important for HBV infection, were analyzed.

[0064] 1.2 Experimental Results

[0065] Depend on Figure 1It can be seen that the NTCP of donkeys is very close to that of primates, and the similarity between donkey NTCP and human NTCP is higher than that of tree shrew NTCP, which is known to support HBV infection. Figure 2 It can be seen that donkey NTCP is consistent with the corresponding sites of human NTCP at amino acids 87, 158, and 165. This shows that donkey NTCP is very similar to human NTCP.

[0066] 2. Donkey NTCP supports virus binding

[0067] 2.1 Experimental Methods

[0068] To confirm that donkey NTCP can support HBV infection, this example used in vitro experiments in the human liver cancer cell line HepG2. First, a donkey NTCP lentiviral overexpression vector was constructed, and the tagRFP fluorescent protein was added to the 3' end of NTCP. Human NTCP was also constructed as a positive control. Next, HepG2 cells were infected with lentivirus to overexpress both donkey NTCP and human NTCP. The binding of the HBV preS1 peptide to donkey NTCP was then investigated.

[0069] 2.2 Experimental Results

[0070] Laser confocal microscopy revealed that HepG2 control cells did not express NTCP, while both human and donkey NTCP were expressed on the cell membrane, showing red fluorescence. The results showed that HBV could bind to the cell surface expressing human and donkey NTCP, and HBV and NTCP had good co-localization ( Figure 3 ). This shows that donkey NTCP supports HBV binding.

[0071] 3. Donkey NTCP supports HBV satellite virus infection

[0072] 3.1 Experimental Methods

[0073] In this example, HepG2 cells were infected with lentivirus to overexpress donkey NTCP and human NTCP, respectively, and then infected with HDV (multiplicity of infection: 500). Five days after infection, immunofluorescence assays were performed to detect HDV antigen HDAg, and statistical analysis was performed across different visual fields.

[0074] 3.2 Experimental Results

[0075] The results are as follows Figure 4 As shown, donkey NTCP expression supports HDV infection ( Figure 4 A), and the ability of donkey NTCP to support HDV infection is comparable to that of human NTCP ( Figure 4 B). Thus, donkey NTCP supports HBV entry into cells and infection by the HBV satellite virus HDV.

[0076] 4. Donkey NTCP supports HBV infection

[0077] 4.1 Experimental Methods

[0078] To further investigate whether donkey NTCP supports HBV infection, in this example, HepG2 cells were infected with lentivirus to overexpress donkey NTCP and human NTCP, respectively. The cells were then pretreated according to the HBV infection protocol with a multiplicity of infection of 4000. 2% DMSO and 4% Peg8000 were added to the infection medium. After elution after infection, the supernatants were collected at different time points and the viral marker HBeAg ( Figure 5 At the end of infection, cells were lysed and the level of cccDNA formed in cells after HBV infection was detected according to the standard HBV cccDNA procedure.

[0079] 4.2 Experimental Results

[0080] ELISA test results showed that donkey NTCP expression can enable HepG2 to support real HBV infection, and HBeAg levels increased with the extension of infection time ( Figure 5 Quantitative results showed that donkey NTCP expression indeed supported HBV infection, with high levels of HBV cccDNA detected compared to human NTCP ( Figure 5 C).

[0081] 5. Donkey Hepatocytes Support HBV Infection

[0082] 5.1 Experimental Methods

[0083] In this example, primary donkey hepatocytes were isolated and cultured in vitro, showing that primary donkey hepatocytes can be cultured well on the wall ( Figure 6 Donkey primary hepatocytes were pretreated with primary cell culture medium containing 0.5% DMSO for 24 hours and then infected with HBV using infection medium (0.5% DMSO, 4% PEG8000) at a multiplicity of infection of 2000. After overnight incubation, cells were washed five times with prewarmed culture medium. Supernatants were collected at different time points and assayed for the viral marker HBeAg using ELISA.

[0084] 5.2 Experimental Results

[0085] The results showed that the level of HBeAg, a virological marker of HBV, in the supernatant of infected cells gradually increased on days 4, 6, and 8 after infection ( Figure 6 B). This shows that donkey hepatocytes can support HBV infection.

[0086] Since HDV is a satellite virus of HBV, and donkey primary hepatocytes already support HBV infection, this suggests that they can also support HDV infection. Furthermore, donkey primary hepatocytes can be co-cultured with non-parenchymal cells to establish a 3D hepatocyte culture system or liver organoids. This culture method generally only increases the duration of hepatocyte culture and maintains the characteristics of primary hepatocytes, and does not affect HBV infection. Therefore, while this example has demonstrated HBV infection in conventional donkey primary hepatocytes, other 3D cultured primary hepatocytes or liver organoids also support HBV infection.

[0087] 6. Donkey Primary Hepatocyte Chimeric Liver Supports HBV and Satellite Virus Infection

[0088] 6.1 Experimental Methods

[0089] In this example, donkey hepatocytes were transplanted into the spleen using HSV-TK-NCG mice and Fah-KO-NCG mice. The specific method involved resuscitating primary donkey hepatocytes, counting them, and then performing a splenic injection. One million donkey hepatocytes were transplanted per mouse. Eight to ten weeks after the transplant, the primary donkey hepatocytes were able to proliferate in the mouse liver and be used for infection with HBV and its satellite viruses.

[0090] 6.2 Experimental Results

[0091] The results showed that donkey hepatocytes introduced into mice formed chimeric livers that supported HBV and its satellite virus infection.

[0092] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of donkey primary hepatocytes in constructing a human HBV infection model, characterized in that: The infection model is a cell model or an animal model. The donkey primary hepatocytes show adhesion after in vitro culture. The construction includes pretreating the donkey primary hepatocytes with a primary cell culture medium containing 0.5% DMSO for 24 hours, and infecting HBV with an infection culture medium. After incubation overnight, the donkey primary hepatocytes are washed with a preheated culture medium, and then the supernatant at different time points is collected to detect the viral marker HBeAg, thereby indicating that the model construction is successful.

2. An organism for simulating human HBV infection, characterized in that: The organism is a cell group, tissue, organoid or organ comprising donkey primary hepatocytes, wherein the donkey primary hepatocytes show adhesion after in vitro culture and are donkey primary hepatocytes pretreated with primary cell culture medium containing 0.5% DMSO for 24 hours.

3. A method for screening drugs for treating HBV infection, characterized in that: The method comprises using the organism according to claim 2 to perform the following steps: (1) measuring the amount of HBV in the organism to obtain a first measurement value; (II) contacting the test drug with the organism; (III) measuring the amount of HBV in the organism after contact with the test drug to obtain a second measurement value; and (IV) comparing the first measurement value and a second measurement value; when the second measurement value is less than the first measurement value, screening the drug to be tested as a candidate drug capable of treating HBV infection; and when the second measurement value is greater than or equal to the first measurement value, screening the drug to be tested as a drug that is not useful for treating HBV infection; The amount of HBV refers to the amount of the virological marker HBeAg.

4. Use of an organism in antiviral research or drug development, characterized in that: The virus includes human HBV, and the organism is the organism according to claim 2.

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