Humanized KRAS G12D orthotopic liver cancer animal model, construction method and application thereof

Abstract

The present application provides a complex model combining a humanized in situ liver cancer nude mouse and a KRAS G12D nude plasmid transgene, which can quickly reach 100% tumorigenesis in 7 weeks from modeling, and presents histological features similar to human clinical hepatocellular carcinoma, is suitable for a complex model for new drug research and development in the fields of precise treatment of liver cancer, PROTAC targeted protein degradation agents, small molecule drugs, small nucleic acid drugs and gene therapy, and can achieve efficient and economical modeling of human hepatocarcinogenic gene-induced liver cancer humanized mice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a humanized in situ liver cancer animal model, its construction method, and its application. Background Technology

[0002] Primary liver cancer (PLC), also known as hepatocellular carcinoma in situ, is the third leading cause of cancer-related death worldwide, and its incidence is on the rise. It is estimated that by 2025, the number of patients with liver cancer worldwide will increase by more than one million each year. Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, accounting for about 90% of cases. It is one of the malignant tumors with a poor prognosis. The most common cause of HCC is infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Sustained virological response (SVR) is achieved by patients using antiviral drugs, accounting for about 60% of cases (Llovet JM, Kelley RK, Villanueva A, Singal AG, Picarsky E, Roayaie S, Lencioni R, Koike K, Zucman-Rossi J, Finn RS. Hepatocellular carcinoma. Nat Rev Dis Primers. 2021; 7(1):6).

[0003] With the development and popularization of high-throughput next-generation sequencing (NGS), cancer driver genes with oncogenic functions can be identified and initially applied to personalized risk prediction of liver cancer. Gain-of-function mutations in KRAS G12D are one of the factors driving primary liver cancer (D'Artista L, Moschopoulou AA, Barozzi I, Craig AJ, Seehawer M, Herrmann L, Minnich M, Kang TW, Rist E, Henning M, Klotz S, Heinzmann F, Harbig J, Sipos B, Longerich T, Eilers M, Dauch D, Zuber J, Wang XW, Zender L. MYC determines lineage commitment in KRAS-driven primary liver cancer development. J Hepatol. 2023; 79(1):141-149). Other primary liver cancer drivers include mutations in TP53, CTNNB1, and TERT. HCC has mutations in approximately 25% of cases, but these mutations have not yet been translated into clinical practice.

[0004] G12D is the most common KRAS activating mutation, not only in primary liver cancer, but also in adenocarcinomas (Zeissig MN, Ashwood LM, Kondrashova O, Sutherland KD. Next batter up! Targeting cancers with KRAS-G12D mutations. Trends Cancer. 2023. pii:S2405-8033(23)00137-1). As KRAS G12D inhibitors enter clinical trials, an effective animal model is crucial to understanding the biology of KRAS G12D cancers and determining how the immune microenvironment affects treatment response. However, most animal models developed for KRAS G12D inhibitors are pancreatic cancer models, and only subcutaneous rather than in situ pancreatic cancer models (Kemp SB, Cheng N, Markosyan N, Sor R, Kim IK, Hallin J, Shoush J, Quinones L, Brown NV, Bassett JB, Joshi N, Yuan S, Smith M, Vostrejs WP, Perez-Vale KZ, Kahn B, Mo F, Donahue TR, Radu CG, Clendenin C, Christensen JG, Vonderheide RH, Stanger BZ. Efficacy of a Small-Molecule Inhibitor of KrasG12D in Immunocompetent Models of Pancreatic Cancer. Cancer Discov. 2023; 13(2):298-311). Currently, humanized KRAS is still lacking. G12D orthotopic liver cancer animal model, however, the orthotopic liver cancer model is more similar to the tumor microenvironment of clinical HCC patients than the subcutaneous tumor model (Qiu R, Murata S, Cheng C, Mori A, Nie Y, Mikami S, Hasegawa S, Tadokoro T, Okamoto S, Taniguchi HA Novel Orthotopic Liver Cancer Model for Creating a Human-like Tumor Microenvironment. Cancers (Basel). 2021; 13(16) pii:cancers13163997).

[0005] Existing mouse models of hepatocellular carcinoma (HCC) induce tumor formation through chemical induction, such as diethylnitrosamine (DEN), take as long as 8 months to a year. Moreover, these are mouse-derived HCC models that are not human and cannot replicate the human tumor microenvironment. Using hepatocyte-specific Alb-Cre; KrasG12D transgenic mouse strains, spontaneous HCC tumor formation occurs in about 10 months, with a modeling rate of 46.2% (Ye H, Zhang C, Wang BJ, Tan XH, Zhang WP, Teng Y, Yang X. Synergistic function of Kras mutation and HBx in initiation and progression of hepatocellular carcinoma in mice. Oncogene. 2014; 33(43):5133-8). If HBV X protein (HBx) is introduced, tumor formation can be advanced to 8 months, with a modeling rate of 62.5%.

[0006] A rat model of induced hepatocellular carcinoma (iHCC) was established by first creating primary human hepatocytes (PHH). Then, lentivirus was used to introduce the MYC, TP53 R249S, and KRAS G12D genes into the PHH genome. The rat livers were then inoculated orally, and the iHCC rat model was established after 2–4 months. (Jiang Z, Cheng L, Wu Z, Zhou L, Wang H, Hong Q, Wu Q, Long Y, Huang Y, Xu G, Yao Y, Tang Z, Zhang Z, Yang L, Luo W, Yang J, Gong L, Liu P, Chen X, Cui S, Zhang Q, Li Y, Li P. Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors. EMBO) Rep.2022Jun7;23(6):e54275) is currently a relatively successful humanized orthotopic liver cancer rat model, but the modeling time from cell preparation, screening, modeling to tumor formation takes at least 6 months, and 3 genes need to be introduced.

[0007] The existing KRAS G12D gene knock-in mouse models use fully immunized mice such as C57BL / 6 (Kras-G12D mice (Strain NO. T007054) on the GemPharmatech (Nanjing, China) website. https: / / www.gempharmatech.com / shop / detail / 11322.html). Fully immunized mice exhibit strong immune rejection of foreign cells, resulting in extremely low tumor formation rates when using cell fluid to create in situ liver cancer models. Generally, secondary modeling is required in immunodeficient mice such as nude mice. Therefore, there is currently a lack of humanized in situ liver cancer models that incorporate the humanized KRAS G12D gene on the market.

[0008] The Department of Oncology at the Icahn School of Medicine, Mount Sinai, established nine humanized transgenic HCC mouse models using HDI naked plasmid DNA to investigate the correlation between MYC and HCC driver factors such as TP53, CTNNB1, TERT, Kmt2b, Kmt2c, Pten, and Axin1 (Molina-Sánchez P, Ruiz de Galarreta M, Yao MA, Lindblad KE, Bresnahan E, Bitterman E, Martin TC, Rubenstein T, Nie K, Golas J, Choudhary S, Bárcena-Varela M, Elmas A, Miguela V, Ding Y, Kan Z, Grinspan LT, Huang KL, Parsons RE, Shields DJ, Rollins RA, Lujambio A. Cooperation Between Distinct Cancer Driver Genes Underlies Intertumor Heterogeneity in Hepatocellular Carcinoma. Gastroenterology. 2020; 159(6):2203-2220.e14), but there is still a lack of humanized KRAS G12D transgenic HCC mouse models. Summary of the Invention

[0009] In view of the problems existing in the prior art and to overcome the defects of existing animal models, the purpose of this invention is to solve the problem of the lack of humanized in situ liver cancer models with tumor microenvironment similar to clinical phenotype. In order to achieve the purpose of this invention, the inventors have constructed a humanized KRAS G12D in situ liver cancer animal model by using plasmid construction, in situ seeding of liver cancer cells into the liver, and HDI secondary modeling technology.

[0010] One aspect of the present invention provides a method for constructing a humanized KRAS G12D orthotopic hepatocellular carcinoma animal model, comprising the following steps:

[0011] Step (1): Design, scale-up, and purification of KRAS G12D naked plasmid DNA;

[0012] Step (2): Huh6 human hepatoblastoma cells were inoculated into the livers of nude mice;

[0013] Step (3): Establish a humanized KRAS G12D transgenic orthotopic liver cancer mouse model by HDI secondary modeling.

[0014] Preferably, in step (1), the KRAS G12D naked plasmid is a KRAS G12D gene obtained by site-directed mutation of the nucleotide sequence GGT to GAT using the human KRAS gene as a reference sequence.

[0015] Furthermore, in step (1), the KRAS G12D naked plasmid is prepared by adding a Kozak sequence before the start codon of the KRAS G12D gene and adding a FLAG tag sequence at the 5' end of the stop codon.

[0016] The Kozak sequence is preferably GCCACC, and the FLAG tag sequence consists of three parts.

[0017] Preferably, in step (1), the restriction enzyme sequences of BamHI with the 5' end sequence of GGATCC and XhoI with the 3' end sequence of CTCGAG are designed, ligated into pLC vector plasmid DNA with mouse liver tissue-specific promoters and enhancers, transformed into DH5α competent cells, and amplified and purified to obtain the KRAS G12D naked plasmid.

[0018] In step (2), the preferred method is to use the human hepatoblastoma cell line Huh6. Huh6 cells are transferred to a culture flask for culture, and cells in the logarithmic growth phase are digested to prepare a cell suspension. The cell suspension is then inoculated into the liver of SPF-grade thymic-agnostic Balb / c-Nu nude mice.

[0019] Preferably, in step (2), cells are digested with 0.25% trypsin to prepare 2 x 10 cells per 20 μPBS suspension. 6 Cell suspension.

[0020] In step (3), after the Huh6 humanized orthotopic liver cancer nude mouse model was established, pLC-KRAS G12D plasmid DNA solution was injected and the humanized KRAS G12D transgenic orthotopic liver cancer mouse model was established by tail vein HDI.

[0021] Preferably, the HDI model was induced in nude mice with humanized hepatocellular carcinoma 4 weeks after the initial modeling with Huh6.

[0022] Preferably, the solvent for the pLC-KRAS G12D solution is 0.9% NaCl physiological saline.

[0023] Preferably, a pLC-KRAS G12D plasmid DNA solution equivalent to 10% of the mouse's body weight is prepared, such as 3 ml for a mouse weighing 30g.

[0024] Preferably, the pLC-KRAS G12D plasmid DNA solution is rapidly delivered into liver cells via mouse tail vein HDI within 5-8 seconds to express the humanized KRAS G12D naked plasmid, thereby establishing a humanized KRAS G12D transgenic orthotopic liver cancer mouse model.

[0025] In another aspect, the present invention provides a humanized KRAS G12D orthotopic liver cancer animal model.

[0026] In one aspect, the present invention provides an application of a humanized KRAS G12D orthotopic liver cancer animal model, which can be used to study the pathogenesis of orthotopic liver cancer or to screen drugs for orthotopic liver cancer.

[0027] To address the problems existing in the prior art, this invention provides a complex model combining humanized orthotopic hepatocellular carcinoma nude mice and KRAS G12D nude plasmid HDI transgene. This model can rapidly achieve 100% tumor formation within 7 weeks from model initiation. The humanized orthotopic hepatocellular carcinoma nude mice retain the original structural characteristics of hepatocellular carcinoma tissue and have a growth environment and biological characteristics more similar to clinical HCC tumors. This model can effectively simulate the tumor microenvironment, including the immune escape and invasive metastatic capabilities of hepatocellular carcinoma cells. It is a complex model suitable for the development of new drugs in the fields of PROTAC-targeted protein degraders, small molecule drugs, small nucleic acid drugs, and gene therapy for precision treatment of hepatocellular carcinoma. It enables efficient and economical modeling of humanized mice with hepatocellular carcinoma induced by various human oncogenes. The technical solution provided by this invention can effectively construct a humanized KRAS G12D gene overexpression in situ hepatocellular carcinoma in nude mice, which is simple, economical, efficient, and rapid to model. This model better simulates the tumor microenvironment of human HCC patients and fills the gap in complex animal models of humanized KRAS G12D gene and orthotopic hepatocellular carcinoma. Preclinical efficacy evaluation using this animal model more realistically reflects clinical trials in human patients. Furthermore, based on this model, it is possible to develop complex animal models of orthotopic hepatocellular carcinoma with various humanized oncogenes related to human HCC-related mutations.

[0028] The humanized KRAS G12D orthotopic liver cancer nude mouse model and its underlying technology extension into a large animal model will be one of the preferred models for preclinical evaluation of KRAS G12D target inhibitors, and will make a significant contribution to the treatment of human healthy liver cancer.

[0029] The beneficial effects of this invention are as follows: First, the model disclosed in this invention can achieve modeling in a short period of 7 weeks, with a 100% tumorigenesis rate in situ hepatocellular carcinoma. This overcomes the limitation that chemical induction takes up to a year and the limitation that existing specific point mutation gene knock-in mice are mostly fully immunized mice, making it difficult to achieve secondary in situ hepatocellular carcinoma inoculation. This achieves short-term modeling, shortening the research and development time, and even shortening the time for preclinical efficacy evaluation of this target hepatocellular carcinoma. Second, the liver pathology of the mouse model presents histological features similar to those of human clinical HCC, such as tumor-infiltrating lymphocytes, necrosis, vascular invasion, Mallory bodies, and cholestasis. Third, the model construction method disclosed in this invention is simple and low-cost, reducing research and development costs. Fourth, the model construction method disclosed in this invention can not only introduce a single oncogenic mutation gene but also has the potential to introduce multiple genes simultaneously. Fifth, the model disclosed in this invention can fill the gap in KRAS G12D plus HCC hepatocellular carcinoma models. Sixth, the model construction method disclosed in this invention has the potential to be applied to large animal models. Seventh, the model disclosed in this invention can be applied to the developed KRAS model. Expanding the indications for liver cancer in the development of new G12D inhibitor drugs.

[0030] In summary, the method for constructing a humanized KRAS G12D orthotopic liver cancer animal model disclosed in this invention can rapidly establish the model within 7 weeks with a tumor formation rate of 100%, and it exhibits histological characteristics similar to human clinical HCC. It can be applied to the preclinical efficacy evaluation of anti-liver cancer drugs targeting KRAS G12D inhibitors, which is beneficial to the study of the pathogenesis of human liver cancer and the development of targeted drug therapy for liver cancer. Attached Figure Description

[0031] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0032] Figure 1 : To efficiently establish a plasmid map of a humanized KRAS G12D orthotopic liver cancer animal model.

[0033] Figure 2 Schematic diagram of the process for creating a complex humanized KRAS G12D orthotopic liver cancer nude mouse model.

[0034] Figure 3 Phenotypic results of nude mice with humanized KRAS G12D orthotopic hepatocellular carcinoma after 7 weeks.

[0035] Figure 4 Phenotypic results of nude mice with humanized KRAS G12D orthotopic hepatocellular carcinoma after 9 weeks.

[0036] Figure 5 Tumor weight phenotypes in nude mice with humanized KRAS G12D orthotopic hepatocellular carcinoma at 7 and 9 weeks of age.

[0037] Figure 6 Expression of KRAS G12D-3xFlag mRNA in nude mice with humanized KRAS G12D orthotopic hepatocellular carcinoma for 7 weeks.

[0038] Figure 7 Liver pathological morphology in nude mice with humanized KRAS G12D orthotopic hepatocellular carcinoma after 9 weeks.

[0039] Explanation of the name:

[0040] HDI (Hydrodynamic Injection): Utilizing high-speed hydrodynamic injection (also known as hydrodynamic tail vein injection or hydrodynamic gene delivery), HDI uses the dynamic pressure generated by rapidly injecting a large volume of fluid into a blood vessel to permeate cell membranes and promote intracellular gene transfer. Within a 5-8 second timeframe, naked plasmid DNA expressing humanized genes is injected via the mouse tail vein into a solution volume equivalent to 10% of the mouse's body weight. The powerful hydrodynamic forces caused by the injection dilate the hepatic pores, allowing genetic material to enter the hepatocytes. Once the transferred genetic material is inside the hepatocyte, the hydrodynamic pressure decreases, the hepatic pores close, and the genetic information remains within the cell. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0042] Example 1: Design and purification of KRAS G12D naked plasmid DNA

[0043] Using the human KRAS gene (Genebank ID: NM_001369787.1) as a reference sequence, a mutation was designed to change the 12th amino acid from glycine (Gly, G) to aspartic acid (Asp, D), with the corresponding nucleotide sequence G. G T-site mutation to G A T designed a Kozak sequence with the sequence GCCACC before the start codon (ATG) of the KRAS G12D gene, and added three FLAG tag sequences (SEQ ID NO.1) to the 5' end of the stop codon (TAA) of the human KRAS G12D gene. Finally, a 657bp restriction enzyme sequence was designed with BamHI (GGATCC) at the 5' end and XhoI (CTCGAG) at the 3' end. After gene synthesis (Nanjing Genscript Biotech Co., Ltd.), it was ligated into a pLC vector through the restriction enzyme sites to become a full-length 4053bp plasmid DNA. See details... Figure 1 .

[0044] Example 2: Modeling of Huh6 orthotopic hepatocellular carcinoma in nude mice

[0045] Human hepatoblastoma cells Huh6 (Zhejiang Meisen Cell Technology Co., Ltd., CTCC-400-0177) were aliquoted into several vials with a base area of ​​75 cm². 2In a culture flask, culture by the conventional method, that is, use DMEM culture medium containing 10% fetal bovine serum and culture in a constant temperature and humidity incubator at 37°C and 5% CO2. When the cells are in the logarithmic growth phase, digest with 0.25% trypsin, collect the digestion solution, centrifuge and remove the supernatant, wash twice with PBS solution and then make a cell suspension, and adjust to suspend 2x10 6 cell solution for subsequent inoculation.

[0046] Use SPF-grade athymic Balb / c-Nu nude mice, female, 4-5 weeks old, weighing 18-22 g [Shanghai Slac, SCXK (Shanghai) 2022-0004], and adaptively raise for several days. After anesthetizing the nude mice, fix them in the supine position on a board. After disinfecting the abdomen, open the abdominal cavity along the midline of the abdomen to expose the liver (one lobe). Use a micro syringe to extract 20 μl (containing 2x10 6 Huh6 cells / 20 μl PBS) cell suspension, with the injection depth of 3 mm (the vertical distance from the surface of the liver lobe), do not penetrate the liver lobe, and keep the injection angle less than 30 degrees. Immerse the needle more than 1 cm and then inject. Slowly inject the cell solution and keep the needle in place for more than 5 seconds. After pulling out the needle, press with a cotton ball to stop bleeding. Suture the wound and disinfect. Place the animal on a 37°C constant temperature heating plate. After waking up, put it in a cage for conventional feeding.

[0047] During this period, weigh the mice every three days and observe the health status of the mice. By the fourth week, palpate the liver and feel a hard mass as if there is a tumor.

[0048] Example 3: Establishment of a humanized KRAS G12D transgenic orthotopic liver cancer nude mouse model

[0049] Four weeks after the establishment of the Huh6 humanized orthotopic liver cancer nude mouse model, each mouse is to be injected with a total volume of 10% of its body weight of liquid. Using 0.9% NaCl normal saline as a solvent, prepare plasmid DNA solutions for the pLC vector negative control group at 20 μg / mouse, the pLC-KRAS G12D low-dose model group at 2 μg / mouse, and the pLC-KRAS G12D high-dose model group at 20 μg / mouse. Fix the mouse with a mouse holder, soak the tail of the nude mouse in warm water at 45°C. After exposing the mouse tail vein, through the mouse tail vein HDI, quickly inject a liquid equivalent to 10% of the mouse body weight volume (for example, the injection volume for a 30 g nude mouse is 3 ml of plasmid solution) within 5-8 seconds, and deliver the KRAS G12D naked plasmid expressing the humanized gene into the liver cells for expression, so as to establish a humanized KRAS G12D transgenic orthotopic liver cancer mouse model. For the detailed secondary modeling process, see Figure 2 .

[0050] Mice were weighed and their health observed every three days. By week 7, palpation of the liver revealed obvious tumors, and some models showed phenotypes such as emaciation and hunched back. Dissection of all 14 models at week 7 showed tumor formation, a tumor formation rate of 100%. Figure 3 The tumor was carefully separated from the liver and weighed. The results were as follows: (1) Huh6+pLC negative control group (n=2): average tumor weight 212.0 mg, average whole liver weight 1733.0 mg; (2) Huh6+pLC-KRAS G12D 2μg low-dose model group (n=6): average tumor weight 471.3 mg, average whole liver weight 2029.5 mg; (3) Huh6+pLC-KRAS G12D 20μg high-dose model group (n=6): average tumor weight 1022.3 mg, average whole liver weight 2652.8 mg.

[0051] Throughout the experiment, mice were weighed and their health monitored every three days. By week 9, palpation of the liver revealed more pronounced tumors, and all models exhibited phenotypes such as emaciation and hunched backs. At the end of the experiment, all 14 dissected models showed tumor formation, a tumor formation rate of 100%. Figure 4 The tumor was carefully separated from the liver and weighed. The following statistics were recorded: (1) Huh6+pLC negative control group (n=2): average tumor weight 362.2 mg, average whole liver weight 1955.9 mg; (2) Huh6+pLC-KRAS G12D 2μg low-dose model group (n=6): average tumor weight 1365.7 mg, average whole liver weight 3007.4 mg; (3) Huh6+pLC-KRAS G12D 20μg high-dose model group (n=6): average tumor weight 2488.2 mg, average whole liver weight 4032.1 mg. The average tumor weight showed that KRasG12D transgene was associated with the in situ hepatocellular carcinoma phenotype. Figure 5 ).

[0052] After homogenizing liver tissue from humanized KRAS G12D orthotopic hepatocellular carcinoma nude mice for 7 weeks, total RNA was extracted using the Trizol method and reverse transcribed into 1st-cDNA. The KRAS G12D-3xFlag mRNA level was specifically identified using the forward primer sequence 5'-ACAAGACAGGGTGTTGATGATGC-3' and the reverse primer sequence 5'-GTCCTTATCGTCGTCATCTTTGTAATCC-3' located on the three FLAG tags (3xFlags). Relative quantification with mouse Actin mRNA showed that the high-dose model group overexpressed humanized KRAS G12D-3xFlag at a level 161.0±13.78 times higher than the vector negative control, while the low-dose model group overexpressed it at a level 107.2±7.20 times higher. The transgenic mRNA level was positively correlated with tumor phenotype. Figure 6 ).

[0053] Nine weeks after fixation of liver tissue from humanized KRAS G12D orthotopic hepatocellular carcinoma nude mice, pathological identification was performed by HE staining. Figure 7 Most liver cancer tumors protrude from the surface of the liver, and the tumor tissue can be clearly seen through dissection. The white appearance is due to the accumulation of large amounts of fat or glycogen in the cytoplasm, giving it a transparent, cell-like appearance. Figure 3 and 4 However, a small percentage of liver cancer tumors grow into the liver from the site of cell inoculation, forming well-defined, mosaic-like solid liver tumors. When the tumor is small, such as less than 1 mm in diameter... Figure 7 A, where the double arrows represent tumor diameter), requires pathological histological examination such as HE staining for identification. Nine-week-old liver tissue from humanized KRAS G12D orthotopic hepatocellular carcinoma nude mice exhibited histological features similar to human clinical HCC, including: ① tumor-infiltrating lymphocytes (TILs); Figure 7 B, arrow), is a type of highly heterogeneous lymphocyte found within tumor tissue; clinically, the number of TILs is closely related to patient prognosis; ② Necrotic area ( Figure 7 C), ③Vascular infiltration or invasion ( Figure 7 D, arrow), ④ Mallory-Denk body ( Figure 7 E (arrow) is a characteristic of hyaluronic acid bodies, a feature of highly differentiated hepatocellular carcinoma; and ⑤ tumor cells secrete bile, causing yellow-green bile stasis ( Figure 7 F (arrow).

Claims

1. A method for constructing a humanized KRAS G12D orthotopic hepatocellular carcinoma animal model, comprising the following steps: Step (1): Design, scale-up, and purification of pLC-KRAS G12D plasmid DNA; Step (2): Inoculate the livers of nude mice with a suspension of Huh6 human hepatoblastoma cells; Step (3): A humanized KRAS G12D transgenic orthotopic liver cancer mouse model was established by secondary modeling via tail vein HDI; The specific steps (1) are as follows: using the human KRAS gene as a reference sequence, the nucleotide sequence GGT is site-directedly mutated to GAT to obtain the KRAS G12D gene. A Kozak sequence is added before the start codon of the KRAS G12D gene, and a FLAG tag sequence is added at the 5' end of the stop codon. A BamHI restriction site with the sequence GGATCC is designed at the 5' end and an XhoI restriction site with the sequence CTCGAG is designed at the 3' end. The fragment containing the KRAS G12D gene is ligated into the pLC vector plasmid DNA with mouse liver tissue-specific promoters and enhancers. The DNA is transformed into DH5α competent cells and obtained after amplification and purification. The specific steps (3) are as follows: After establishing the Huh6 humanized orthotopic liver cancer nude mouse model in step (2), the humanized KRAS G12D transgenic orthotopic liver cancer mouse model is established by secondary modeling through tail vein HDI injection of pLC-KRAS G12D plasmid DNA solution; the solvent of the pLC-KRAS G12D plasmid DNA solution is physiological saline, the injection volume is equivalent to 10% of the mouse body weight, and the injection is completed within 5 to 8 seconds.

2. The construction method according to claim 1, wherein the Kozak sequence is GCCACC and the FLAG tag sequence is three.

3. According to the construction method of claim 1, in step (2), the human hepatoblastoma cell line Huh6 is used. Huh6 cells are transferred to a culture flask for culture, and cells in the logarithmic growth phase are digested to prepare a cell suspension. The cell suspension is then inoculated into the liver of SPF-grade thymus-agnostic Balb / c-Nu nude mice.

4. According to the construction method of claim 3, after Huh6 cells are transferred to culture flasks and cultured, the cells are digested with 0.25% trypsin to prepare 2 x 10 cells per 20 μl PBS suspension. 6 Cell suspension.

5. The construction method according to claim 1, wherein the Huh6 humanized orthotopic hepatocellular carcinoma nude mouse model is subjected to HDI secondary modeling 4 weeks after modeling.

6. A humanized KRAS G12D orthotopic liver cancer animal model prepared according to any one of claims 1-5.

7. The application of the humanized KRAS G12D orthotopic liver cancer animal model as described in claim 6, in the study of the pathogenesis of orthotopic liver cancer or in the screening of drugs for orthotopic liver cancer.

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