Construction method and application of hHer2 gene humanized mouse EMT-6 tumor cell line animal model

Abstract

The invention belongs to the field of biological medicine, and particularly discloses a construction method and application of an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, and the construction method comprises the following steps: 1) constructing an hHer2 overexpression lentiviral vector, and constructing the hHer2 overexpression lentiviral vector according to the gene sequence of hHer2; (2) constructing an EMT-6 / hHer2 cell line, transfecting the EMT-6 cell line by using the overexpression lentiviral vector of the hHer2, carrying out cell monoclonal screening, detecting the expression condition of the hHer2 by using the obtained EMT-6 / hHer2 monoclonal cells by using an FACS method, and constructing the EMT-6 / hHer2 cell line; 3) establishment of an EMT-6 / hHer2 tumor model: inoculating the EMT-6 / hHer2 cell line to a BALB / C mouse to establish a mouse tumor model, the tumor model can be used for pharmacodynamic evaluation research of hHer2 immunization and targeted combined therapy, and research results can help research of hHer2 drug combined therapy and research and development of related antibody drugs.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically discloses a method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model and an application thereof. Background Art

[0002] HER2 receptor (also known as c-erbB-2, HER2 / neu) is a transmembrane protein with a molecular weight of 185kD and tyrosine kinase activity. Its encoding gene is located on human chromosome 17q21 and is a member of the epidermal growth factor receptor family. Overexpression of the HER2 gene can lead to excessive cell proliferation and phenotypic malignant transformation. Studies have found that 30% of breast cancer patients have overexpression of the HER2 gene. These patients have high tumor malignancy, early recurrence and metastasis, poor prognosis, and resistance to certain chemotherapy drugs. It has also been found that overexpression of the HER2 gene is associated with the patient's disease-free survival and overall survival. Using tumor biomarkers as important predictors of tumor treatment response is one of the main purposes of tumor biology research.

[0003] HER2 is a member of the HER receptor family and is overexpressed in a variety of cancers, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, liver cancer, lung cancer, ovarian cancer, and salivary gland cancer [1]. The complete HER2 protein consists of three functional regions: (1) an extracellular end (ECD) that can bind to HER2 family members; (2) a lipophilic transmembrane segment; and (3) an intracellular end with tyrosine kinase activity. Previous studies have shown that HER2 gene amplification and overexpression are present in 15%-20% of breast cancer patients [2]. In addition, a large amount of evidence shows that HER2 is overexpressed in breast cancer patients, and that patients with HER2 overexpression have a poor prognosis, including resistance to chemotherapy, hormone therapy, and radiotherapy. It is also found that patients have a high probability of metastasis and recurrence. Currently, HER2 overexpression is considered an independent prognostic factor for breast cancer, and HER2 expression levels have been used as an independent indicator for evaluating tumor biological behavior. HER2 is also considered an important therapeutic target for the treatment of HER2-positive breast cancer [3].

[0004] Treatments for breast cancer include surgery, hormone receptor endocrine therapy, radiotherapy, molecular targeted therapy, chemotherapy, and immunotherapy. Currently, surgical resection is still the main treatment method [4], among which targeted therapy has gradually become a hot research field. Human epidermal growth factor receptor 2 (HER2)-positive breast cancer refers to ERBB2 / neu proto-oncogene amplification or HER2 transmembrane receptor protein overexpression, which accounts for 15% to 20% of the five molecular types of breast cancer [5]. Compared with other types of breast cancer, HER2-positive breast cancer is a special subtype of breast cancer that is more aggressive, prone to early recurrence and metastasis, and has a poor prognosis [6-7]. Among Chinese breast cancer patients, 2-3 out of every 10 are HER2-positive [8].

[0005] Currently, HER2 overexpression is considered an independent prognostic factor for breast cancer, and HER2 expression levels have been used as an independent indicator for evaluating tumor biological behavior. HER2 is also considered an important therapeutic target for the treatment of HER2-positive breast cancer [9]. In recent years, molecular targeted therapy for HER2 breast cancer has made rapid progress and has become one of the effective treatments for breast cancer.

[0006] Due to the rapid development of immunotherapy in recent years, more and more studies have focused on the combined immune and targeted therapy of hHer2. The main purpose of this study is to construct a mouse tumor model that has a complete immune system and can perform hHer2 targeted therapy, which can be used for the pharmacodynamic evaluation of hHer2 immune and targeted combined therapy. Syngenic tumor transplantation model is a type of mouse tumor model that can be used for immune drug efficacy evaluation, and it is also the earliest animal model used for the study of immune drugs. However, it has no human drug targets, and cannot conduct research on humanized targeted drugs, and thus cannot conduct research on immune and targeted combined therapy.

[0007] References:

[0008] [1]IQBAL N,IQBAL N.Human epidermal growth factor receptor 2(HER2)incancers:overexpression and therapeutic implications[J]. Mol Biol Int, 2014, 2014:852748.

[0009] [2]CAMERON D,PICCART-GEBHART MJ,GELBER RD,et al.11years'

[0010] HER2阳性早期乳腺癌辅助化疗后曲妥珠单抗的随访:HERceptin辅助(HERA)试验的最终分析[J]。《柳叶刀》,2017年,389(10075):1195 - 1205。

[0011] [3]达伍德S,布罗利奥K,布兹达尔AU等。HER2状态和曲妥珠单抗治疗对转移性乳腺癌女性患者预后的影响:一项基于机构的综述[J]。《临床肿瘤学杂志》,2010年,28(1):92 - 98。

[0012] [4]哈贝克N,佩诺 - 洛卡F,科尔特斯J等。乳腺癌[J]。《自然综述:疾病引物》,2019年,5(1):66。

[0013] [5]《2020CSCO乳腺癌诊疗指南》[S]。2020年。中国临床肿瘤学会(CSCO)乳腺癌诊疗指南[S]。2020年。

[0014] [6]哈贝克N,格纳特M。乳腺癌[J]。《柳叶刀》,2017年,389(10074):1134 - 1150。

[0015] [7]克罗宁KA,哈兰LC,多德KW等。美国早期患者HER - 2阳性乳腺癌肿瘤患病率的基于人群的估计[J]。《癌症研究》,2010年,28(9):963 - 968。

[0016] [8] Zheng Rongshou, Sun Kexin, Zhang Siwei, et al. Analysis of the prevalence of malignant tumors in China in 2015[J]. Chinese Journal of Oncology, 2019, 41(1): 19-28.

[0017] [9]Zheng RS, Sun KX, Zhang SW, et al. Report of cancer epidemiology in China, 2015[J]. Chin J Oncol, 2019, 41(1):19-28. Summary of the invention

[0018] To solve the above problems, a method for constructing an animal model of hHer2 gene humanized mouse EMT-6 tumor cell line and its application are disclosed. We added hHer2 target to humanize it on the basis of homologous tumor transplantation model. On the basis of its complete immune system, the target of hHer2 targeted drug was added, and the research of immune and targeted combined therapy can be carried out. We selected mouse tumor cell line EMT-6 for hHer2 humanization (EMT-6 / hHer2), and constructed a humanized mouse tumor model based on it. The tumor model can be used for pharmacodynamic evaluation research of hHer2 immune and targeted combined therapy. Our research results can help the research of hHer2 drug combination therapy and the development of related antibody drugs.

[0019] The present invention includes the following technical solutions:

[0020] A method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model comprises the following steps:

[0021] 1) Construction of hHer2 overexpression lentiviral vector

[0022] According to the gene sequence of hHer2, a hHer2 overexpression lentiviral vector was constructed;

[0023] 2) Construction of EMT-6 / hHer2 cell line

[0024] The hHer2 overexpression lentiviral vector was used to transfect the EMT-6 cell line and perform cell monoclonal screening. The expression of hHer2 in the obtained EMT-6 / hHer2 monoclonal cells was detected by FACS to construct the EMT-6 / hHer2 cell line;

[0025] 3) Establishment of EMT-6 / hHer2 tumor model

[0026] The EMT-6 / hHer2 cell line was inoculated into BALB / C mice to establish a mouse tumor model.

[0027] Furthermore, in the above-mentioned method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, in the step 1), the hHer2 gene sequence is shown in NM_004448.3.

[0028] Furthermore, in the above-mentioned method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, the step 1) comprises the following steps:

[0029] S1: inserting the hHer2 gene fragment into the cloning vector pcDNA3.1 to form the cloning plasmid pcDNA3.1 / hHer2;

[0030] S2: The pLVX-EF1α-IRES-Puro vector plasmid and the pcDNA3.1 / hHer2 plasmid were double-digested with EcoR I and Xho I restriction sites, and then the pLVX-EF1α-IRES-Puro vector fragment and the hHer2 gene fragment were recovered and ligated in the presence of T4 DNA ligase. After identification, the plasmid pLVX-EF1α-IRES-Puro-hHer2 was obtained;

[0031] S3: The pLVX-EF1α-IRES-Puro-hHer2 plasmid was transfected into 293T cells by liposome method for virus packaging to obtain lentiviral particles.

[0032] Furthermore, in the above-mentioned method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, the step 2) comprises the following steps:

[0033] a: pLVX-EF1α-IRES-Puro-hHer2 lentivirus infected EMT-6 cells:

[0034] a1: EMT-6 cells were inoculated in a 6-well plate one day before lentiviral infection and cultured in a 5% CO2, 37°C constant temperature incubator;

[0035] a2: On the day of the experiment, the cell confluence reached 30-50%;

[0036] a3: Place the pLVX-EF1α-IRES-Puro-hHer2 lentiviral particles on ice to dissolve;

[0037] a4: Take 100 μL of pLVX-EF1α-IRES-Puro-hHer2 lentiviral solution and add it to EMT-6 cells and continue culturing;

[0038] a5: After 72 hours of culture, add the screening antibiotic Puromicin to screen for cell resistance;

[0039] a6: After the screening is completed, the obtained cells are digested into single cells, and the cell monoclonal clones are picked using the limiting dilution method. After one week, after the monoclonal cell amplification is completed, the monoclonal cells are identified by FACS to confirm whether hHer2 is successfully introduced into EMT-6 cells.

[0040] Furthermore, the method for constructing the hHer2 gene humanized mouse EMT-6 tumor cell line animal model also includes:

[0041] b: FACS identification of EMT-6 / hHer2 monoclonal cells:

[0042] b1: Collect EMT-6 / hHer2 monoclonal cells into a 5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0043] b2: Add 2 mL FACS buffer (PBS + 2% FBS) to resuspend the cells, mix well by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0044] b3: Add 2 μL PE-hHer2 antibody, incubate at 4°C for 30 min, centrifuge at 1000 rpm for 10 min, discard the supernatant, and wash twice with FACS buffer;

[0045] b4: Add 500 μL FACS buffer, resuspend the cells to make a single cell suspension, and transfer it into the marked flow cytometry loading tube;

[0046] b5: set up another tube of wild-type EMT-6 cell suspension as a negative control;

[0047] b6: Detection by flow cytometry.

[0048] Furthermore, in the above-mentioned method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, the step 3) comprises the following specific steps:

[0049] c1: cell culture

[0050] EMT-6 / hHer2 cells were cultured in DMEM medium containing 10% fetal bovine serum, and EMT-6 / hHer2 cells in the exponential growth phase were collected and resuspended to a suitable concentration for subcutaneous tumor inoculation in mice;

[0051] c2: Tumor inoculation

[0052] The experimental mice were subcutaneously inoculated with EMT-6 / hHer2 cells on the right back, with a cell inoculation volume of 1×10 6 / mouse, the inoculation volume was 0.1mL / mouse, and the tumor growth was observed regularly. After the tumor was formed, regular observation was performed;

[0053] c3: FACS identified EMT-6 / hHer2 tumors expressing hHer2.

[0054] Furthermore, in the above method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, the c3: FACS identification of EMT-6 / hHer2 tumors expressing hHer2 comprises:

[0055] EMT-6 and EMT-6 / hHer2 tumors grew to approximately 500 mm 3 When the cells were grown, the tumor tissues were removed and the cells were made into single cell suspensions; the expression of hHer2 in EMT-6 and EMT-6 / hHer2 tumor single cells was detected by FACS.

[0056] Furthermore, the method for constructing the hHer2 gene humanized mouse EMT-6 tumor cell line animal model also includes 4) evaluating hHer2 drugs in the EMT-6 / hHer2 tumor model, comprising the following steps:

[0057] Wild-type EMT-6 and modified EMT-6 / hHer2 cell lines were inoculated into BALB / C mice to establish a mouse tumor model. When the tumor grew to about 80 mm 3 When the tumor size was about 100 μg / cm2, each model was divided into 2 groups and treated with drugs. One group was given the isotype control antibody Isotype control at a dose of 10 mpk twice a week; the other group was given the hHer2 antibody drug Trastuzumab at a dose of 10 mpk twice a week. The patients were observed regularly and the tumor volume was measured.

[0058] The present invention also discloses the application of the animal model prepared by the above construction method in evaluating hHer2 related antibody drugs.

[0059] Furthermore, in the above application, the antibody drug includes Trastuzumab.

[0060] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0061] The present invention discloses a method for constructing an animal model of hHer2 gene humanized mouse EMT-6 tumor cell line and its application. Most of the existing tumor models are constructed by inoculating human tumor cells with immunodeficient mice. Such tumor models cannot be used to study immune drugs due to the lack of human immune system. In addition, there is a class of immune reconstruction mouse tumor models, such as PBMC (Peripheral blood mononuclear cell) immune reconstruction mouse tumor model and CD34+ immune reconstruction mouse model. Such models can only reconstruct the immune function of T cells, and cannot fully reflect the response of the immune system to immune drugs, and these models are very difficult to model and costly. The tumor model established by the present invention has a complete immune system and a human hHer2 target, and can be used for testing hHer2 targeted drugs, and can be used for the pharmacodynamic evaluation study of immune and targeted combined therapy of hHer2 targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a map of hHer2 overexpression lentiviral vector;

[0063] Figure 2 FACS identification of EMT-6 / hHer2 cell lines: wild-type EMT-6 cells do not express hHer2 (left panel); EMT-6 / hHer2 cells express hHer2 (right panel);

[0064] Figure 3 This is the growth curve of the EMT-6 / hHer2 tumor model. Wild-type EMT-6 and monoclonal EMT-6 / hHer2 cells were inoculated subcutaneously in BALB / C mice to establish a tumor model.

[0065] Figure 4 FACS was used to identify the level of hHer2 expression in EMT-6 / hHer2 tumors. Wild-type EMT-6 tumors do not express hHer2 (left panel); EMT-6 / hHer2 tumors express hHer2 (right panel);

[0066] Figure 5 hHer2 drugs were evaluated in the EMT-6 / hHer2 tumor model. Wild-type EMT-6 was treated with hHer2 drugs (Trastuzumab), and modified EMT-6 / hHer2 was treated with hHer2 drugs (Trastuzumab). DETAILED DESCRIPTION

[0067] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] Example 1

[0069] Construction of pLVX-EF1α-IRES-Puro-hHer2 vector

[0070] 1.1 Synthesis of hHer2 gene fragment

[0071] According to the hHer2 gene sequence (NM_004448.3) on the NCBI website, the gene fragment was synthesized by GeneWeiZhi Biotechnology Co., Ltd. The synthesized hHer2 gene fragment was inserted into the cloning vector

[0072] pcDNA3.1 to form the cloning plasmid pcDNA3.1 / hHer2.

[0073] 1.2 pLVX-EF1α-IRES-Puro vector plasmid and pcDNA3.1 / hHer2 plasmid digestion:

[0074]

[0075] 1.3 Reagent digestion of pLVX-EF1α-IRES-Puro vector fragment and hHer2 gene fragment recovery:

[0076] Purify the digested plasmid product using a gel recovery kit according to the instructions.

[0077] 1.4 Connect the pLVX-EF1α-IRES-Puro vector fragment and the hHer2 gene fragment:

[0078]

[0079] 1.5 Transformation of ligation products:

[0080] The ligated plasmid was transformed into competent cells DH5α, evenly spread on Amp-resistant LB solid culture medium plates, and cultured in a 37°C incubator for 12-16 hours until a single colony appeared.

[0081] 1.6 Identification of vector plasmid:

[0082] A single colony was picked and expanded for culture and plasmid was extracted. The extracted plasmid was sequenced and identified, and the correctly identified plasmid was named pLVX-EF1α-IRES-Puro-hHer2.

[0083] Example 2

[0084] pLVX-EF1α-IRES-Puro-hHer2 for viral packaging

[0085] 2.1 Lipofectamine transfection of pLVX-EF1α-IRES-Puro-hHer2 plasmid into 293T cells

[0086] (1) One day before transfection, 293T cells were seeded in a 6-well plate and cultured in a 5% CO2, 37°C constant temperature incubator.

[0087] (2) On the day of the experiment, the cell confluence reached 30-50%.

[0088] Each well is transfected as follows:

[0089] A. Dissolve 4 μg of the plasmid DNA extracted above in 250 μL Opti-mem serum-free medium.

[0090] B. Dissolve 10 μL of lipo3000 in 250 μL of Opti-mem serum-free medium, mix well and leave at room temperature for 5 minutes.

[0091] Mix tubes AB and let stand for 20 minutes.

[0092] (3) Replace the culture medium in the 6-well plate with serum-free culture medium, 2000 μL culture medium per well. Add the mixture in tubes AB to the corresponding wells of the 6-well plate and replace it with serum-containing culture medium after 4-6 hours.

[0093] (4) After 48 hours of culture, the cell supernatant was collected, which contained the packaged lentiviral particles.

[0094] (5) The collected cell supernatant was centrifuged at 12,000 rpm for 10 minutes, the supernatant was dispensed into EP tubes, and stored in a -80°C refrigerator.

[0095] Example 3

[0096] Humanization of hHer2 in EMT-6 cells

[0097] 3.1 Infection of EMT-6 cells with pLVX-EF1α-IRES-Puro-hHer2 lentivirus

[0098] (1) One day before lentiviral infection, EMT-6 cells were inoculated in a 6-well plate and cultured in a 5% CO2, 37°C constant temperature incubator.

[0099] (2) On the day of the experiment, the cell confluence reached 30-50%.

[0100] (3) Place the pLVX-EF1α-IRES-Puro-hHer2 lentiviral particles obtained in step 3.1 on ice to dissolve.

[0101] (4) Take 100 μL of pLVX-EF1α-IRES-Puro-hHer2 lentiviral solution and add it to EMT-6 cells and continue culturing.

[0102] (5) After 72 hours of culture, the selection antibiotic Puromicin was added to screen for cell resistance.

[0103] (6) After the screening is completed, the obtained cells are digested into single cells, and the cell monoclonal clones are picked using the limiting dilution method. After about one week, when the monoclonal cell amplification is completed, the monoclonal cells are identified by FACS to confirm whether hHer2 is successfully introduced into EMT-6 cells.

[0104] 3.2 FACS identification of EMT-6 / hHer2 monoclonal cells

[0105] (1) Collect EMT-6 / hHer2 monoclonal cells into a 5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0106] (2) Add 2 mL of FACS buffer (PBS + 2% FBS) to resuspend the cells, mix well by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0107] (3) Add 2 μL of PE-hHer2 antibody, incubate at 4°C for 30 min, centrifuge at 1000 rpm for 10 min, discard the supernatant, and wash twice with FACS buffer.

[0108] (4) Add 500 μL FACS buffer and resuspend the cells to make a single-cell suspension. Transfer the suspension into the labeled flow cytometry loading tube.

[0109] (5) Set up another tube of wild-type EMT-6 cell suspension as a negative control.

[0110] (6) Detection by flow cytometry.

[0111] (7) Experimental results are as follows Figure 2 As shown, hHer2 has been successfully introduced into EMT-6 cells, and the EMT-6 / hHer2 cell line has been successfully constructed.

[0112] Example 4

[0113] Establishment of EMT-6 / hHer2 tumor model

[0114] 4.1 Cell culture

[0115] EMT-6 / hHer2 cells were cultured in DMEM medium containing 10% fetal bovine serum. EMT-6 / hHer2 cells in the exponential growth phase were collected and resuspended to a suitable concentration for subcutaneous tumor inoculation in mice.

[0116] 4.2 Tumor inoculation

[0117] The experimental mice were subcutaneously inoculated with EMT-6 / hHer2 cells on the right back.

[0118] 1×10 6 / mouse, the inoculation volume was 0.1mL / mouse, and the tumor growth was observed regularly. Figure 3 As shown, wild-type EMT-6 and EMT-6 / hHer2 were inoculated

[0119] BALB / C mice were able to form tumors subcutaneously, proving that the EMT-6 / hHer2 tumor model was successfully established.

[0120] Example 5

[0121] FACS identification of EMT-6 / hHer2 tumors expressing hHer2

[0122] EMT-6 and EMT-6 / hHer2 tumors grew to approximately 500 mm 3 When the tumor tissue was removed, the cells were made into single cell suspension. FACS was used to detect the expression of hHer2 in EMT-6 and EMT-6 / hHer2 tumor single cells. The FACS experimental process was as described in 3.2. The experimental results are shown in Figure 4 As shown: Wild-type EMT-6 tumors do not express hHer2; EMT-6 / hHer2 tumors express hHer2, and the hHer2 gene is not lost during the model establishment process.

[0123] Example 6

[0124] EMT-6 / hHer2 Tumor Model for Evaluation of hHer2 Drugs

[0125] 6.1 Cell culture

[0126] Wild-type EMT-6 and modified EMT-6 / hHer2 cells were cultured in DMEM medium containing 10% fetal bovine serum. EMT-6 and EMT-6 / hHer2 cells in the exponential growth phase were collected and resuspended to a suitable concentration for subcutaneous tumor inoculation in mice.

[0127] 6.2 Tumor inoculation

[0128] The experimental mice were subcutaneously inoculated with EMT-6 and EMT-6 / hHer2 cells on the right back, with a cell inoculation volume of 1×10 6 / mouse, the inoculation volume was 0.1mL / mouse, and the tumor growth was observed regularly.

[0129] 6.3 Drug treatment

[0130] When the tumor grows to about 80 mm 3 When the tumor size was about 100 μg / cm2, each model was divided into two groups and treated with drugs. One group was given an isotype control antibody at a dose of 10 mpk twice a week; the other group was given an hHer2 antibody drug (Trastuzumab) at a dose of 10 mpk twice a week. The patients were observed regularly and the tumor volume was measured. Figure 5 As shown, the results showed that the modified EMT-6 / hHer2 model can evaluate the hHer2 antibody drug (Trastuzumab), while the wild-type EMT-6 model has no effect.

[0131] As can be seen from the above examples, we added hHer2 targets for humanization on the basis of homologous tumor transplantation models. On the basis of its complete immune system, the target of hHer2 targeted drugs was added, and research on immune and targeted combined therapy can be carried out. We selected the mouse tumor cell line EMT-6 for hHer2 humanization (EMT-6 / hHer2), and constructed a humanized mouse tumor model, which can be used for pharmacodynamic evaluation of hHer2 immune and targeted combined therapy. Our research results can help the research on hHer2 drug combination therapy and the development of related antibody drugs.

[0132] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model, characterized in that: The following steps are involved: 1) Construction of hHer2 overexpression lentiviral vector According to the gene sequence of hHer2, a hHer2 overexpression lentiviral vector was constructed; 2) Construction of EMT-6 / hHer2 cell line The hHer2 overexpression lentiviral vector was used to transfect the EMT-6 cell line and perform cell monoclonal screening. The expression of hHer2 in the obtained EMT-6 / hHer2 monoclonal cells was detected by FACS to construct the EMT-6 / hHer2 cell line; 3) Establishment of EMT-6 / hHer2 tumor model The EMT-6 / hHer2 cell line was inoculated into BALB / C mice to establish a mouse tumor model.

2. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 1, characterized in that: In the step 1), the hHer2 gene sequence is shown in NM_004448.

3.

3. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 1, characterized in that: Step 1) The following steps are involved: S1: inserting the hHer2 gene fragment into the cloning vector pcDNA3.1 to form the cloning plasmid pcDNA3.1 / hHer2; S2: The pLVX-EF1α-IRES-Puro vector plasmid and the pcDNA3.1 / hHer2 plasmid were double-digested with EcoR I and Xho I restriction sites, and then the pLVX-EF1α-IRES-Puro vector fragment and the hHer2 gene fragment were recovered and ligated in the presence of T4 DNA ligase. After identification, the plasmid pLVX-EF1α-IRES-Puro-hHer2 was obtained; S3: The pLVX-EF1α-IRES-Puro-hHer2 plasmid was transfected into 293T cells by liposome method for virus packaging to obtain lentiviral particles.

4. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 3, characterized in that: The step 2) comprises the following steps: a: pLVX-EF1α-IRES-Puro-hHer2 lentivirus infected EMT-6 cells: a1: EMT-6 cells were inoculated in a 6-well plate one day before lentiviral infection and cultured in a 5% CO2, 37°C constant temperature incubator; a2: On the day of the experiment, the cell confluence reached 30-50%; a3: Place the pLVX-EF1α-IRES-Puro-hHer2 lentiviral particles on ice to dissolve; a4: Take 100 μL of pLVX-EF1α-IRES-Puro-hHer2 lentiviral solution and add it to EMT-6 cells and continue culturing; a5: After 72 hours of culture, add the screening antibiotic Puromicin to screen for cell resistance; a6: After the screening is completed, the obtained cells are digested into single cells, and the cell monoclonal clones are picked using the limiting dilution method. After one week, after the monoclonal cell amplification is completed, the monoclonal cells are identified by FACS to confirm whether hHer2 is successfully introduced into EMT-6 cells.

5. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 4, characterized in that: Also includes: b: FACS identification of EMT-6 / hHer2 monoclonal cells: b1: Collect EMT-6 / hHer2 monoclonal cells into a 5 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant; b2: Add 2 mL FACS buffer (PBS + 2% FBS) to resuspend the cells, mix well by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant; b3: Add 2 μL PE-hHer2 antibody, incubate at 4°C for 30 min, centrifuge at 1000 rpm for 10 min, discard the supernatant, and wash twice with FACS buffer; b4: Add 500 μL FACS buffer, resuspend the cells to make a single cell suspension, and transfer it to the marked flow cytometry loading tube; b5: set up another tube of wild-type EMT-6 cell suspension as a negative control; b6: Detection by flow cytometry.

6. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 5, characterized in that: The step 3) includes the following specific steps: c1: cell culture EMT-6 / hHer2 cells were cultured in DMEM medium containing 10% fetal bovine serum, and EMT-6 / hHer2 cells in the exponential growth phase were collected and resuspended to a suitable concentration for subcutaneous tumor inoculation in mice; c2: Tumor inoculation The experimental mice were subcutaneously inoculated with EMT-6 / hHer2 cells on the right back, with a cell inoculation volume of 1×10 6 / mouse, the inoculation volume was 0.1mL / mouse, and the tumor growth was observed regularly. After the tumor was formed, regular observation was performed; c3: FACS identified EMT-6 / hHer2 tumors expressing hHer2.

7. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 6, characterized in that: c3: FACS identification of EMT-6 / hHer2 tumors expressing hHer2, including: EMT-6 and EMT-6 / hHer2 tumors grew to approximately 500 mm 3 When the cells were grown, the tumor tissues were removed and the cells were made into single cell suspensions; the expression of hHer2 in EMT-6 and EMT-6 / hHer2 tumor single cells was detected by FACS.

8. The method for constructing an hHer2 gene humanized mouse EMT-6 tumor cell line animal model according to claim 1, characterized in that: It also includes 4) EMT-6 / hHer2 tumor model to evaluate hHer2 drugs. The following steps are involved: Wild-type EMT-6 and modified EMT-6 / hHer2 cell lines were inoculated into BALB / C mice to establish a mouse tumor model. When the tumor grew to about 80 mm 3 When the tumor size was about 100 μg / cm2, each model was divided into two groups and treated with drugs. One group was given the isotype control antibody Isotype control at a dose of 10 mpk twice a week; the other group was given the hHer2 antibody drug Trastuzumab at a dose of 10 mpk twice a week. The patients were observed regularly and the tumor volume was measured.

9. Use of the animal model prepared by the construction method according to any one of claims 1 to 8 in evaluating hHer2-related antibody drugs.

10. The use according to claim 9, characterized in that: The antibody drugs include Trastuzumab.

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