A method for constructing a Huh-7-ATP7BKO-MRE-Luc cell line and its application

Abstract

The present application discloses a method for constructing a Huh‑7‑ATP7BKO‑MRE‑Luc cell line and its application, which belongs to the field of biomedicine. The construction of the Huh‑7‑ATP7BKO‑MRE‑Luc cell line is achieved by transfection of a gene knockout plasmid and a gene insertion plasmid. Therefore, the cell line is a quantifiable cell model with the same mechanism of action that can be used for the in vitro biological activity evaluation of AAV therapeutic vectors for Wilson's disease.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a construction method and application of a Huh-7-ATP7BKO-MRE-Luc cell line. Background Art

[0002] Wilson disease (WD) is a rare autosomal recessive genetic disease that mainly affects the liver, nervous system, and other tissues. The root cause of the disease is a mutation in the ATP7B gene located on chromosome 13, which leads to abnormal function of the ATP7B protein. The ATP7B gene encodes a P-type copper-transporting ATPase that is mainly expressed in hepatocytes and is used for transmembrane transport of copper. Abnormal function of the ATP7B protein causes hepatocytes to reduce copper excretion into the bile, leading to copper accumulation in the liver and other tissues, which in turn leads to liver, nervous, and related systemic clinical symptoms.

[0003] As a cutting-edge biomedical field, gene therapy aims to treat a range of genetic and chronic diseases by adjusting, repairing or replacing defective genes in patients. Adeno-associated virus (AAV) vectors have become one of the most widely studied and applied vectors in the field of in vivo gene therapy due to their structural and biological advantages such as stable physical and chemical properties, weak pathogenicity, low integration risk, and persistent expression of exogenous genes. Using AAV vectors to carry the ATP7B gene and introduce the ATP7B protein that expresses normal function into the patient's body is expected to achieve a one-time administration and lifelong benefit treatment for Wilson's disease.

[0004] Regulations require the establishment of biological activity analysis methods that are identical or related to the product's in vivo mechanism of action for the quality evaluation of gene therapy products. However, there is currently a lack of cell models that can effectively evaluate the biological activity of Wilson disease gene therapy vectors. The construction of a cell model with ATP7B gene knockout or silenced expression has important application value for studying the development mechanism of cell models of ATP7B gene and Wilson disease gene therapy vector biological activity, targeted drug treatment mechanism, etc. Summary of the invention

[0005] The content of this application is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this application is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0006] In order to solve the technical problems mentioned in the above background technology section, some embodiments of the present application provide

[0007] A method for constructing a Huh-7-ATP7BKO-MRE-Luc cell line, wherein the cell line is derived from a human Huh-7 cell line; the cell is constructed by knocking out the ATP7B gene of the Huh-7 cell line and inserting a metal response element (MRE)-luciferase reporter system.

[0008] Furthermore, ATP7B gene knockout was achieved by gene knockout plasmid transfection, and the knockout plasmid carried elements including: sgRNA transcription sequence specifically targeting ATP7B gene, Cas9 protein-puromycin resistance expression element; metal response element (MRE)-luciferase reporter system insertion was achieved by gene insertion plasmid transfection, and the gene insertion plasmid carried elements including: PiggyBac5' inverted terminal repeat sequence (ITR), 4×MRE, E1b minimal promoter, luciferase coding sequence and PiggyBac3'ITR sequence, and a sequential combination of puromycin resistance expression element.

[0009] Furthermore, the sgRNA sequence specifically targeting the ATP7B gene is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0010] Furthermore, the PiggyBac 5' inverted terminal repeat sequence is SEQ ID NO.4; the 4×MRE sequence is SEQ ID NO.5; the E1b minimal promoter sequence is SEQ ID NO.6; the luciferase coding sequence is SEQ ID NO.7; and the PiggyBac 3'ITR sequence is SEQ ID NO.8.

[0011] A Huh-7-ATP7BKO-MRE-Luc cell line is used to evaluate the function of a gene therapy vector, characterized in that after being infected with a gene therapy vector, the Huh-7-ATP7BKO-MRE-Luc cells can reflect the function of the vector through changes in luciferase expression regulated by metal ion concentration.

[0012] A Huh-7-ATP7BKO-MRE-Luc cell line is used to evaluate the function of a Wilson disease therapeutic drug, characterized in that the Huh-7-ATP7BKO-MRE-Luc cell can reflect the effectiveness of the Wilson disease therapeutic drug through changes in the luciferase expression regulated by metal ion concentration.

[0013] Application of a Huh-7-ATP7BKO-MRE-Luc cell line in drug evaluation for diseases related to copper ion abnormalities.

[0014] A use of a Huh-7-ATP7BKO-MRE-Luc cell line in copper ion concentration detection, including clinical sample copper ion detection or diagnosis and environmental copper ion detection.

[0015] The beneficial effects of the present application are: the Huh-7-ATP7BKO-MRE-Luc cell line has knocked out the ATP7B gene and cannot express normal ATP7B protein, which is similar to the patient's phenotype. In addition, the cell line contains a luciferase reporter system regulated by the intracellular copper ion concentration (metal response element, MRE), which can quantitatively evaluate the copper transport biological function of the ATP7B protein. Therefore, the cell line is a quantifiable cell model with the same mechanism of action that can be used to evaluate the in vitro biological activity of AAV therapeutic vectors for Wilson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings constituting a part of this application are used to provide a further understanding of this application, so that other features, purposes and advantages of this application become more obvious. The illustrative embodiment drawings and their descriptions of this application are used to explain this application and do not constitute an improper limitation on this application.

[0017] In addition, throughout the drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that the components and elements are not necessarily drawn to scale.

[0018] In the attached picture:

[0019] Figure 1 is the map of the knockout plasmid (LYM3P357-sg1);

[0020] Figure 2 is a map of the gene insertion plasmid (LYM3P355);

[0021] Figure 3 This is the confirmation diagram of ATP7B gene mutation;

[0022] Figure 4 This is a graph of monoclonal gene sequencing results;

[0023] Figure 5 This is the ATP7B protein knockout identification diagram;

[0024] Figure 6 is the luciferase-copper ion concentration response curve;

[0025] Figure 7 is the luciferase-AAV infection MOI response curve;

[0026] Figure 8 It is a table of denaturation and annealing procedures for PCR amplification products;

[0027] Fig. 9 is a table of monoclonal luciferase assay results. DETAILED DESCRIPTION

[0028] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0029] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0030] The present disclosure will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0031] Reference Figure 1-Figure 9 , Example 1

[0032] A method for constructing a Huh-7-ATP7BKO-MRE-Luc cell line. The Huh-7-ATP7BKO-MRE-Luc cell line is derived from a human Huh-7 cell line.

[0033] The cell line was constructed by knocking out the ATP7B gene in the Huh-7 cell line and inserting the metal response element (MRE)-luciferase reporter system.

[0034] Knocking out the ATP7B gene in the Huh-7 cell line includes: transfecting the Huh-7 cell line with a gene knockout plasmid containing a sgRNA transcription sequence specifically targeting the ATP7B gene and a Cas9 protein-puromycin resistance expression element, knocking out the ATP7B gene through the CRISPR system, and screening monoclonal strains; the map of the gene knockout plasmid is as follows Figure 1 shown.

[0035] The insertion metal response element (MRE)-luciferase reporter system includes: using a gene insertion plasmid containing a sequential combination of PiggyBac5' inverted terminal repeat sequence (ITR), 4×MRE, E1b minimal promoter, luciferase coding sequence and PiggyBac3'ITR sequence and a puromycin resistance expression element to transfect Huh-7-ATP7BKO cells, inserting the metal response element-luciferase reporter system into the genome of the cells through the PiggyBac transposon system, and screening single clones; the map of the gene insertion plasmid is shown in Figure 2 shown.

[0036] The sgRNA transcription sequence specifically targeting the ATP7B gene includes at least one of the three sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. Preferably, the sgRNA transcription sequence specifically targeting the ATP7B gene is SEQ ID NO.3.

[0037] The PiggyBac 5' inverted terminal repeat sequence is SEQ ID NO.4; the 4×MRE sequence is SEQ ID NO.5; the E1b minimal promoter sequence is SEQ ID NO.6; the luciferase coding sequence is SEQ ID NO.7; and the PiggyBac 3'ITR sequence is SEQ ID NO.8. Example 2

[0038] A method for constructing a Huh-7-ATP7BKO-MRE-Luc cell line in which the ATP7B gene is knocked out and a metal response element-luciferase reporter system is inserted, comprising:

[0039] Huh-7 cells were transfected with the knockout plasmid and monoclonal screening was performed by adding puromycin to obtain the Huh-7-ATP7BKO cell line;

[0040] Huh-7-ATP7BKO cells were transfected with the gene insertion plasmid, and monoclonal screening was performed by adding puromycin to obtain the Huh-7-ATP7BKO-MRE-Luc cell line.

[0041] Huh-7 cells were transfected with gene knockout plasmids to obtain Huh-7-ATP7BKO cell lines, including:

[0042] The continuously cultured Huh-7 cells were seeded into T25 culture flasks;

[0043] The gene knockout plasmid, DMEM and FectoVIR transfection reagent were mixed to prepare a transfection complex;

[0044] When the confluence in the T25 flask reaches 50%-70%, add the transfection complex to the T25 flask;

[0045] After puromycin selection, the cells were diluted and plated in 96-well plates;

[0046] A monoclonal Huh-7-ATP7BKO cell line was screened out.

[0047] The Huh-7-ATP7BKO cells were transfected with the gene insertion plasmid to obtain the Huh-7-ATP7BKO-MRE-Luc cell line, including:

[0048] Continuously cultured Huh-7-ATP7BKO cells were seeded into T25 culture flasks;

[0049] The gene insertion plasmid, DMEM and FectoVIR transfection reagent were mixed to prepare a transfection complex;

[0050] After puromycin selection, the cells were diluted and plated in 96-well plates;

[0051] The monoclonal Huh-7-ATP7BKO-MRE-Luc cell line was screened out.

[0052] Specifically, Huh-7 cell seeds were cultured for 4 generations and then inoculated into T25 culture flasks; specifically, the number of inoculated cells was 1.5×10 6 ; Continue to culture for 24 hours after inoculation.

[0053] The gene knockout plasmid, DMEM and FectoVIR transfection reagent are mixed to obtain a transfection complex; specifically, 3 μg of a plasmid containing a sgRNA transcription sequence specifically targeting the ATP7B gene is added to 500 μL of DMEM, mixed well, and then 6 μL of FectoVIR transfection reagent is added, mixed well, and allowed to stand at room temperature for 25 minutes; the gene knockout plasmid contains a sgRNA sequence specifically targeting the ATP7B gene, and most preferably, the sgRNA sequence is SEQ ID NO.3.

[0054] When the cell confluence reaches 50%-70%, add the transfection complex to a T25 culture flask and place it in a CO2 incubator for continued culture; 24 hours after transfection, replace it with a selection medium containing puromycin.

[0055] After 5 days of screening, the drug was withdrawn and replaced with complete medium without puromycin. After 24 hours of recovery, monoclonal plating was performed and DNA was extracted from the remaining cells for sgRNA cutting confirmation.

[0056] The target region DNA was amplified by PCR using primer pairs on both sides of each sgRNA targeting site. Figure 8 The program performs denaturation and gradient annealing. If the cell genome mutates, a hybrid double strand will be formed during the denaturation and annealing process. Add T7EndonucleaseI (T7E1) to this system, and after enzyme digestion at 37℃ for 5 minutes, the hybrid double strand can be cut. Observe the enzyme-cut fragments through agarose gel electrophoresis to confirm the cutting. The results are as follows Figure 3 As shown, all three sgRNAs can cause mutations in the ATP7B gene.

[0057] After the plated cells were digested with trypsin and the complete medium was terminated, single cell suspension was formed by pipetting and sampling was performed. According to the counting results, the cell suspension was diluted to 7 cells / mL with complete medium.

[0058] After mixing, inoculate into 96-well plates, 100 μL per well, and spread 2 96-well plates for each plasmid. Place the 96-well plates in a CO2 incubator, observe the cell growth status under a microscope regularly, find the monoclonal cell wells and mark them.

[0059] When the monoclonal cells are expanded to a confluence of ~30%, they are digested with trypsin and re-plated in the original wells to make the cells evenly distributed and continue to be cultured until they fill the wells. The monoclonal cells in the 96-well plate are digested and inoculated into the 24-well plate for expansion. When the cells in the 24-well plate are full, they are digested and inoculated into the 6-well plate for expansion.

[0060] After the cells in the 6-well plate are fully grown, digest and resuspend them, and half of the cells are inoculated into T25 for expansion culture; DNA is extracted from the other half of the cells, and the primer pairs on both sides of each sgRNA targeting site are used to amplify the target region DNA by PCR, and the genotype is identified by sequencing. According to the sequencing results ( Figure 4 ), a homozygous single base (G) insertion cell line (SG1-A1) was screened. The sequencing results of the remaining cell lines all showed overlapping peaks, and more than two bases appeared at a single site, indicating that they may be non-monoclonal cell lines, or that Cas9 continued to edit genes in different cells after cell division, so these cell lines were discarded.

[0061] After the cells in the T25 culture flask are fully grown, digest and resuspend them, and take 1.5×10 6The protein expression level was detected by cells, and the remaining cells were inoculated into T75 for expansion culture. After rinsing the cells used for protein detection with PBS, the cell pellet was added with urea lysis solution containing protease inhibitors and ultrasonically lysed for 20 minutes. The lysed sample was centrifuged in a centrifuge at 4°C and 16000g for 10 minutes, and the supernatant was taken to a new centrifuge tube and incubated at 95°C for 10 minutes. The treated cell lysate was subjected to SDS-PAGE electrophoresis, and the positive control (cell lysate overexpressing ATP7B-LY, ATP7B-LY is a truncated form of the natural ATP7B protein) and Huh-7 mother cell control (Huh-7 cell lysate that has not been gene-edited) were loaded at the same time. After transfer, it was divided into two parts. The large molecular weight part was detected with ATP7B-specific antibody (Anti-ATP7BRabbitMonoclonalAntibody), and the small molecular weight part was detected with β-Actin antibody. The results showed that ( Figure 5 ), the expression of ATP7B protein could not be detected, and a Huh-7-ATP7BKO monoclonal cell line was obtained.

[0062] After the cells in the T75 culture flask are fully grown, they are further expanded and cultured to T175; after full growth, some cells are taken to construct the Huh-7-ATP7BKO-MRE-Luc cell line, and the remaining cells are frozen.

[0063] Huh-7-ATP7BKO cells were transfected with the gene insertion plasmid, and monoclonal screening was performed after adding puromycin to obtain Huh-7-ATP7BKO-MRE-Luc cell lines, including:

[0064] The gene insertion plasmid comprises a sequential combination of PiggyBac5'ITR, 4xMRE, E1b minimal promoter, luciferase coding sequence and PiggyBac3'ITR sequence, and a puromycin resistance expression element, wherein the PiggyBac5' inverted terminal repeat sequence is SEQ ID NO.4, the 4xMRE sequence is SEQ ID NO.5, the E1b minimal promoter sequence is SEQ ID NO.6, the luciferase coding sequence is SEQ ID NO.7, and the PiggyBac3'ITR sequence is SEQ ID NO.8.

[0065] Huh-7-ATP7BKO cells were seeded into T25 culture flasks; specifically, the number of cells seeded was 1.5×10 6 ; Continue to culture for 24 hours after inoculation.

[0066] The gene insertion plasmid, DMEM and FectoVIR transfection reagent were mixed to obtain a transfection complex; specifically, 3 μg of the gene insertion plasmid was added to 500 μL of DMEM, mixed well, and then 6 μL of FectoVIR transfection reagent was added, mixed well, and allowed to stand at room temperature for 25 minutes;

[0067] When the cell confluence reaches 50%-70%, add the transfection complex to a T25 culture flask and place it in a CO2 incubator for continued culture; 24 hours after transfection, replace it with a selection medium containing puromycin.

[0068] After 5 days of screening, the drug was withdrawn and replaced with complete medium without puromycin. Monoclonal plating was performed after 24 hours of recovery.

[0069] After the plated cells are digested with trypsin and the complete medium is terminated, a single cell suspension is formed by blowing, and samples are taken for counting. According to the counting results, the cell suspension is diluted to 8 cells / mL with complete medium. After mixing, it is inoculated into a 96-well plate, 100 μL per well, and a total of 6 96-well plates are plated. The 96-well plate is cultured in a CO2 incubator, and the cell growth status is observed regularly under a microscope. The monoclonal cell wells are found and marked. When the monoclonal cells are expanded to a confluence of ~30%, they are digested with trypsin, and the original wells are re-plated to make the cells evenly distributed, and the culture is continued until the wells are full. After the monoclonal cells in the 96-well plate are digested, they are inoculated into a 24-well plate for expansion.

[0070] After the cells in the 24-well plate are fully grown, digest and resuspend them, take 1 / 2 of the cells and inoculate them into a new 24-well plate as a test plate, and take another 1 / 4 of the cells and inoculate them into another new 24-well plate as a seed plate. From this step on, maintenance medium is used for cell expansion culture. The test plate was taken out after 3 days of culture, and complete culture medium containing a final concentration of 100μM CuSO4 was added to each well. After continuing to culture for 24 hours, discard the supernatant, rinse with DPBS, and then add 100μL CellLysisBuffer, and oscillate at room temperature and 450rpm for lysis for 20 minutes. Transfer the lysate to a 96-well U-bottom plate, centrifuge at 4°C and 4000rpm for 5 minutes, take 20μL of supernatant, add 100μL Luciferase Reaction Solution, and detect the chemiluminescent signal. The test results are as follows. Fig. 9 As shown, clone 1A1 had very strong luciferase expression, 3A1 had a higher luciferase signal, and the remaining clones had lower signals and were abandoned for cultivation.

[0071] After the 1A1 and 3A1 clones in the seed plate have grown to full size, digest them and inoculate them into 6-well plates for expansion. After the cells in the 6-well plates have grown to full size, digest them and inoculate them into T25 culture flasks for expansion. After the cells in the T25 culture flask have grown to full size, digest them and inoculate them into T75 culture flasks for expansion. Select Huh-7-ATP7BKO-MRE-Luc cells. After the cells in the T75 culture flask have grown to full size, digest them and inoculate them into T175 culture flasks for expansion. After they have grown to full size, freeze the cells.

[0072] Among them, complete culture medium: 89% DMEM medium + 10% fetal bovine serum + 1% double antibody;

[0073] Screening medium: complete medium + final concentration 2 μg / mL puromycin;

[0074] Maintenance medium: complete medium + puromycin at a final concentration of 1 μg / mL. Example 3

[0075] Application of Huh-7-ATP7BKO-MRE-Luc cells in evaluating the potency of gene therapy vectors carrying ATP7B.

[0076] Verification that the luciferase expression level of Huh-7-ATP7BKO-MRE-Luc cells is regulated by copper ion concentration:

[0077] Huh-7-ATP7BKO-MRE-Luc cells were plated in 24-well plates at 1.2 × 10 cells per well. 6 Cells. After 72 hours of culture, complete culture medium containing final concentrations of 0, 10, 20, 40, 60, 80, and 100 μM CuSO4 was added. After 24 hours of continued culture, the supernatant was discarded, the cells were rinsed with DPBS, and then 100 μL Cell Lysis Buffer was added. The cells were shaken at room temperature and 300 rpm for 20 minutes. The lysate was transferred to a 96-well U-bottom plate and centrifuged at 4°C and 4000 rpm for 5 minutes. 20 μL of supernatant was taken, 100 μL of Luciferase Reaction Solution was added, and the chemiluminescent signal was detected. The results are as follows: Figure 6 As shown, the copper ion concentration in the range of 10-100 μM (log10) conforms to an S-shaped curve with the luciferase signal value, and when fitted in a 4-parameter manner, the correlation coefficient R2 is 0.9808.

[0078] A use of Huh-7-ATP7BKO-MRE-Luc cells in the development of drugs for Wilson's disease;

[0079] Huh-7-ATP7BKO-MRE-Luc cells are used for AAV in vitro biological activity detection:

[0080] Huh-7-ATP7BKO-MRE-Luc cells were seeded in 24-well plates at a density of 1.2×10 6 cells per well. After culturing for 24 hours, AAV8-ATP7B was added for infection. Another two groups of cell wells were not infected with AAV as controls. 48 hours after infection, complete medium containing 75 μM CuSO4 at the final concentration was added to the AAV-infected wells and one group of control wells (background), and medium without CuSO4 was added to the other group of control wells (blank). After continuing to culture for 24 hours, the supernatant was discarded, and the cells were rinsed once with DPBS. Then, 100 μL of Cell Lysis Buffer was added, and the cells were lysed by shaking at 300 rpm at room temperature for 20 minutes. The lysate was transferred to a 96-well U-bottom plate and centrifuged at 4000 rpm for 5 minutes at 4°C. 20 μL of the supernatant was taken and added to 100 μL of Luciferase Reaction Solution to detect the chemiluminescence signal. The in vitro biological activity of AAV8-ATP7B can be quantified by the inhibition rate of the reaction with excess copper, and the calculation formula is as follows:

[0081]

[0082] Specificity: When the virus was infected, another group of cell wells was set up, and a preparation solution without AAV (negative control) was added. The results of luciferase detection were in line with RFU blank (12243) < RFU test sample (294258) < RFU negative control (418933) < RFU background (483267), indicating that the signal inhibition was specific to AAV infection.

[0083] Linearity: The MOIs of AAV8-ATP7B infection were set to 1×10 4 , 3×10 4 , 1×10 5 , 3×10 5 , 1×10 6 , 3×10 6 , respectively. After infection, copper was added and the luciferase signal was detected. The results were as Figure 7 shown. When the MOI was in the range of 1×10 4 -3×10 6 (log10), it was in line with an S-shaped curve with the luciferase signal value. When fitted by the four-parameter method, the correlation coefficient R2 was 0.9708.

[0084] Precision: The same sample was repeatedly detected 5 times on different days. The inhibition rates of the reaction with excess copper were 38.94%, 42.73%, 45.49%, 42.63%, and 28.31% respectively, and the coefficient of variation RSD was 17.0%.

[0085] The above results indicate that the analytical method established using the Huh-7-ATP7BKO-MRE-Luc cell line performs well in determining the in vitro biological activity of AAV8-ATP7B.

[0086] Application of a Huh-7-ATP7BKO-MRE-Luc cell line in drug evaluation for diseases related to copper ion abnormalities.

[0087] Application of Huh-7-ATP7BKO-MRE-Luc cell line in copper ion concentration detection; including clinical sample copper ion detection or diagnosis and environmental copper ion detection

[0088] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the embodiments of the present disclosure.

Claims

1. A method for constructing a Huh-7-ATP7BKO-MRE-Luc cell line, characterized in that: The cell line is derived from human Huh-7 cell line; The cell was constructed by knocking out the ATP7B gene in the Huh-7 cell line and inserting the metal response element (MRE)-luciferase reporter system; ATP7B gene knockout was achieved by transfection with a knockout plasmid, which carried elements including: a sgRNA transcription sequence specifically targeting the ATP7B gene, and a Cas9 protein-puromycin resistance expression element; The metal response element (MRE)-luciferase reporter system insertion was achieved by transfection with a gene insertion plasmid carrying the following elements: a sequential combination of the PiggyBac5' inverted terminal repeat (ITR), 4×MRE, E1b minimal promoter, luciferase coding sequence and PiggyBac3'ITR sequence, and a puromycin resistance expression element; The PiggyBac5' inverted terminal repeat sequence is SEQ ID NO.4; The 4×MRE sequence is SEQ ID NO. 5; The E1b minimal promoter sequence is SEQ ID NO.6; The luciferase coding sequence is SEQ ID NO.7; The PiggyBac 3' ITR sequence is SEQ ID NO.

8.

2. The method for constructing the Huh-7-ATP7BKO-MRE-Luc cell line according to claim 1, characterized in that: The sgRNA sequence specifically targeting the ATP7B gene is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

3. A use of the Huh-7-ATP7BKO-MRE-Luc cell line according to claim 1 in evaluating the function of a gene therapy vector, characterized in that: After Huh-7-ATP7BKO-MRE-Luc cells were infected with gene therapy vectors, the expression of luciferase was regulated by metal ion concentration, reflecting the function of the vector.

4. A use of the Huh-7-ATP7BKO-MRE-Luc cell line according to claim 1 in evaluating the function of a drug for treating Wilson's disease, characterized in that: Huh-7-ATP7BKO-MRE-Luc cells reflect the effectiveness of Wilson disease therapeutic drugs by changes in the expression of luciferase regulated by metal ion concentrations.

5. Use of the Huh-7-ATP7BKO-MRE-Luc cell line as claimed in claim 1 in detecting environmental copper ion concentration.

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