A recombinant brain glioma cell with stable knockout of METTL7B gene and a construction method thereof

Abstract

The present invention discloses a recombinant cell for glioma with stable knockout of METTL7B gene and a construction method thereof, belonging to the technical field of genetic engineering. The technical problem to be solved by the present invention is: how to construct a glioma cell model with METTL7B knockout. In order to solve the technical problem, the present invention provides a recombinant cell, wherein the recombinant cell does not contain a gene encoding a METTL7B protein or the expression amount of the METTL7B protein is reduced or the activity of the METTL7B protein is reduced, and the METTL7B protein is a protein with an amino acid sequence of SEQ ID No. 1. The present invention also improves the construction method of the recombinant cell. The recombinant cell obtained by the present invention provides an available cell model for the study of the function and mechanism of action of human METTL7B protein, not only for studying its molecular mechanism, but also for discovering it as a new drug target and providing an important research tool for drug discovery.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and specifically to a brain glioma recombinant cell with a stably knocked-out METTL7B gene and a construction method thereof. Background Art

[0002] Glioma is a general term for tumors originating from neuroepithelium. It is the most common intracranial tumor, accounting for 40% to 50% of brain tumors and the most common primary intracranial tumor. Its malignancy rate is 2% to 3% of systemic tumors, and its annual incidence rate is 3 to 8 people per 100,000 population. Glioma can be divided into low-grade glioma and high-grade glioma. High-grade glioma has biological characteristics such as rapid invasive growth, abnormal vascular proliferation, and easy destruction of brain tissue, which makes malignant glioma difficult to treat and has a poor prognosis. After systemic treatment, its median survival time is still less than 15 months. At present, the treatment of glioma is still mainly surgical resection, supplemented by radiotherapy and chemotherapy after surgery. However, due to short-term recurrence and drug resistance, the treatment effect is unsatisfactory. Therefore, the situation of glioma prevention and treatment is still very severe. Therefore, exploring the molecular mechanism of the occurrence and development of glioma is very critical for in-depth understanding of its pathogenesis, as well as disease prevention and treatment.

[0003] METTL7B (methyltransferase like 7B) is a member of the methyltransferase-like family. Its protein structure contains an S-adenosylmethionine binding site (SAMbinding site). METTL7B is involved in the regulation of invasion and migration of various malignant tumors. METTL7B is involved in the genomic stability and tumor invasion of breast cancer cells. METTL7B can promote the transformation of tumor epithelial cells to mesenchymal cells, thereby leading to the invasion and migration of thyroid cancer cells. The latest study shows that the expression of METTL7B is most significantly increased in brain glioma tissues compared with other members and is negatively correlated with survival. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to construct a METTL7B knockout brain glioma cell model.

[0005] In order to solve the above technical problems, in a first aspect, the present invention provides a recombinant cell, wherein the recombinant cell does not contain a gene encoding a METTL7B protein or the expression level of the METTL7B protein is reduced or the activity of the METTL7B protein is reduced, and the METTL7B protein is any one of the following:

[0006] A1), a protein having an amino acid sequence of SEQ ID No. 1;

[0007] A2) A protein related to the METTL7B protein obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A1) having more than 80% identity with the protein shown in A1);

[0008] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] Among them, SEQ ID No. 1 consists of 244 amino acid residues.

[0010] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0011] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0012] Furthermore, in the above-mentioned recombinant cell, the recombinant cell is constructed according to the following method.

[0013] Furthermore, the recipient cells of the above-mentioned recombinant cells are animal cells.

[0014] Furthermore, the recipient cells of the above recombinant cells are human cells.

[0015] Furthermore, the recipient cells of the above-mentioned recombinant cells are human glioma cells.

[0016] Furthermore, the recipient cell of the above recombinant cell is U251 cell.

[0017] In a second aspect, the present invention provides a method for constructing the above-mentioned recombinant cell, the method comprising knocking out the METTL7B gene in a recipient cell to obtain the recombinant cell, wherein the METTL7B gene is any one of the following:

[0018] g1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2;

[0019] g2), the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.3;

[0020] g3), a DNA molecule that has 80% or more identity with the nucleotide sequence defined in g1) or g2), and encodes the METTL7B protein.

[0021] Furthermore, in the above method, knocking out the METTL7B gene in the recipient cell includes: infecting the recipient cell with a lentivirus, wherein the lentivirus expresses gRNA and Cas9 protein targeting the METTL7B gene.

[0022] Furthermore, in the above method, the target sequence of the gRNA is SEQ ID No. 4 (5'-GTTCTACCCACCGGGCTGCA-3').

[0023] In one embodiment of the present invention, the lentivirus is prepared by co-transfecting host cells with the recombinant plasmid pLV-U6-gRNA1-METTL7B-puro, plasmid psPAX2 and plasmid pMD2G.

[0024] The recombinant plasmid pLV-U6-gRNA1-METTL7B-puro can express sgRNA and Cas9 protein targeting the METTL7B gene, and the recombinant plasmid pLV-U6-gRNA1-METTL7B-puro is constructed based on the backbone plasmid pLV-U6-puro-Cas9.

[0025] Furthermore, in the above method, the knocking out of the METTL7B gene in the recipient cell is to mutate the METTL7B gene in the recipient cell as follows: inserting a nucleotide T between the 293rd and 294th nucleotides of SEQ ID No.3.

[0026] Furthermore, in the above method, the recipient cell is any one of the following:

[0027] C1), animal cells;

[0028] C2), mammalian cells;

[0029] C3), human cells;

[0030] C4), human glioma cells;

[0031] C5) and U251 cells.

[0032] In one embodiment of the present invention, relative to the wild-type U251, in the two chromosomes of the U251 cells in which METTL7B (gRNA1) was knocked out, the METTL7B gene added a nucleotide T (located in the first exon) between the 293rd and 294th nucleotides of SEQ ID No. 3, resulting in a frameshift mutation in METTL7B, thereby causing METTL7B to be unable to express protein normally, thereby achieving the knockout of the METTL7B gene.

[0033] In a third aspect, the present invention provides a biological material related to the above-mentioned recombinant cell, wherein the biological material is selected from any one of the following:

[0034] B1), a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the METTL7B protein or the activity of the METTL7B protein;

[0035] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0036] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0037] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0038] Furthermore, in the above-mentioned biological material, B1) the nucleic acid molecule is a gRNA targeting the METTL7B gene in the above-mentioned recombinant cell, or the nucleic acid molecule is a DNA molecule expressing a gRNA targeting the METTL7B gene in the above-mentioned recombinant cell.

[0039] Furthermore, in the above-mentioned biological material, the target sequence of the gRNA is SEQ ID No. 4 (5'-GTTCTACCCACCGGGCTGCA-3').

[0040] Furthermore, in the above-mentioned biological materials, the recombinant microorganism described in B4) may be a recombinant lentivirus.

[0041] In the present invention, the recombinant cell may be an in vitro cell. The recombinant cell may be a human cell or an animal cell. The human cell is not a reproductive cell or a fertilized egg.

[0042] The animal cell is not a germ cell or a fertilized egg or an embryonic stem cell.

[0043] Furthermore, the recombinant cell in the present invention may be a U251 cell line in which METTL7B is stably knocked out.

[0044] In one embodiment of the present invention, the construction process of the stable cell line is as follows: the monoclonal cells after METTL7B protein knockout verification are cultured in a high-glucose DMEM (Gibco, C11995500BT) medium containing 10% FBS (Cellbox, AUS-01S-02), and the U251 cell line with stable METTL7B knockout is obtained after amplification.

[0045] In a fourth aspect, the present invention provides an application, wherein the application is any one of the following:

[0046] A1) Use of the above-mentioned cells or the above-mentioned biological materials in the study of the function and / or mechanism of action of METTL7B protein;

[0047] A2) Use of the above-mentioned cells or the above-mentioned biological materials in the preparation of products for studying the function and / or mechanism of action of METTL7B protein;

[0048] A3) Application of the above cells or the above biological materials in the study of METTL7B protein and the pathogenesis of brain glioma;

[0049] A4) Use of the above-mentioned cells or the above-mentioned biological materials in the preparation of products for studying the METTL7B protein and the pathogenesis of brain glioma;

[0050] A5) Application of the above cells or the above biological materials in screening of drugs for treating brain glioma;

[0051] A6) Use of the above cells or the above biological materials in the preparation of products for screening drugs for treating brain gliomas.

[0052] The beneficial technical effects achieved by the present invention are as follows:

[0053] 1) The present invention obtains stable recombinant cells by knocking out METTL7B in recipient cells U251.

[0054] 2) The present invention also provides a method for constructing the above-mentioned recombinant cell. The present invention screens out a target sequence with high editing efficiency and high specificity through a large amount of creative labor to achieve efficient and specific knockout of the METTL7B gene.

[0055] 3) The recombinant cells obtained by the present invention provide a usable cell model for the study of the function and mechanism of action of human METTL7B, which not only provides an important research tool for studying its molecular mechanism, but also for discovering it as a novel drug target and drug discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the map of the recombinant plasmid pLV-U6-gRNA1-METTL7B-puro.

[0057] Figure 2 The monoclonal cells obtained in experimental group 1 were sequenced after PCR amplification using primers designed and verified upstream and downstream of the knockout target site according to the human METTL7B genome. Figure 2METTL7B WT is the sequencing result of METTL7B of wild-type U251, METTL7B KO1 and METTL7B KO2 are monoclonal cells in experimental group 1, i.e., METTL7B gene was knocked out using gRNA1 as the target sequence, and the arrows point to the mutated bases in the knocked-out cells.

[0058] Figure 3 The monoclonal cells obtained from experimental group 2 were sequenced after PCR amplification using primers designed and verified upstream and downstream of the knockout target site according to the human METTL7B genome. Figure 3 METTL7B WT is the sequencing result of METTL7B of wild-type U251, METTL7B KO1 and METTL7B KO2 are monoclonal cells in experimental group 2, i.e., METTL7B gene was knocked out using gRNA2 as the target sequence.

[0059] Figure 4 The monoclonal cells obtained from experimental group 3 were sequenced after PCR amplification using primers designed and verified upstream and downstream of the knockout target site according to the human METTL7B genome. Figure 4 METTL7B WT is the sequencing result of METTL7B of wild-type U251, METTL7B KO1 and METTL7B KO2 are monoclonal cells in experimental group 3, i.e., METTL7B gene was knocked out using gRNA3 as the target sequence.

[0060] Figure 5 The monoclonal cells obtained from experimental group 4 were sequenced after PCR amplification using primers designed and verified upstream and downstream of the knockout target site according to the human METTL7B genome. Figure 5 METTL7B WT is the sequencing result of METTL7B of wild-type U251, METTL7B KO1 and METTL7B KO2 are monoclonal cells in experimental group 4, i.e., METTL7B gene was knocked out using gRNA4 as the target sequence.

[0061] Figure 6 Western blot was used to detect the expression of METTL7B protein in monoclonal cells after gRNA1 knocked out METTL7B. Figure 6 WT is the measurement result of the monoclonal cell NC in the control group, and KO1 and KO2 are the expression of METTL7B protein in the monoclonal cells after METTL7B was knocked out by gRNA1. The results show that there is no METTL7B protein expression in the monoclonal cells after METTL7B was knocked out by gRNA1. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0063] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0064] The backbone plasmid Cas9-Puro was purchased from Guangzhou Yuanjing Biotechnology Co., Ltd., with the catalog number YV-Cas-LV001-1000;

[0065] Lentiviral packaging plasmid psPAX2 was purchased from Nanjing Jiangyuan Biotechnology Co., Ltd., catalog number JY03029;

[0066] Lentivirus packaging plasmid pMD2.G was purchased from Nanjing Jiangyuan Biotechnology Co., Ltd., with the catalog number JY03027;

[0067] Escherichia coli Stbl3 chemically competent cells were purchased from Shanghai Zeye Biotechnology Co., Ltd., with the catalog number ZY-121112;

[0068] Human embryonic kidney 293T (HEK-293) cells were purchased from Guangzhou Saiku Biotechnology Co., Ltd., with the catalog number CC4002;

[0069] Human glioma cell line U251 (U-251MG) was purchased from Guangzhou Saiku Biotechnology Co., Ltd. with the catalog number CC1701.

[0070] Example 1: A method for constructing a U251 cell line with stable knockout of the METTL7B gene

[0071] The amino acid sequence of METTL7B protein is SEQ ID No.1, which consists of 244 amino acids. Its coding sequence is a DNA molecule with a nucleotide sequence of SEQ ID No.2, which is 675 bp in size. Its genomic sequence is a DNA molecule with a nucleotide sequence of SEQ ID No.3, which is 1316 bp in size.

[0072] 1) METTL7B gRNA sequence design

[0073] According to the GenBank number of METTL7B in the database submitted by NCBI: NM_152637.3 (14-AUG-2022), the target sequence was designed and synthesized through the online design website (crispr.mit.edu / ) and following the gRNA design principles of CRISPR / Cas9. The nucleotide sequence is as follows:

[0074] gRNA1: 5′-GTTCTACCCACCGGGCTGCA-3′, located in exon 1 of the METTL7B gene;

[0075] gRNA2: 5′-CCACCACAAACCGCTCATAT-3′, located in exon 1 of the METTL7B gene;

[0076] gRNA3: 5′-CAGTAGGGCCACTTTCCCGG-3′, located in exon 1 of the METTL7B gene;

[0077] gRNA4: 5'-GTCAGCACGGCCATCAGGTA-3', located in exon 1 of the METTL7B gene.

[0078] 2) Construction of pLV-U6-gRNA-METTL7B-puro recombinant plasmid

[0079] The gRNA1 sequence with EcoRI and ApaLI restriction enzyme recognition sites at both ends was synthesized, and it was double-digested with EcoRI (purchased from Biolabs, catalog number: #B0101S) and ApaLI (purchased from Biolabs, catalog number: #R0507V) with the backbone plasmid Cas9-Puro. The gRNA1 sequence after restriction enzyme digestion was ligated with the backbone plasmid Cas9-Puro after restriction enzyme digestion overnight using T4 ligase (purchased from ThermoFisher, catalog number: EL0011) and then transformed into the competent Escherichia coli Stbl3. The positive single colony was picked and shaken for sequencing verification to obtain the recombinant plasmid pLV-U6-gRNA1-METTL7B-puro ( Figure 1 As shown in the figure, the structure of the recombinant plasmid pLV-U6-gRNA1-METTL7B-puro is to replace the fragment between the EcoRI and ApaLI restriction enzyme recognition sites of the backbone plasmid Cas9-Puro (the small fragment between the EcoRI and ApaLI restriction enzyme recognition sites) with the target sequence of gRNA1. The recombinant plasmid pLV-U6-gRNA1-METTL7B-puro expresses Cas9 protein and gRNA1 targeting the METTL7B gene, and the target sequence of gRNA1 is 5'-GTTCTACCCACCGGGCTGCA-3'.

[0080] The preparation method of the recombinant plasmid pLV-U6-gRNA2-METTL7B-puro, pLV-U6-gRNA3-METTL7B-puro or pLV-U6-gRNA3-METTL7B-puro refers to pLV-U6-gRNA1-METTL7B-puro, the only difference being that the target sequence fragment of gRNA1 constructed into the backbone vector pLV-U6-puro-Cas9 is replaced with the target sequence corresponding to gRNA2, gRNA3 or gRNA4.

[0081] The recombinant plasmid pLV-U6-gRNA2-METTL7B-puro expresses Cas9 protein and gRNA2 targeting the METTL7B gene, and the target sequence of gRNA2 is 5'-CCACCACAAACCGCTCATAT-3'. The recombinant plasmid pLV-U6-gRNA3-METTL7B-puro expresses Cas9 protein and gRNA3 targeting the METTL7B gene, and the target sequence of gRNA3 is 5'-CAGTAGGGCCACTTTCCCGG-3'. The recombinant plasmid pLV-U6-gRNA4-METTL7B-puro expresses Cas9 protein and gRNA4 targeting the METTL7B gene, and the target sequence of gRNA4 is 5'-GTCAGCACGGCCATCAGGTA-3'.

[0082] 3) Cell culture and transfection

[0083] 293T cells were cultured in high-glucose DMEM (Gibco, C11995500BT) containing 10% FBS (Cellbox, AUS-01S-02) at 37°C and 5% CO 2 Incubator. In this study, the Lipofectamine 2000 method was used to transfect 293T cells with the correctly sequenced recombinant plasmids obtained in step 2). The specific steps are: 293T cells with vigorous growth and good growth status were plated into 6-well plates one day in advance, and when the cell density reached 60%-70%, Lipofectamine 2000 transfection reagent (Thermo, 11668019) and Opti-MEM (Thermo, 11058021) were used for transfection. The ratio of transfection plasmid to packaging plasmid in each well was: pLV-U6-gRNA1-METTL7B-puro:psPAX2:pMD2G=5:3:2. The medium was changed 6 hours after transfection, and the supernatant was collected after culturing with high-glucose DMEM medium containing 10% FBS for 48 hours. The supernatant was centrifuged at 4°C and 2000g for 30 minutes, and the supernatant was collected. After filtering with a 0.45μm filter membrane, it was frozen at -80°C to obtain the lentivirus METTL7B-gRNA1-KO for METTL7B knockout.

[0084] The preparation method of the lentivirus METTL7B-gRNA2-KO for METTL7B knockout refers to the lentivirus METTL7B-gRNA1-KO, with the only difference being that the plasmid pLV-U6-gRNA1-METTL7B-puro for transfecting 293T cells is replaced with pLV-U6-gRNA2-METTL7B-puro.

[0085] The preparation method of the lentivirus METTL7B-gRNA3-KO for METTL7B knockout refers to the lentivirus METTL7B-gRNA1-KO, with the only difference being that the plasmid pLV-U6-gRNA1-METTL7B-puro for transfecting 293T cells is replaced with pLV-U6-gRNA3-METTL7B-puro.

[0086] The preparation method of the lentivirus METTL7B-gRNA4-KO for METTL7B knockout refers to the lentivirus METTL7B-gRNA1-KO, with the only difference being that the plasmid pLV-U6-gRNA1-METTL7B-puro for transfecting 293T cells is replaced with pLV-U6-gRNA4-METTL7B-puro.

[0087] The preparation method of the lentivirus METTL7B-NC used in the control group was similar to that of METTL7B-gRNA1-KO, with the only difference being that the plasmid pLV-U6-gRNA1-METTL7B-puro for transfecting 293T cells was replaced with the backbone plasmid pLV-U6-puro-Cas9.

[0088] 4) Lentiviral infection

[0089] The experiment was divided into 4 experimental groups and a control group. In the experimental group 1, 1 mL of the lentivirus METTL7B-gRNA1-KO obtained above for METTL7B knockout was added with 5 ug / mL of polybrene (purchased from MedChemExpress, HY-112735) and then added to the U251 cells in a 6-well plate (the 6-well plate was cultured with high-glucose DMEM containing 10% FBS for U251 cell adherence culture one day in advance), and replaced with virus-free high-glucose DMEM medium (Gibco, C11995500BT) after 24 hours. After 72 hours, puromycin (puromycin, purchased from Selleck, S7417) was added for screening, and the complete culture medium containing puromycin at a final concentration of 2 μg / μL was replaced every two days until all the untransfected U251 cells in the control group died; the screened cells were then digested with 0.25% trypsin, and the cells were plated on a 96-well plate by limiting dilution to clone the knockout cells, and 20 monoclonal cells were randomly selected and numbered KO-METTL7B(gRNA1)-1 to KO-METTL7B(gRNA1)-20.

[0090] The preparation method of monoclonal cell KO-METTL7B (gRNA2) in Experimental Group 2 was similar to that in Experimental Group 1, with the only difference being that the lentivirus METTL7B-gRNA1-KO for METTL7B knockout added to the 6-well plate was replaced with METTL7B-gRNA2-KO.

[0091] The preparation method of monoclonal cell KO-METTL7B (gRNA3) in Experiment 3 group was referred to that in Experiment 1 group, with the only difference being that the lentivirus METTL7B-gRNA1-KO for METTL7B knockout added to the 6-well plate was replaced with METTL7B-gRNA3-KO.

[0092] The preparation method of monoclonal cell KO-METTL7B (gRNA4) in Experiment 4 group was referred to that in Experiment 1 group, with the only difference being that the lentivirus METTL7B-gRNA1-KO for METTL7B knockout added to the 6-well plate was replaced with METTL7B-gRNA4-KO.

[0093] The preparation method of monoclonal cell NC in the control group was similar to that in the experimental group, with the only difference being that the METTL7B knockout lentivirus added to the 6-well plate was replaced with an equal volume of lentivirus METTL7B-NC in the control group.

[0094] 5) Knockout cell sequencing and verification

[0095] Take 20 wells of monoclonal cells from each of the four experimental groups and 1 well of cells from the control group, wash the cells twice with PBS, add 0.25% trypsin to digest the cells, and collect the cells by centrifugation at 1000r / min. Extract the genome using a genomic DNA extraction kit. According to the human METTL7B genome, the primers cas9KO-7B-F / cas9KO-7B-R (the primers can determine the mutation of the four targets) were designed and verified upstream and downstream of the knockout target site. The genome of wild-type U251 cells was used as a control, and 100ng of genomic DNA was added to the 50μL system to obtain a PCR product containing the target fragment.

[0096] The specific reaction system is as follows: 2×Taq PCR MasterMix (purchased from Tiangen Biochemical Technology Co., Ltd., KT201), 25 μL; cas9KO-7B-F, 1 μL; cas9KO-7B-R, 1 μL; gDNA, 100 ng; ddH 2 O, add to a total volume of 50 μL. The nucleotide sequences of primers cas9KO-7B-F / cas9KO-7B-R are as follows:

[0097] cas9KO-7B-F: 5'-ATCCTGGTCCCACTC CTGCAG-3';

[0098] cas9KO-7B-R: 5'-GACCTCCTGCAGGACCTTCCTTG-3'.

[0099] The reaction conditions are as follows:

[0100]

[0101] The PCR products were detected by 1.8% agarose gel electrophoresis. The target fragments were recovered and sent for sequencing. Figure 2-Figure 5 The sequencing results showed that, compared with the wild-type U251, in the two chromosomes of the U251 cell KO-METTL7B(gRNA1) in which METTL7B(gRNA1) was knocked out, the METTL7B gene added a nucleotide T between the 293rd and 294th nucleotides of SEQ ID No.3 (located in the first exon), resulting in a frameshift mutation in METTL7B, which in turn caused METTL7B to be unable to express protein normally, thus achieving the knockout of the METTL7B gene.

[0102] However, there was no mutation in the METTL7B gene of the other three target gRNAs, gRNA2, gRNA3 or gRNA4, corresponding to the METTL7B knockout U251 cells KO-METTL7B(gRNA2), KO-METTL7B(gRNA3) and KO-METTL7B(gRNA4).

[0103] The above results show that the target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors.

[0104] 6) Protein verification of knockout cell lines: Monoclonal cells from experimental group 1 and control group (1 well in the control group and 2 wells in the experimental group 1, which had been sequenced and confirmed to be expressed in the METTL7B gene (SEQ ID No.3) One nucleotide T was added between the 293rd and 294th nucleotides, and the cells were lysed with RAPI lysis buffer. TUBULIN was used as the internal reference protein, and Western blot was used to detect the expression of METTL7B protein in U251 cells. The specific detection method is: prepare 1 plate of 12.5% ​​SDS-PAGE protein gel (protein gel preparation kit was purchased from Shanghai Yazyme Pharmaceutical Technology Co., Ltd., PG113), load 20ug of protein from the experimental group 1 and the control group and 5μL of protein marker (protein marker was purchased from Shanghai Yazyme Pharmaceutical Technology Co., Ltd., WJ106), and perform protein electrophoresis (80V constant voltage electrophoresis for 30 minutes, 120V constant voltage electrophoresis for 1 hour). After the electrophoresis, transfer the membrane (PVDF membrane is activated with methanol for 1 minute in advance, and placed in the order of black plate-fiber pad-filter paper-gel-PVDF membrane-filter paper-fiber pad-white plate, clamp the plate and put it in the wet transfer electrophoresis tank, with one side of the black plate facing Black negative electrode, 200mA constant current transfer for 1h). After the transfer, the PVDF membrane was placed on a cyclotron and rinsed for 1min, and the rinse was repeated 2-3 times. After the rinse, the PVDF membrane was soaked in 5% skim milk powder in TBST and blocked on a shaker at room temperature for 2h. After the blocking, the PVDF membrane was placed on a cyclotron and rinsed for 10min, and the rinse was repeated 2-3 times. After the rinse, the METTL7B antibody (purchased from Abclonal, A7200) and the TUBULIN antibody (purchased from After incubation, place the PVDF membrane on a rotary oscillator and rinse for 10 minutes, and repeat the rinse 2-3 times. After rinsing, incubate with anti-rabbit secondary antibody (purchased from CST, #7074S) at room temperature for 1 hour. After incubation, place the PVDF membrane on a rotary oscillator and rinse for 10 minutes, and repeat the rinse 2-3 times. After rinsing, use ECL developer (purchased from Bio-Rad, 1705060) for development and photography. The results are shown in Figure 6As shown, the results showed that: METTL7B protein could not be detected in the monoclonal cells obtained in Experimental Group 1, indicating that the monoclonal cells obtained in Experimental Group 1 had knocked out METTL7B protein.

[0105] 7) Construction of stable cell lines: The monoclonal cells of the experimental group 1 and the control group after protein verification were cultured in high-glucose DMEM (Gibco, C11995500BT) medium containing 10% FBS (Cellbox, AUS-01S-02). After amplification, the U251 cell line with stable knockout of METTL7B was obtained, which was cryopreserved for subsequent functional experimental studies. During the cryopreservation process, 1×10 6 For each cell, add 1 mL of freezing solution (FBS:DMSO=9:1), put the freezing solution containing cells into a cryovial, write down the cell type, freezing date, and cell generation, put it in a program cooling box, place it at -80℃ for 2 days, and then transfer it to liquid nitrogen.

[0106] In summary, the present invention uses CRISPR / cas9 technology to construct U251-METTL7B - / - The cell line provides available experimental materials for further research on the function and mechanism of action of human METTL7B, not only for studying its molecular mechanism, but also for discovering it as a new drug target and providing important research ideas for drug discovery.

[0107] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for constructing a recombinant cell, characterized in that: The method comprises knocking out the METTL7B gene in the recipient cell to obtain the recombinant cell, wherein the recipient cell is a U251 cell, and the METTL7B gene is g1) and / or g2): g1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2; g2), the nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.3; The knocking out of the METTL7B gene in the recipient cell comprises: infecting the recipient cell with a lentivirus, wherein the lentivirus expresses a gRNA and a Cas9 protein targeting the METTL7B gene, and the target sequence of the gRNA is SEQ ID No.

4.

2. A method for constructing a recombinant cell, characterized in that: The method comprises knocking out the METTL7B gene in a recipient cell to obtain the recombinant cell, wherein the recipient cell is a U251 cell, and the METTL7B gene is a DNA molecule whose nucleotide sequence of the coding chain is shown in SEQ ID No.3; The knocking out of the METTL7B gene in the recipient cell is to perform the following mutation on the METTL7B gene in the recipient cell: inserting one nucleotide T between the 293rd and 294th nucleotides of SEQ ID No.

3.

3. A nucleic acid molecule that inhibits the expression of a gene encoding a METTL7B protein or reduces the content of the METTL7B protein; The amino acid sequence of the METTL7B protein is SEQ ID NO.1; The nucleic acid molecule is a gRNA targeting the METTL7B protein encoding gene or the nucleic acid molecule is a DNA molecule expressing a gRNA targeting the METTL7B protein encoding gene; The target sequence of the gRNA is SEQ ID No.

4.

4. An expression cassette, characterized in that The expression cassette contains the nucleic acid molecule of claim 3.

5. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 3 or the expression cassette according to claim 4.

6. A recombinant microorganism, characterized in that The recombinant microorganism contains the nucleic acid molecule according to claim 3, the expression cassette according to claim 4 or the recombinant vector according to claim 5.

7. A recombinant cell, characterized in that The recombinant cell is constructed according to the method described in claim 1 or 2.

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