CN115490769B

CN115490769B - Monoclonal antibody against SLC7A11 protein, hybridoma cell line and application

Info

Publication number: CN115490769B
Application number: CN202210683139.5A
Authority: CN
Inventors: 陈善稳; 刘向征; 李吉昌; 王鹏远
Original assignee: Peking University First Hospital
Current assignee: Peking University First Hospital
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-09-01
Anticipated expiration: 2042-06-15
Also published as: CN115490769A

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody resisting SLC7A11 protein, a hybridoma cell line and application thereof. The monoclonal antibody provided by the invention can specifically identify and bind with the solute carrier family 7 member 11 protein, and can be used for detecting SLC7A11 protein in biological materials with high sensitivity and high specificity, thereby being used for diagnosis or auxiliary diagnosis of cancers up-regulated by SLC7A11 protein; in addition, the monoclonal antibody can also effectively block SLC7A11 protein-mediated glutamic acid and cystine transportation, inhibit cancer cell proliferation and promote cancer cell apoptosis by combining and inhibiting SLC7A11 protein activity in cancer cells, so that the monoclonal antibody has better anticancer property and can be used for treating or assisting in treating cancers up-regulated by SLC7A11 protein.

Description

Monoclonal antibody against SLC7A11 protein, hybridoma cell line and application

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody resisting SLC7A11 protein, a hybridoma cell line and application thereof.

Background

Solute carrier family 7 member 11 protein (SLC 7a 11) is the 7 th member of the heterodimeric amino acid transporter family of light subunits, with homology in all vertebrates. The human SLC7A11 gene is located on 4q28.3 chromosome, SLC7A11 protein has 12 transmembrane domains in human body, and consists of 501 amino acids, and the N terminal and the C terminal of the SLC7A11 gene are located in cytoplasm. The SLC7A11 protein mainly mediates the reverse transport of cystine and glutamic acid, and forms an Xc-system together with the SLC3A2 protein to regulate intracellular glutamic acid and cystine contents. Most cells rely on Xc-system to take up cystine from extracellular environment, and input cystine is reduced to cysteine to participate in synthesis of Glutathione (GSH), so SLC7A11 protein is closely related to electrophile detoxification, maintenance of redox balance in cells, storage of cysteine and other cell functions.

Since the discovery of SLC7A11 by Bannai and Kitamura in 1980, there have been a number of reports indicating that SLC7A11 is ubiquitously highly expressed in various cancers. The SLC7A11 with high expression can reversely transfer cystine and glutamic acid, and improve the content of intracellular cystine and extracellular glutamic acid. The large amount of input cystine is reduced into cysteine in cells, and is used for synthesizing GSH, and the antioxidation characteristic of GSH can inhibit iron death of tumor cells, thereby promoting the development of tumors. Glutamate, which is exported in large amounts, acts as an important oncogenic signaling molecule through glutamate receptors on cancer and non-cancer cells, promoting malignant transformation, proliferation, invasive metastasis of tumors, and inhibiting the immune system. Meanwhile, the SLC7A11 protein can be used for commonly generating chemotherapy resistance in various cancer cells through regulating oxidative stress, such as cisplatin resistance of gastric cancer cells, geldanamycin resistance of lung cancer cells, temozolomide resistance of glioblastoma, gemcitabine resistance of pancreatic cancer cells and the like. In addition, the SLC7a11 protein is also capable of mediating drug resistance by extracellular export of glutamate to enhance the inhibitory function of regulatory T cells (tregs).

Therefore, the SLC7A11 can become a potential tumor treatment target, and a monoclonal antibody for detecting and targeting the SLC7A11 is necessary to be developed, so that a new idea and tool are provided for diagnosis and treatment of cancers. However, there are still few monoclonal antibodies used to detect, target SLC7a11 in the related art.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defect that the monoclonal antibody for detecting and targeting SLC7A11 in the prior art is still less, so as to provide a monoclonal antibody for resisting SLC7A11 protein, a hybridoma cell line and application.

For this purpose, the invention provides a monoclonal antibody from the hybridoma cell strain 1A4-9-3 which secretes xct antibody and has the preservation number of CGMCC No.45147.

Alternatively, the monoclonal antibody specifically recognizes and binds to solute carrier family 7 member 11 protein.

Alternatively, the subtype of the monoclonal antibody is the IgG2b type.

Optionally, the heavy chain of the monoclonal antibody comprises an amino acid sequence shown in SEQ ID NO. 1;

the light chain of the monoclonal antibody comprises an amino acid sequence shown in SEQ ID NO. 2.

The invention also provides a hybridoma cell strain 1A4-9-3 for secreting xct antibodies, which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 45147.

Alternatively, the secretory xct antibody hybridoma cell line 1A4-9-3 uses a solute carrier family 7 member 11 protein as an immunogen.

Alternatively, the hybridoma cell strain 1A4-9-3 secreting xct antibodies is obtained by fusing spleen cells of an antigen-immunized Balb/C mouse with myeloma cells SP 2/0;

alternatively, the antigen immunized Balb/C mice are Balb/C mice immunized with cells that overexpress the solute carrier family 7 member 11 protein.

The invention also provides the use of a monoclonal antibody according to any one of the preceding claims or a secreted xct antibody hybridoma cell line 1A4-9-3 according to any one of the preceding claims in any one of the following:

A. use in the preparation of a product for detecting SLC7a11 protein in a biological material;

B. use in the manufacture of a product for diagnosing, alleviating, treating or adjunctively treating a cancer in which SLC7a11 protein expression is up-regulated;

C. use in the manufacture of a product for diagnosing, alleviating, treating or aiding in the treatment of colon, lung and breast cancer;

D. use in the preparation of a product for inhibiting SLC7a11 protein activity in a biological material;

E. use in the manufacture of a product for inhibiting or blocking SLC7a11 protein-mediated glutamate and/or cystine transport;

F. use in the manufacture of a product for inhibiting proliferation of cancer cells;

G. use in the manufacture of a product for promoting apoptosis of cancer cells;

H. use in the manufacture of a product for alleviating or eliminating cancer cell chemoresistance caused by the SLC7a11 protein;

optionally, the product comprises a reagent, a test strip or a kit.

The invention also provides a product for diagnosis, auxiliary diagnosis, alleviation, treatment or auxiliary treatment of cancer, which contains the monoclonal antibody of any one of the above, wherein the cancer is the cancer with up-regulated SLC7A11 protein expression.

Optionally, the product comprises a reagent, a test strip or a kit;

alternatively, the cancer includes colon cancer, lung cancer and breast cancer.

The technical scheme of the invention has the following advantages:

1. the monoclonal antibody provided by the invention can specifically identify and bind with the solute carrier family 7 member 11 protein, and can be used for detecting SLC7A11 protein in biological materials with high sensitivity and high specificity, thereby being used for diagnosing or assisting diagnosis of cancers with up-regulated SLC7A11 protein expression; in addition, the monoclonal antibody can also effectively block SLC7A11 protein-mediated glutamic acid and cystine transportation, inhibit cancer cell proliferation and promote cancer cell apoptosis by combining and inhibiting SLC7A11 protein activity in cancer cells, so that the monoclonal antibody has better anticancer property and can be used for treating or assisting in treating cancers up-regulated by SLC7A11 protein.

2. The hybridoma cell strain secreting xct antibody is obtained by fusing spleen cells of an antigen immunized Balb/C mouse with myeloma cells SP2/0, wherein the genetic background of the Balb/C mouse is clear, the immune effect is good, the immune operation is convenient, the fusion efficiency of spleen cells of the Balb/C mouse and myeloma cells SP2/0 is high, and the rejection is small, so that the hybridoma cell strain secreting xct antibody can secrete monoclonal antibody resisting SLC7A11 protein efficiently and stably.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a map of the recombinant plasmid SLC7A11-pcDNA3.1 according to example 1 of the present invention;

FIG. 2 is a graph showing the identification of the over-expression effect of SLC7A11 protein in 293F cells after transfection in example 1 of the present invention;

FIG. 3 is a graph showing the results of a flow assay for a second subclone of rescreened cells in example 2 of the present invention;

FIG. 4 is a graph showing the results of a flow assay for third subclone rescreened cells in example 2 of the present invention;

FIG. 5 is a graph showing the results of Western Blot experiments to detect that the SLC7A11 monoclonal antibody specifically recognizes the endogenous SLC7A11 protein in colon cancer cells in example 4 of the present invention;

FIG. 6 is a graph showing the results of purity detection of two monoclonal antibodies in example 2 of the present invention;

FIG. 7 is a graph showing the effect of two monoclonal antibodies in example 3 of the present invention on inhibiting intestinal cancer cell proliferation;

FIG. 8 is a graph showing the inhibitory effect of the monoclonal antibody of example 5 on reverse transport of glutamic acid;

FIG. 9 is a graph showing the inhibitory effect of the monoclonal antibody of example 5 of the present invention on the reverse transport of cystine;

FIG. 10 is a graph showing the effect of monoclonal antibodies in example 6 of the present invention on promoting apoptosis of intestinal cancer cells;

FIG. 11 is a graph showing the inhibitory effect of the monoclonal antibodies of example 7 on proliferation of lung cancer cells and breast cancer cells.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

The biological material or reagent related to the embodiment of the invention is as follows:

pcDNA3.1 vector (source: hangzhou Hua An Biotechnology Co., ltd.); high purity plasmid extraction kit (source: axygen, model: AP-MX-P-10); 293F cells (Source: hangzhou Hua' an Biotechnology Co., ltd.); fetal bovine serum (source: zhejiang Tianzhou biotechnology Co., ltd., lot: 11011-8611); DMEM medium (Source: shanghai source culture Biotechnology Co., ltd., lot number: L110 KJ); SPF Balb/C female mice (source: hangzhou university animal center); BSA (Source: biological engineering (Shanghai) Co., ltd., lot number: E66103); PBS (Source: shanghai Source culture Biotechnology Co., ltd., lot number: B310 KJ); igG control antibody (source: jackson ImmunoResearch Inc., lot number: 115-005-003); anti-mouse-FITC secondary antibody (source: hangzhou Hua' an Biotechnology Co., ltd., lot number: HA 1009); SP2/0 cells (source: hangzhou Hua's Biotechnology Co., ltd.); thymocytes from 4 week old mice (source: the university of Hangzhou animal center); intestinal cancer cell SW480 (origin: ATCC); intestinal cancer cell LOVO (source: ATCC); lung cancer cell A549 (source: ATCC); breast cancer cell MCF7 (source: ATCC).

The apparatus or device according to the embodiment of the present invention is as follows:

flow cytometry (manufacturer: BD, type: C6).

EXAMPLE 1 construction and transfection of SLC7A11 recombinant expression plasmid

The SLC7a11 Open Reading Frame (ORF) nucleotide sequence was obtained from the NCBI website under gene accession No. 23657. The SLC7A11 gene is artificially synthesized by general biology (Anhui) Co., ltd, the synthesized SLC7A11 gene is inserted into pcDNA3.1 vector, positive clone is selected and cultured in large quantity, and then the recombinant plasmid SLC7A11-pcDNA3.1 is extracted by a high-purity plasmid extraction kit according to the use instruction of the kit. The map of the recombinant plasmid SLC7A11-pcDNA3.1 is shown in figure 1, and the nucleotide sequence is shown in SEQ ID NO. 3.

The recombinant plasmid SLC7A11-pcDNA3.1 was transfected into 293F cells and 293F cells were transfected with pcDNA3.1 empty vector as a negative control by the following method:

(1) Preparation of 293F cell suspension with cell density of (1.0-1.2). Times.10 ⁶ Individual/ml;

(2) According to 1.0X10 ⁶ Concentration of individual/ml 293F cells, 2 ml/well were added in 6 well plates;

(3) Preparing a DNA transfection system: taking 160 mu L of Opti-MEM, adding 2.8 mu g of recombinant plasmid SLC7A11-pcDNA3.1, mixing uniformly, adding 8 mu L of PEI (polyethylenimine), mixing uniformly, and standing for 20 minutes at room temperature;

(4) Adding the mixture obtained in the operation (3) into one well of the 6-well plate obtained in the operation (2), repeating the operation (3) to 6 pieces Kong Jiaman, and placing the 6-well plate into a Infos shaker at 115rpm and 37 ℃ and 5% CO ₂ Culturing for 2 days under the condition;

(5) After the culture in operation (4), all samples were collected, centrifuged at 1000rpm, and the pellet was obtained after centrifugation.

After the transfection, the pellet obtained in the step (5) was collected and cultured in DMEM medium containing 10% fetal bovine serum at 37℃and 5% CO ₂ Is cultured for 48 hours under the condition of (2). After the culture is finished, 293F cells successfully transfected are screened out by using an SLC7A11 antibody (Hua' an organism, HA 600097) through a flow cytometry method, the over-expression effect of SLC7A11 protein is identified, the identification result is shown in figure 2, and as can be seen from figure 2, cells positive for SLC7A11 antibody flow exist in cell precipitation after the transfection, so that the transfection is successful.

EXAMPLE 2 screening and preparation of anti-SLC 7A11 monoclonal antibodies

1. Immunization of animals

Experimental group: 293F cells overexpressing SLC7A11 prepared in example 1 were taken according to 1X 10 ⁶ Dose of individual cells/mice 5 SPF-grade Balb/C females (mice numbered 1#, 2#, 3#, 4#, 5 #) were subjected to subcutaneous multiple injections to form primary immunizations, followed by booster immunizations every two weeks, each booster immunization dose being 1X 10 ⁶ Four total immunizations were boosted per cell/mouse.

Control group: the negative control 293F cells transfected with pcDNA3.1 empty vector prepared in example 1 were used for mouse immunization according to the same immunization method as the experimental group.

2. Cell flow type serum immune titer detection

Serum immunotiters of mice in the experimental and control groups were tested, respectively, according to the following procedure:

2.1 cell pretreatment: 293F cells transfected with SLC7A11 (hereinafter referred to as "SLC7A11-293F cells") prepared in example 1 were centrifuged at 1500rpm for 5 minutes with PBS buffer, and the cell pellet was washed 3 times with PBS; after washing, the washed cells were added to a 96-well V-plate at 50000 cells/well and blocked with a PBS buffer containing 1wt% bsa, left at 4 ℃ for 30 minutes, then resuspended in PBS buffer, centrifuged at 1500rpm for 5 minutes, and the cell pellet was washed 1 time with PBS;

2.2 sample treatment: performing orbital blood collection on the immunized mice obtained in the step 1, separating serum, and diluting the serum with PBS buffer solution containing 1wt% BSA according to the volume ratio of 1:100, 1:1000 and 1:10000 respectively;

2.3 primary anti-reaction: respectively adding serum samples with various dilution gradients into the 96-well V-shaped plate pretreated in the step 2.1, wherein the drop adding amount of each well is 50 mu l; after the dripping is finished, placing the refrigerator at the temperature of 4 ℃ for 1 hour;

2.4 secondary antibody reaction: diluting anti-mouse-FITC secondary antibody by using PBS buffer solution according to a volume ratio of 1:1000, then adding the diluted anti-mouse-FITC secondary antibody into the 96-well V-shaped plate obtained in the step 2.3, dropwise adding 50 μl of the diluted anti-mouse-FITC secondary antibody into each well, placing a refrigerator at 4 ℃ for 30 minutes after dropwise adding, then re-suspending cells by using the PBS buffer solution, centrifuging at 1500rpm for 5 minutes, washing cell sediment by using PBS for 3 times, and re-suspending the cell sediment by using 150 μl of PBS buffer solution for standby;

2.5 flow cytometer detection: and (3) pre-heating the flow cytometer, performing on-machine detection on the cells obtained in the step (2.4) after washing, selecting an FL1 channel, and recording an MFI value.

The serum immune titers of mice in the experimental group or the control group are detected, and the titers of the cells obtained in the step 2.1, the cells obtained in the step 2.1 and the anti-mouse-FITC secondary antibodies and the goat anti-mouse IgG control antibodies (1 mug/ml) are detected by using a flow cytometer. The test results are shown in Table 1.

TABLE 1 cell flow assay results

As can be seen from table 1, the serum immune titers of the mice in the experimental group were significantly higher than those of the mice in the control group, indicating that the immunization of 5 mice in the experimental group was successful. Comparing the 5 mice in the experimental group, the serum immune titers of the 3# mice and the 4# mice are higher than those of other mice, and the 3# mice are selected in the subsequent experiments to continue the experiments, and the 4# mice are used as backup mice.

3. Cell fusion

3.1 preparation work

3.1.1 preparing complete medium: IMDM (Iscove's modified Dulbecco's medium) medium containing 10wt% fetal bovine serum, 1 Xgreen-streptomycin diabody;

3.1.2 preparation of hybridoma selection media: IMDM medium containing 1 XHAT medium additive, 15wt% fetal bovine serum, 1 Xgreen-streptomycin diabody;

3.1.3 preparation of trophoblasts: the thymus cells of the mice with the age of 4 weeks are freshly prepared and resuspended in IMDM culture medium for later use;

3.1.4 preparation of myeloma (hereinafter referred to as SP 2/0) cells: the SP2/0 cells were passaged to log phase and resuspended in IMDM medium for use.

3.2 preparation of mouse spleen cells

3.2.1 taking the 3# mice after four times of booster immunizations of the experimental group in the step 1, and taking spleens;

3.2.2 placing the spleen of the mouse in a sterile plate with the diameter of 10cm, cutting off redundant adhesion tissues, placing the plate in a cell filter screen, and grinding the plate by using a sterile 2mL syringe core;

3.2.3 after milling, the cell filter screen was rinsed with IMDM medium, the cells were allowed to enter a 50mL centrifuge tube, centrifuged at 1500rpm for 3min, and the cell pellet was washed twice with IMDM medium for further use.

3.3 cell fusion

3.3.1 mixing SP2/0 cells with the washed mouse spleen cells in step 3.2.3 according to the ratio of 1:1 of cell precipitation volume;

3.3.2 after mixing the cells evenly, adding IMDM culture medium to make the volume of liquid in the tube 30mL, centrifuging at 1500rpm for 5min, discarding the supernatant, and taking cell sediment;

3.3.3 after careful knockdown, the cell pellet was transferred to a beaker containing warm water at 37 ℃;

3.3.4 adding 1mL of PEG dropwise into the knocked-out cells at a constant speed within 1min, and standing for 30s in a water bath at 37 ℃ to obtain fused cells;

3.3.5 adding 10mL of IMDM culture medium to the fused cells at a rate of 5mL (2 drops/s) followed by 5mL (5 drops/s), then adding IMDM culture medium in time to make the total volume 30mL, centrifuging at 1200rpm for 3min, discarding the supernatant, and taking cell pellet;

3.3.6 cell pellet is tapped up, slowly transferred into hybridoma selection medium, and trophoblast is added into the selection medium, and fused cells and trophoblast are gently mixed, and the volume ratio of the fused cells to the trophoblast is 1:1.

4. hybridoma cells (fused cells) first subcloning

4.1 preparation of subclone medium: IMDM medium containing 15wt% fetal calf serum, 1 XHT medium additive, 1 XQing-streptomycin double antibody, subcloning medium is pre-incubated in 37 deg.C water bath;

4.2 observing the growth condition of cells in the cell culture hole under a microscope, and determining the number of cells required to be taken for making a plate of subcloning;

4.3 plating: adding 6.5mL of subcloning culture medium into a sample adding groove, and then adding counted positive hybridoma cells to make the cell concentration be 125/200 mu l, so as to obtain hybridoma cell suspension; adding hybridoma cell suspension into a 96-well cell culture plate according to the volume of 200 μl/well, adding 41 wells in total, sequentially numbering 12 wells of a first row as 1A1-1A12, 12 wells of a second row as 1B1-1B12, 12 wells of a third row as 1C1-1C12, and 5 wells of a fourth row as 1D1-1D5;

4.4 culturing: placing the cell culture plate obtained in step 4.3 at 37deg.C and 5% CO ₂ After 7 days, the supernatant was collected and ELISA tested to screen out clone wells positive for SLC7A11-293F cells. The ELISA detection method comprises the following steps:

1) Coating the plates with purified SLC7A11 protein at 4℃overnight at a protein concentration of 2. Mu.g/ml;

2) After coating, adding 2wt% BSA, and blocking for 2 hours at 37 ℃;

3) After the end of the closure, the washing solution (manufacturer: hangzhou Union biotechnology Co., ltd., product number: e0281 3 times, adding cell supernatant, wherein a blank control (blank) is PBS, and a negative control (negative) is hybridoma culture medium;

4) Washing 3 times with washing solution, adding PBS to dilute 10000 times of GAM-HRP secondary antibody, 100 μl/well, incubating in incubator at 37deg.C for 1 hr;

5) Taking out, and washing with washing liquid for 3 times;

6) Color development, color development liquid (manufacturer: shanghai Biyun biotechnology Co., ltd., product number: p0209) 100 μl/well, the development time was about 10min, and each well was terminated by adding 50 μl of stop solution (manufacturer: hangzhou Union biotechnology Co., ltd., product number: e0301 A) is provided;

7) Absorbance was measured at two wavelengths (450 nm,630 nm), and the data were recorded and stored, and the results are shown in table 2.

Table 2: ELISA test results of supernatants from wells of first subclones

As can be seen from Table 2, the first subclones Kong Zhongshang numbered 1A4, 1A10, 1B8, 1C1, 1C7, 1D2 and 1D3 have higher absorbance values, indicating that the supernatants of these wells contained anti-SLC 7A11 antibodies, and that ELISA detection results were positive, thus the hybridoma cells in these wells were subcloned a second time.

5. Secondary subcloning of hybridoma cells

6.5mL of subcloning medium was added to each of the sample-adding tanks, and positive hybridoma cells in the first subcloning wells numbered 1A4, 1A10, 1B8, 1C1, 1C7, 1D2 and 1D3 were then added, respectively, to prepare hybridoma cell suspensions having cell concentrations of 125/200. Mu.L. Adding the hybridoma cell suspension into a 96-hole cell culture plate according to the amount of 200 mu l/hole for each hybridoma cell suspension, adding 4 holes in total, adding fresh subcloning culture medium into the rest hybridoma cell suspension for 5 times, adding into the 96-hole cell culture plate according to the amount of 200 mu l/hole, and adding 4 holes in total; the procedure was then repeated sequentially, and hybridoma cell suspensions were added to finally form 4 cell concentration gradients, 125, 25, 5 and 1 per well, 4 wells per concentration gradient, numbered sequentially 1-16. The above procedure was repeated until the 7 hybridoma cell suspensions were completely inoculated into the cell culture plates.

After inoculation, the cell culture plate was placed at 37℃in 5% CO ₂ After 7 days, the supernatants of each well were collected, and the supernatants of each well were ELISA-detected by the method of 4.4, and the results of the detection of the second subclone well positive to ELISA were screened as shown in Table 3.

TABLE 3 ELISA assay results for supernatants from each well of the second subclones

As can be seen from Table 3, the second subclones Kong Zhongshang numbered 1A4-9, 1A4-10, 1A10-2, 1A10-7, 1A10-15, 1B8-3, 1B8-8, 1B8-9, 1C1-7, 1C1-13 and 1D2-7 had higher absorbance values, and the wells were subjected to cell flow assay, as shown in FIG. 3, and the supernatants of the wells contained anti-SLC 7A11 antibodies, and the ELISA assay was positive, so that the hybridoma cells in the wells were subjected to the third subcloning.

6. Third subcloning of hybridoma cells

According to the second subcloning method in procedure 5, hybridoma cells in the second subcloning wells numbered 1A4-9, 1A4-10, 1A10-2, 1A10-7, 1A10-15, 1B8-3, 1B8-8, 1B8-9, 1C1-7, 1C1-13 and 1D2-7 were subcloned, respectively, and ELISA assays were performed, and the results are shown in Table 4.

TABLE 4 ELISA detection results of supernatants from third subclones of wells

As can be seen from Table 4, the immune titers of the supernatant on SLC7A11 in the third subcloning sieve openings numbered 1A4-9-3, 1A4-10-1, 1A10-2-5, 1A10-7-1, 1A10-7-3, 1B8-3-4, 1C1-7-1, 1C1-7-3 and 1C1-7-4 were significantly higher, and the cell flow assays were performed on these wells, as in operation 2, and the assay results are shown in FIG. 4 and Table 5.

TABLE 5 results of flow-through detection of third subclone supernatant

As can be seen from FIGS. 4 and 5, the fluorescence intensities of the third subclone wells numbered 1A4-9-3 and 1A4-10-1 were significantly higher than those of the other wells, so that the hybridoma cells in the third subclone wells numbered 1A4-9-3 and 1A4-10-1 remained and were designated as hybridoma cells 1A4-9-3 and 1A4-10-1, respectively.

7. Identification of monoclonal hybridoma cell subtypes

Subtype identification was performed on hybridoma cells 1A4-9-3 and 1A4-10-1 as follows:

the coated antibody (goat anti-mouse IgG antibody) was diluted to 0.5. Mu.g/ml with 100mM PBS buffer (pH 7.4), added dropwise to the plates, and added at 0.1ml per well overnight at 4 ℃; after 2 washes with PBST (phosphate Tween buffer), 200. Mu.l of blocking solution (1 wt% BSA) was added to each well and incubated at 37℃for 2h; after 3 times of PBST washing, 100 μl of hybridoma supernatant was added to each well, and incubated at 37deg.C for 1 hr; after 3 PBST washes, blocked solution was added according to 1:1000 (κ, λ) or 1:2000 (other) volume ratio of HRP-labeled goat anti-mouse subtype antibody added at 0.1ml per well and incubated at 37 ℃ for 1h; PBST was washed 3 times, 50. Mu.l TMB was added to each well, absorbance was measured at two wavelengths (450 nm,630 nm) for 10-20min, and the data were recorded. Subtype identification results are shown in table 6.

TABLE 6 identification of subtype 1A4-9-3 and 1A4-10-1 of hybridoma cells

As can be seen from Table 6, the subtypes of the antibodies secreted by the hybridoma cells 1A4-9-3 and 1A4-10-1 are both of the IgG2b type.

8. Purification of monoclonal antibodies

The purified 1A4-9-3 monoclonal antibodies and 1A4-10-1 monoclonal antibodies were prepared as follows:

8.1 preparation of 8-12 week old SPF-grade Balb/C mice, each pre-sensitized by intraperitoneal injection of 300. Mu.l of Freund's incomplete adjuvant;

8.2 preparation of cell lines (hybridoma 1A4-9-3 or 1A 4-10-1) for ascites preparation, expansion culture and maintenance, and injection of 1X 10 per mouse ⁶ Individual cells, meterCalculating the required cell suspension volume;

8.3 transferring the desired volume of the cell suspension into a 15ml centrifuge tube, centrifuging at 1200rpm for 5min, removing the cell supernatant, adding serum-free IMDM medium to suspend so that the concentration per cell is 1X 10 ⁶ Every 500 mu l, packaging into 1.5ml centrifuge tube after blowing, and sealing with sealing film;

8.4 mice were intraperitoneally injected with the cell suspension obtained in step 8.3 at the injection amount of 500. Mu.l/mouse on the 7 th day after pre-sensitization;

8.5, after the injection is finished, the mice are continuously fed, whether the abdomen of the mice is raised or not is observed from the 10 th day, when the abdomen of the mice is obviously raised, the ascites is collected, and the ascites is collected once every other day;

8.6, after the collected mouse ascites is subjected to water bath in a water bath kettle at 37 ℃ for 30min, centrifuging for 5min at 12000rpm, taking the upper layer ascites into a new centrifuge tube, and marking. Purification was performed using Protein A/G.

The purified 1A4-9-3 monoclonal antibody and 1A4-10-1 monoclonal antibody were subjected to purity detection by SDS-PAGE, respectively, and the detection results are shown in FIG. 6. As can be seen from FIG. 6, the two antibodies have clear bands in the heavy chain region (55 kDa) and the light chain region (25 kDa).

EXAMPLE 3 cytotoxicity of monoclonal antibodies against intestinal cancer cells

Intestinal cancer tumor cells (SW 480, LOVO) were seeded in 96-well plates, and approximately 5000 tumor cells were seeded per well, and 6 groups of 6 wells each were divided. After the cells had been attached, the medium was discarded, and after washing the cells with PBS, 100. Mu.l of DMEM medium was added to each well so that each group contained different concentrations of monoclonal antibodies (0. Mu.g/ml, 10. Mu.g/ml, 20. Mu.g/ml, 30. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml) and placed in an incubator for cultivation for 24 hours. After the culture is finished, adding a culture medium containing CCK8 into each hole (the volume ratio of the culture medium to the CCK8 is 10:1), putting the mixture back into an incubator for continuous culture, taking out the 96-well plate after 2 hours, reading the value in an enzyme-labeled instrument at the wavelength of 450nm, and detecting the killing effect of monoclonal antibodies with different concentrations on cells, wherein the result is shown in figure 7.

As can be seen from FIG. 7, both monoclonal antibodies 1A4-9-3 and 1A4-10-1 kill intestinal cancer cells, but the killing effect of 1A4-9-3 is more remarkable, so monoclonal antibody 1A4-9-3 was selected.

EXAMPLE 4 specificity verification of the SLC7A11 monoclonal antibody

(1) Respectively washing NMC356 cells and LOVO cells twice with precooled PBS, then adding RIPA lysate (containing protease inhibitor), blowing and sucking, and mixing;

(2) Mixing, placing on ice, cracking for 30min, centrifuging at 12000rpm and 4deg.C for 15min, transferring supernatant into new tube, and marking;

(3) Protein quantification is carried out according to the BCA protein quantification kit, and each sample is added with a protein loading buffer solution (company: biyundian, product number: P0015) and then subjected to metal bath at 100 ℃ for 5min;

(5) Performing 12% PAGE gel electrophoresis after the metal bath is finished, and performing 300mA constant current rotation for 60min;

(6) Blocking with TBST solution containing 5wt% BSA for 1h at room temperature;

(7) Monoclonal antibodies 1A4-9-3 or 1A4-10-1 (SLC 7A11 antibody Xct of Huaan is used as positive control and GAPDH is used as internal reference) were added and incubated overnight at 4 ℃;

(8) The TBST buffer solution is washed for 3 times, after 5min of each washing, the membrane is incubated for 1h at room temperature (goat anti-mouse IgG antibody 1:5000), and the membrane is exposed after TBST washing.

As a result, as shown in FIG. 5, it can be seen from FIG. 5 that monoclonal antibody 1A4-9-3 can detect SLC7A11 protein highly expressed in LOVO cells with a high degree of specificity.

EXAMPLE 5 blocking Effect of SLC7A11 monoclonal antibody on reverse transport of glutamic acid and cystine

(1) LOVO cells were plated in 24-well plates and split equally into two groups, with 15. Mu.l of monoclonal antibody 1A4-9-3 (100. Mu.g/ml) added to the experimental group and equal amounts of IgG to the control group.

(2) Culturing at 37deg.C in cell incubator for 24 hr, discarding supernatant, and washing with PBS for 3 times;

(3) Adding 100 μl of RIPA lysate into each well, incubating on ice for 30min, and completely lysing cells;

(4) Centrifuging at 12000rpm and 4deg.C for 15min, transferring supernatant into new tube, and marking;

(5) Detecting intracellular glutamic acid content by using a glutamic acid detection kit, detecting intracellular cysteine content by using a cysteine detection kit, and marking by using protein concentration.

As shown in FIGS. 8 and 9, it can be seen that SLC7A11 monoclonal antibody 1A4-9-3 can prevent the reverse transport of glutamic acid and cystine, raise intracellular glutamic acid concentration and reduce intracellular GSH content.

EXAMPLE 6 Proapoptotic Effect of SLC7A11 monoclonal antibodies on intestinal cancer cells

(1) LOVO cells were plated in 24-well plates and split equally into two groups, with 15. Mu.l 1A4-9-3 (100. Mu.g/ml) added to the experimental group and equal amounts of IgG to the control group.

(3) 100 μl of pancreatin was added per well to digest cells;

(4) Cells were washed 2 times with PBS;

(5) Adding annexin V-FITC and PI, and incubating for 5min at room temperature;

(6) And (5) detecting on-line in a streaming mode.

As shown in FIG. 10, it can be seen that SLC7A11 monoclonal antibody 1A4-9-3 can promote apoptosis of intestinal cancer cells.

EXAMPLE 7 inhibition of proliferation of lung cancer cells and breast cancer cells by monoclonal antibodies

Lung cancer cells (a 549) and breast cancer cells (MCF 7) were seeded in 96-well plates, and then the cells were treated with different concentrations of monoclonal antibodies 1A4-9-3 (0 μg/ml,10 μg/ml,20 μg/ml,30 μg/ml,50 μg/ml,100 μg/ml) for 24 hours, respectively, and proliferation of the cells was examined with CCK8, see example 3.

As shown in FIG. 11, it can be seen that the purified monoclonal antibody 1A4-9-3 is effective in inhibiting the proliferation of lung cancer cells (A549) and breast cancer cells (MCF 7).

Example 8

Sequencing the monoclonal antibody 1A4-9-3, wherein the sequencing result shows that the full-length sequence of the heavy chain of the monoclonal antibody 1A4-9-3 is shown as SEQ ID NO. 1:

DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGTISYNPSLTSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWRFDGAWFSYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK；

the full-length sequence of the light chain of the monoclonal antibody 1A4-9-3 is shown as SEQ ID NO. 2:

DIVMTQAAFSNPVTLGTSASISCRSSESLLHTNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLQLPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

it is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Sequence listing

<110> Beijing university first Hospital

<120> monoclonal antibody against SLC7A11 protein, hybridoma cell line and use thereof

<130> HA202201080

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 443

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Thr Ile Ser Tyr Asn Pro Ser Leu

50 55 60

Thr Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Trp Arg Phe Asp Gly Ala Trp Phe Ser Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 2

<211> 219

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Leu His Thr

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Gln Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 3

<211> 6901

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcatg 960

gtcagaaagc ctgttgtgtc caccatctcc aaaggaggtt acctgcaggg aaatgttaac 1020

gggaggctgc cttccctggg caacaaggag ccacctgggc aggagaaagt gcagctgaag 1080

aggaaagtca ctttactgag gggagtctcc attatcattg gcaccatcat tggagcagga 1140

atcttcatct ctcctaaggg cgtgctccag aacacgggca gcgtgggcat gtctctgacc 1200

atctggacgg tgtgtggggt cctgtcacta tttggagctt tgtcttatgc tgaattggga 1260

acaactataa agaaatctgg aggtcattac acatatattt tggaagtctt tggtccatta 1320

ccagcttttg tacgagtctg ggtggaactc ctcataatac gccctgcagc tactgctgtg 1380

atatccctgg catttggacg ctacattctg gaaccatttt ttattcaatg tgaaatccct 1440

gaacttgcga tcaagctcat tacagctgtg ggcataactg tagtgatggt cctaaatagc 1500

atgagtgtca gctggagcgc ccggatccag attttcttaa ccttttgcaa gctcacagca 1560

attctgataa ttatagtccc tggagttatg cagctaatta aaggtcaaac gcagaacttt 1620

aaagacgcct tttcaggaag agattcaagt attacgcggt tgccactggc tttttattat 1680

ggaatgtatg catatgctgg ctggttttac ctcaactttg ttactgaaga agtagaaaac 1740

cctgaaaaaa ccattcccct tgcaatatgt atatccatgg ccattgtcac cattggctat 1800

gtgctgacaa atgtggccta ctttacgacc attaatgctg aggagctgct gctttcaaat 1860

gcagtggcag tgaccttttc tgagcggcta ctgggaaatt tctcattagc agttccgatc 1920

tttgttgccc tctcctgctt tggctccatg aacggtggtg tgtttgctgt ctccaggtta 1980

ttctatgttg cgtctcgaga gggtcacctt ccagaaatcc tctccatgat tcatgtccgc 2040

aagcacactc ctctaccagc tgttattgtt ttgcaccctt tgacaatgat aatgctcttc 2100

tctggagacc tcgacagtct tttgaatttc ctcagttttg ccaggtggct ttttattggg 2160

ctggcagttg ctgggctgat ttatcttcga tacaaatgcc cagatatgca tcgtcctttc 2220

aaggtgccac tgttcatccc agctttgttt tccttcacat gcctcttcat ggttgccctt 2280

tccctctatt cggacccatt tagtacaggg attggcttcg tcatcactct gactggagtc 2340

cctgcgtatt atctctttat tatatgggac aagaaaccca ggtggtttag aataatgtcg 2400

gagaaaataa ccagaacatt acaaataata ctggaagttg taccagaaga agataagtta 2460

tgatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 2520

ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2580

gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2640

ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 2700

gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg 2760

gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 2820

agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 2880

tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 2940

ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 3000

cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 3060

tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 3120

tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 3180

caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc 3240

caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt 3300

gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 3360

cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg 3420

cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct 3480

ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca 3540

aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat 3600

cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga 3660

ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc 3720

ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga 3780

atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg 3840

cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc 3900

cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg 3960

atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga 4020

aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc 4080

tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca 4140

tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg 4200

tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct 4260

atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg 4320

accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc 4380

gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac 4440

gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 4500

cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga 4560

gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag 4620

catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 4680

actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta 4740

atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 4800

acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 4860

aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 4920

atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 4980

gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 5040

ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 5100

aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5160

ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5220

aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 5280

gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 5340

tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 5400

tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 5460

gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 5520

cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5580

cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 5640

agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt ttgtttgcaa 5700

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 5760

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 5820

aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 5880

atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 5940

gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 6000

acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 6060

ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 6120

tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 6180

ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 6240

ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 6300

atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 6360

taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 6420

catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 6480

atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 6540

acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 6600

aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 6660

ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 6720

cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 6780

atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 6840

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 6900

c 6901

Claims

1. A monoclonal antibody, which is characterized in that the monoclonal antibody is from a secreted xct antibody hybridoma cell strain 1A4-9-3 with the preservation number of CGMCCNO.45147.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes and binds to solute carrier family 7 member 11 protein.

3. The monoclonal antibody of claim 1, wherein the subtype of the monoclonal antibody is IgG2b type.

4. A monoclonal antibody according to any one of claims 1-3, wherein the heavy chain of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID No. 1;

5. Hybridoma cell strain 1A4-9-3 secreting xct antibody is preserved in China general microbiological culture collection center with a preservation number of CGMCC NO.45147.

6. The secreted xct antibody hybridoma cell line 1A4-9-3 of claim 5, wherein the secreted xct antibody hybridoma cell line 1A4-9-3 is immunogenic against a solute carrier family 7 member 11 protein.

7. The antibody secreting xct hybridoma cell line 1A4-9-3 of claim 5 or 6, wherein said antibody secreting xct hybridoma cell line 1A4-9-3 is obtained by fusion of spleen cells of an antigen immunized Balb/C mouse with myeloma cells SP 2/0;

the Balb/C mice immunized with the antigen are Balb/C mice immunized with cells that overexpress the solute carrier family 7 member 11 protein.

8. Use of the monoclonal antibody of any one of claims 1-4 or the secretory xct antibody hybridoma cell line 1A4-9-3 of any one of claims 5-7 in any one of the following:

A. use in the manufacture of a product for detecting a solute carrier family 7 member 11 protein in a biological material;

B. use in the manufacture of a product for diagnosing, ameliorating, treating or adjunctively treating a cancer in which expression of a solute carrier family 7 member 11 protein is up-regulated;

D. use in the manufacture of a product for inhibiting the activity of a solute carrier family 7 member 11 protein in a biological material;

E. use in the manufacture of a product for inhibiting or blocking solute carrier family 7 member 11 protein-mediated transport of glutamate and/or cystine;

G. use in the manufacture of a product for promoting apoptosis of cancer cells;

H. use in the manufacture of a product for alleviating or eliminating cancer cell chemoresistance caused by a solute carrier family 7 member 11 protein.

9. A product for diagnosis, co-diagnosis, alleviation, treatment or co-therapy of a cancer, characterized in that it comprises a monoclonal antibody according to any one of claims 1-4, said cancer being a cancer in which the expression of a solute carrier family 7 member 11 protein is up-regulated.

10. The product of claim 9, wherein the product comprises a reagent, a test strip, or a kit;

the cancers include colon cancer, lung cancer and breast cancer.