CN114085818A

CN114085818A - Anti-panda Thy1 monoclonal antibody, hybridoma cell strain and application thereof

Info

Publication number: CN114085818A
Application number: CN202111398728.0A
Authority: CN
Inventors: 蔡开来; 安俊辉; 张名岳; 张梦诗; 李非平; 胡贤彪; 王神飞; 王涓; 刘玉良; 侯蓉; 兰景超; 吴永胜
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-02-25

Abstract

The invention discloses a giant panda Thy1 resistant monoclonal antibody, a hybridoma cell strain and application thereof. The preservation number of the hybridoma cell strain Thy1-2 is as follows: CCTCC NO: c202170, preserved in China Center for Type Culture Collection (CCTCC) at 28 months 10 and 2021. The invention utilizes a cell fusion technology to establish hybridoma cell strains secreting anti-Thy 1 monoclonal antibodies, collects and purifies cell strain culture supernatants to obtain high-specificity Thy1 monoclonal antibodies, establishes high-specificity Thy1 monoclonal antibodies to be applied to Western Blot protein detection, is positioning and tissue expression information of Thy1 protein, is applied to identification and sorting of giant panda MGSCs, and utilizes the Thy1 high-specificity monoclonal antibodies to carry out deep research on ligands, antagonists and anti-antagonists of Thy1 receptors.

Description

Anti-panda Thy1 monoclonal antibody, hybridoma cell strain and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a giant panda Thy1 resistant monoclonal antibody, a hybridoma cell strain and application thereof.

Background

Thymocyte differentiation antigen 1(Thy 1), also known as CD90, is a highly conserved glycoprotein and a member of the immunoglobulin superfamily of cell adhesion molecules. It is expressed on the surface of nerve cell, thymocyte, fibroblast and hemopoietic stem cell.

Thy1 was originally identified as a mouse thymocyte differentiation marker, but was found to be expressed in other tissues in subsequent studies. It was found that Thy1 is mainly expressed in serum, cerebrospinal fluid, wound tissue of venous ulcer, joint fluid of rheumatoid arthritis, etc. The expression of the gene is different in the same cell type, for example, the fibroblast can be divided into two subgroups according to the existence of Thy1 on the cell surface, including a Thy1 positive subgroup and a Thy1 negative subgroup. Normal human and animal lung tissue fibroblasts predominate the Thy1 positive subset, whereas in fibroblasts the Thy1 negative subset. Functionally, Thy1 is mainly involved in apoptosis, inflammation, myofibroblast differentiation, and fibrotic signaling, for example, methylation of the Thy1 sequence induces a change in fibroblast phenotype, resulting in myofibroblast transformation, and synthesis of a large amount of extracellular matrix including collagen i and collagen iii. In application, Thy1 has been used as a surface marker for rodent, bull and non-human primate male germ line stem cells (MGSCs), and is widely used in cell isolation and identification studies. In rodents, Thy1 is expressed predominantly in undifferentiated spermatogonial populations at very low levels, and mouse MGSCs can be enriched 30-fold by selecting cells expressing Thy1 by fluorescence live cell screening (FACS) using the Thy1 antibody.

However, the research of Thy1 in pandas still leaves a blank. Due to species differences, there is currently no specific anti-panda Thy1 antibody. Therefore, it is urgently needed to establish a high-specificity Thy1 monoclonal antibody, apply the monoclonal antibody to Western Blot protein detection, provide positioning and cell expression information of the Thy1 protein, apply identification and sorting of giant panda MGSCs, and use the Thy1 high-specificity monoclonal antibody to deeply research ligands, antagonists and anti-antagonists of a Thy1 receptor, further discuss Thy1 secretion and further regulate and control genes, reveal reproductive physiology rules, and provide new data and ideas.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a monoclonal antibody against panda Thy1, a hybridoma cell strain thereof and application thereof, and provides a specific antibody against panda Thy1 and a hybridoma cell strain for producing the antibody.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a hybridoma cell strain Thy1-2 of a monoclonal antibody against giant panda Thy1 has a preservation number of: CCTCC NO: c202170, which was preserved in China Center for Type Culture Collection (CCTCC) at 28/10/2021. The preservation unit: wuhan university. And (4) storage address: wuhan, Wuhan university, China.

An anti-panda Thy1 monoclonal antibody, which is produced by the hybridoma cell line Thy 1-2.

Further, the subtype of monoclonal antibody is IgG 1.

The hybridoma cell strain or the monoclonal antibody is applied to the preparation of a detection reagent.

The monoclonal antibody is applied to preparation of products for detecting Thy1 protein.

Further, the detection reagent or product is used for performing western blot detection.

A kit for auxiliary diagnosis of a fibroblast disease comprises the monoclonal antibody or the monoclonal antibody secreted by a hybridoma cell strain.

The monoclonal antibody or the hybridoma cell strain is applied to separation, identification and/or enrichment of panda MGSCs cells.

The preparation method of the anti-panda Thy1 monoclonal antibody comprises the following steps:

(1) the specific sequence SEQ ID NO of the giant panda Thy1 is designed to be shown in 1, and the amino acid sequence is as follows: PEHSYRSRTNFTSKY, which is synthesized artificially and coupled with KLH by adding a cysteine residue at the N-terminal, and is used as immunogen Thy1-KLH for producing specific antibody;

(2) immunizing a mouse with the immunogen Thy1-KLH prepared in step (1);

(3) cell fusion to obtain a hybridoma cell strain Thy1-2 of the monoclonal antibody against panda Thy 1;

(4) culturing the hybridoma cell strain Thy1-2, collecting cell supernatant, purifying and concentrating to obtain the anti-panda Thy1 monoclonal antibody.

The invention has the beneficial effects that:

the invention utilizes cell fusion technology to establish hybridoma cell strains secreting anti-Thy 1 monoclonal antibodies, collects and purifies cell strain culture supernatants to obtain high-specificity Thy1 monoclonal antibodies, establishes high-specificity Thy1 monoclonal antibodies to be applied to Western Blot protein detection, and provides positioning and cell expression information of Thy1 protein, application to identification and sorting of giant panda MGSCs, and deep research on ligands, antagonists and anti-antagonists of Thy1 receptors by utilizing Thy1 high-specificity monoclonal antibodies, further discusses Thy1 secretion and further gene regulation, reveals reproductive physiological rules, and provides new data and ideas.

Drawings

FIG. 1 is a standard graph of the concentration of Thy1 protein in the cells of example 2;

FIG. 2 shows the result of Western-blot detection in example 2.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

EXAMPLE 1 preparation of anti-panda Thy1 monoclonal antibody

1. Design and synthesis of specific polypeptide fragments

According to NCBI database panda Thy1 amino acid sequence, accession number: XP — 019648701.1, sequence: MNPTIGFALLLTVLQVARGQKVTSLTACLVDQSLRLDCRHENTTTSPIQYEFSL TREKKKHVLYGTMGVPEHSYRSRTNFTSKYDIKVLYLSSFTTKDEGTYTCELR LSGQPPITSSKNVSVLRDKLVKCEGISLLAQNTSGLLLLLLSLPLLQAMDFISL, and referring to sequence analysis of other species, the panda Thy1 specific sequence PEHSYRSRTNFTSKY (Thy1-1) (SEQ ID NO.1) was designed, which was artificially synthesized and coupled to KLH by adding a cysteine residue at the N-terminus, as immunogen Thy1-KLH for producing specific antibodies. For the synthesis of polypeptide fragments, the process is mature, and the polypeptide fragments Thy1-1 and the immunogen Thy1-KLH can be produced by the sequence disclosed in the application and the company of Biotechnology engineering (Shanghai).

2 antigen mouse immunization

With an adjuvant: the mixed solution of antigen 1:1 is inoculated, (the antigen is not enough to be mixed with sodium chloride and then emulsified with adjuvant). The first immunization was performed with Freund's complete adjuvant, and the subsequent immunization was performed with Freund's incomplete adjuvant. Sterile syringes, three-way tubing, disposable syringes were prepared prior to immunization. The antigen and NaCl were first aspirated into the syringe (total 400. mu.L, four doses) using a sterilized syringe, and then the adjuvant was aspirated into the syringe (total 400. mu.L, four doses) using a sterilized syringe; and finally, connecting the sterilized syringe in a three-way pipe for emulsification (the emulsification is about 10 minutes and only needs to be carried out until the mixture is in a water-in-oil state), and finally transferring the fused mixed solution into a disposable syringe for injection.

Day 1: intraperitoneal injection, 200 μ L of one. (antigen amount 100. mu.g/egg)

Day 14: intraperitoneal injection, 200 μ L of one. (thereafter, the antigens were all 50. mu.g/mouse)

Day 21: intraperitoneal injection, 200 μ L of one.

Day 27: intraperitoneal injection, 200 μ L of one.

…………………

(3 to 4 days after the third immunization, blood can be collected from the tail of the mouse, 12000rmp is carried out for 8min, serum is taken to measure the titer, Thy1-1 is used as an antigen with the coating concentration of 2 mug/mL, the titer of the serum is detected by an ELISA method, the fusion can be prepared after the titer reaches the standard, and the immunization is continued until the titer reaches the standard if the titer is not high enough.)

3. Cell fusion:

(1) recovery of myeloma cells (SP 2): firstly, taking out the frozen cells from liquid nitrogen, and quickly putting the cells in a water bath kettle at 37 ℃ for melting to loosen the cells; placing the cells into a 15mL centrifuge tube, taking PBS (5 mL), mixing uniformly, washing at 1000rpm for 5min, centrifuging, discarding the supernatant, washing SP2 cells twice, culturing the cells in a culture bottle, marking, and placing the culture bottle at 37 ℃ and 5% CO₂Culturing in an incubator.

(2) Passage of SP2 cells: when the bottom of the culture bottle is about 80% full of cells, the cells can be passaged. Blowing down the cells by using a gun head, sucking out the culture solution into a 15mL centrifugal tube for 1000r/min, and centrifuging for 5 minutes; discard the supernatant, add PBS 5mL, blow and mix well, centrifuge again and discard the supernatant, repeat PBS washing step 2 times. After washing, 2mL of 10% complete culture medium is added to resuspend the cells, an appropriate amount of the cells are taken out to a culture bottle, and the culture bottle is placed into a carbon dioxide incubator for culture.

(3) Preparation of trophoblast macrophages (ready the day before fusion):

the mouse takes off the cervical vertebra to die, the compression to the abdominal cavity is reduced as much as possible when the cervical vertebra is cut off, the blood vessel in the abdominal cavity is prevented from being damaged, and a great amount of blood cells are prevented from being contained in the feeder cells. The mice were soaked in 75% alcohol for 5 minutes, then the mouse tails were held by hand and rinsed in alcohol up and down several times. Placed in a sterile plate. The skin was cut from the back abdomen with sterile scissors, and both sides of the skin were torn open by hand to expose the abdomen, taking care not to damage the peritoneum. The peritoneum was wiped with an alcohol cotton ball. 6-8mL of the incomplete culture medium containing the double antibody was aspirated by a syringe and injected into the abdominal cavity (double antibody: incomplete culture medium: 1:100), and the peritoneum was carefully lifted by forceps during injection to avoid the needle sticking to the abdominal organs such as the intestinal canal. Gently massaging the abdomen for one minute with a cotton ball, sucking out the injected culture solution, and transferring into a centrifuge tube. Centrifuging at 1000r/min for 5min, and discarding the supernatant. And washed four more times with PBS. Cells were resuspended in 10% complete medium.

Adding the cell suspension into a 96-well plate, adding 100 mu L of the cell suspension into each well, and finally putting the 96-well plate into CO₂The incubator of (2) for cultivation. (macrophages are less abundant and a portion of the collected cells can be discarded as appropriate.)

(4) Preparation of immune spleen cells:

1) taking the spleen of a mouse

And taking the mice meeting the immunity requirement. First, the handling was performed aseptically to prevent cell contamination, and after the mice were sacrificed, they were immersed in 75% alcohol for about five minutes, placed in a sterile dish, and placed in a position (super clean bench) convenient for self-handling, and dissected. The tail of the mouse is cut with a small opening by scissors, the skin and hair layer is cut open by hands, the cut part is lightly wiped by an alcohol cotton ball, the layer of semitransparent film wrapping the internal organs is picked up by forceps, the internal organs are cut open, the spleen is exposed, the spleen is taken out lightly, the upper adipose tissues are removed as much as possible, and the taken spleen is placed in PBS for washing.

2) Spleen cell suspension preparation

Washing the spleen with PBS for about 3 times, placing the spleen in a plate, shearing the spleen as much as possible with scissors, adding PBS for washing and filtering, not organizing cells, collecting separated spleen cell suspension, centrifuging at 1000r/min for 5 minutes, and discarding the supernatant; washing with 5mL PBS at 1000r/min, and centrifuging for 5 min; repeating the steps for three times, adding 2mL of incomplete culture medium (DMEM) to resuspend the cells after washing, diluting the cell suspension by 100 times or 1000 times for counting the cells, and placing the rest cells in a water bath kettle at 37 ℃ for later use.

3) Preparation of SP2 cell suspension

Sucking cell sap by a rubber head suction pipe, blowing a thin film (cells are suspended or slightly attached to the wall to grow) at the bottom of the culture bottle, transferring the cell sap into a 15mL centrifugal tube by using a sample adding gun for 1000r/min, and centrifuging for 5 minutes; discarding the supernatant, adding 5mL of PBS, mixing uniformly, centrifuging for 5 minutes at 1000r/min, repeatedly washing twice, adding 2mL of incomplete culture medium (DMEM) to resuspend the cells after washing, diluting the cell suspension by 100 times or 1000 times for counting the cells, and placing the rest cells in a water bath kettle at 37 ℃ for later use.

(5) Cell fusion with PEG

SP2 was mixed with splenocytes at a ratio of 1:4 (between 1:10 and 1: 4), and centrifuging at 600rpm for 3 min; the supernatant was discarded. Lightly flick the bottom of the tube to loosen the cell pellet. 0.6mL of 50% PEG solution preheated to 37 ℃ was slowly added over 1min with gentle shaking and tapping. Standing for 1min after the addition. 10mL of the preheated incomplete culture solution at 37 ℃ is used for stopping the action of PEG, the incomplete culture solution is beaten while dripping, a centrifugal tube is rotated, the incomplete culture solution is added at a constant speed, and the mixture is kept stand for 2min after the addition. Centrifuging at 800rpm for 5min, and discarding the supernatant; the PEG was removed by washing 2 times with PBS or incomplete medium.

After washing, the supernatant was discarded and the cells were resuspended in 10mL HAT selection medium. The cells are applied to a 96-well plate containing a layer of feeder cells (using previously prepared macrophage plates, with no need to aspirate the liquid from the wells, and no use of incomplete wells)Washing the culture solution once, sucking out the liquid), and adding 100 mu L of culture solution into each well; placing the culture plate in CO₂Culturing in an incubator. After 4 hours, add HAT-containing complete medium (19.6mL 10% complete medium +0.4mL HAT) to wells at 100. mu.L per well; placing the culture plate in CO₂Culturing in an incubator.

(6) Selection culture

(1) HAT culture solution is changed by a half-liquid method on the fourth day of fusion culture: the supernatant in the 96-well plate was pipetted 100. mu.L per well by using a sample-feeding gun, discarded, and a new 100. mu.L per well of LHAT culture solution (HAT culture solution preparation: 10% complete culture solution: HAT 1:50) was added, and 200. mu.L of 50 XHAT was added to 10mL of complete culture solution per plate.

(2) On the seventh day of fusion culture, the HT culture solution is changed by a half-liquid method: the supernatant in the 96-well plate was pipetted 100. mu.L per well using a sample gun, discarded, and a new 100. mu.L LHT medium (HT medium formulation: 10% complete medium: HT 1:50) per well was added, requiring 200. mu.L 50 XHT per plate to be added to 10mL of complete medium.

(7) Positive clone screening

The clone cells are visible in the wells after about 7 days of culture, and when the clone cells grow enough (about 12 days), the culture solution can be sucked to detect whether the cells secrete antibodies.

1) The previously coated corresponding ELISA (Thy1-1 coated at 2. mu.g/mL) plates were removed from the freezer and allowed to return to room temperature, 100. mu.L of each well of the medium to be tested was added to the plates, and two wells served as negative and positive controls, 10% medium for the negative control and 10000-fold diluted serum for the positive control (serum collected from the orbital well of the mice before fusion).

2) And (3) incubation: the plates were sealed with a sealing plate and incubated in an incubator at 37 ℃ for 1 hour.

3) Washing: carefully uncovering the sealing plate film, washing for 4 times, and finally draining water as much as possible.

4) Adding double antibodies: the diabodies were diluted 10000 times, 50. mu.L per well.

5) And (3) incubation: plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.

6) Washing: carefully uncovering the sealing plate film, washing for 4 times, and finally draining water as much as possible.

7) Color development: adding 100 μ L of TMB into each well, mixing by gentle shaking, and developing in incubator for about 10 min.

8) And (3) colorimetric determination: 50. mu.L of stop solution (concentrated sulfuric acid (21.5 mL) was added to each well to a volume of 200mL), the mixture was gently shaken and mixed, the wavelength of the microplate reader was set to 450nm, and the value of each well was measured. The positive result (at least 4 times of the negative control) is selected as a positive clone hole.

(8) Method for screening hybridoma cell strain of anti-panda Thy1 monoclonal antibody by limiting dilution method

1) Counting of positive well cells: the positive cloning wells obtained by screening can be subjected to limiting dilution. Transferring the cells in the holes into a 15mL centrifuge tube (rotating while blowing to suspend the cells), and supplementing 10% of complete culture solution to 2 mL; the plates were counted and the cell fluid containing only 1000 cells was taken for the next experiment after counting (about 100 cells for each 96-well plate and 1000 for each 10-well plate since only one cell was required for 1 well).

2) Adding the cell fluid into 200mL of complete culture solution, mixing uniformly, loading the 96-well plate with 200 mu L/well, and obtaining ten 96-well plates in total.

3) Finally placing the culture plate in CO₂Culturing in an incubator.

4) After 4-5 days of culture, small cell clones were visualized on an inverted microscope, the growth of the cells was observed, and wells where single cells grew together were recorded.

5) Wells with single cell growth and aggregation recorded were replated on day 5 of culture with 100 μ L/well of 10% complete medium.

6) And (3) at 8-9 days, visually observing cell clones, detecting the antibody in time, and detecting the culture solution (ELSIA) in the holes formed by the growth and aggregation of single cells and with better growth conditions, wherein the holes with strong positive are the anti-panda Thy1 monoclonal antibody hybridoma cell strains. The invention finally obtains a hybridoma cell strain Thy1-2 for resisting the giant panda Thy1 monoclonal antibody through screening.

(9) Subtype identification

Subtype identification was performed using Pierce Rapid ELISA Mouse mAb Isotyping Kit 37503 Kit.

Preparation work: TBS in the kit is dissolved in 500mL of double distilled water for diluting a sample, 870mL of double distilled water is uniformly mixed with 30mL of 30X Wash Buffer for washing a plate, the number of plates needed is determined according to the amount of the sample, the rest is put back into a refrigerator at 4 ℃ for storage, 450 mu L of sample diluent is prepared, 20 mu L of cell culture solution is absorbed and added into 980 mu L of TBS for uniform mixing.

The experimental steps are as follows: the plate is balanced to the room temperature, a sample to be detected is added into each hole, 50 mu L/hole is formed, each sample needs to be added with 8 holes, namely one sample, 50 mu L/hole Goat Anti-Mouse IgG + IgA + IgM HRP is added, the plate is gently shaken and evenly mixed, a plate sealing film is covered, the room temperature incubation is carried out for one hour, the plate is washed for 4 times, the moisture is dried, 75 mu L/hole TMB color development liquid is added for color development, the liquid in the hole is seen to turn into blue, the 75 mu L/hole stop liquid is added after the color development is carried out for 5-15min to stop the reaction, and the liquid turns from blue into yellow. The antibody subtype secreted by the anti-panda Thy1 monoclonal antibody hybridoma cell strain Thy1-2 obtained by identification and screening is IgG1 type.

(10) And (5) performing amplification culture, purification and concentration on the monoclonal antibody.

1) Batch culture: the wells with the subtype identified monoclonal antibody were transferred to a 24-well plate (cell suspension by pipetting while rotating, complete transfer was performed) and cultured in 600. mu.L of 10% complete medium.

2) The growth of the cells was observed, after a relatively large number of cells had grown, titer measurement was performed, and cells having a high titer were transferred to a small culture flask and cultured (cells were first blown out from a 24-well plate to suspend the cells, cells were transferred to the culture flask with a sample application gun, and 7mL of 10% complete medium was added).

3) And observing the growth condition of the cells, and transferring the cells to a large culture bottle for culture after the cells grow well. One small flask was transferred to two large flasks for culture (cell passaging).

4) Culturing in several culture bottles, freezing some cells, and purifying antibody column with culture liquid. The column was packed with Pierce Protein G Agarose, and the purified anti-panda Thy1 monoclonal antibody was concentrated in a 10000kda ultrafiltration tube and stored at-20 ℃ until use.

(11) Freezing and storing monoclonal antibody cell strain

After the identified hybridoma cell line Thy1-2 of the anti-panda Thy1 monoclonal antibody is stably cultured, the cells in the culture flask are blown to suspend in the culture solution (the cells are generally suspended in the culture solution or grow adherently), and then the cells are transferred to a 15mL centrifuge tube. Centrifuge at 1000rpm for 5 minutes. Wash twice with PBS: the supernatant in the centrifuge tube was first aspirated, discarded, PBS added, mixed well, centrifuged at 1000 rpm/5 min, and repeated once. Finally, the supernatant was aspirated by a sample application gun, and an appropriate amount of the frozen solution (5 mL serum +4mLDMEM +1 mldmmso) was added to the cells, and the mixture was inverted, filtered, and mixed. Finally, add to the cryopreserved tubes, 1mL of cell sap per tube. Putting the cell strain into a freezing storage box, putting the cell strain into liquid nitrogen for long-term storage for later use at-80 ℃ overnight, and then putting the cell strain Thy1-2 into the liquid nitrogen.

Example 2 anti-panda Thy1 monoclonal antibody used in western blot for detecting Thy1 protein

1. Extraction of Total protein of target cell

The application of the protein extraction kit: c510004-0020, supplied by Biotechnology engineering (Shanghai) Co., Ltd., extracts the total protein of the giant panda bone marrow mesenchymal stem cell, and measures the concentration of the total protein by BCA method.

2. Preparing glue: preparing polyacrylamide gel, 5% of concentrated gel and 12% of separation gel.

3. Preparing a sample: the loading of protein was 80. mu.g.

4. Electrophoresis: concentrating the gel at 80V for 30 min; the gel was separated at 120V for 90 min.

5. Film transferring: 250mA, 40 min.

6. And (3) sealing: 5% skimmed milk, and slowly shaking at 37 deg.C for 2 h.

7. Incubating the primary antibody: the antibodies of the hybridoma cell line Thy1-2 are diluted 1:1000, slowly shaken at 4 ℃ overnight, and rewarmed at 37 ℃ for 1h the next day.

8. Incubation of secondary antibody: 1:8000 dilution, and incubating for 1h at 37 ℃.

9. And (4) ECL exposure.

The results of western blot detection are as follows:

1. cellular protein concentration

(1) Standard curve of

The standard graph is shown in fig. 1.

(2) Protein concentration

(3) The Western-blot detection results are shown in FIG. 2.

The anti-panda Thy1 monoclonal antibody can accurately identify a target protein Thy1, provides information for positioning of Thy1 protein and cell expression, and provides conditions for finding specific ligands, antagonists and anti-antagonists by utilizing the Thy1 high-specificity monoclonal antibody to carry out deep research on ligands, antagonists and anti-antagonists of a Thy1 receptor.

Sequence listing

<110> research base for breeding pandas in Chengdu province

<120> anti-panda Thy1 monoclonal antibody, hybridoma cell strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Pro Glu His Ser Tyr Arg Ser Arg Thr Asn Phe Thr Ser Lys Tyr

1 5 10 15

Claims

1. A hybridoma cell strain Thy1-2 of a monoclonal antibody against giant panda Thy1 has a preservation number of: CCTCC NO: C202170.

2. an anti-panda Thy1 monoclonal antibody produced by the hybridoma cell line Thy1-2 of claim 1.

3. The anti-panda Thy1 monoclonal antibody according to claim 2, wherein the subtype of the monoclonal antibody is IgG 1.

4. Use of the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2 or 3 in the preparation of a test agent.

5. Use of the monoclonal antibody of claim 2 or 3 in the preparation of a product for detecting Thy1 protein.

6. Use according to claim 4 or 5, wherein the detection reagent or product is for use in a western blot assay.

7. A kit for auxiliary diagnosis of a fibroblast lesion disease, comprising the monoclonal antibody of claim 2 or 3, or the monoclonal antibody secreted by the hybridoma cell strain of claim 1.

8. The monoclonal antibody of claim 2 or 3, or the hybridoma cell line of claim 1, for use in isolation, identification and/or enrichment of panda MGSCs cells.

9. The method for preparing the anti-giant panda Thy1 monoclonal antibody according to claim 2 or 3, comprising the steps of: culturing the hybridoma cell line Thy1-2 of claim 1, collecting cell supernatant, purifying and concentrating to obtain the anti-giant panda Thy1 monoclonal antibody.