Monoclonal antibody and hybridoma cell line PLM1 that recognize porcine liver mesenchymal stem cells

Abstract

The present invention discloses a monoclonal antibody for identifying porcine liver mesenchymal stem cells and a hybridoma cell line PLM1. Using porcine liver mesenchymal stem cells as antigens, a hybridoma cell line PLM1 is constructed by applying monoclonal antibody technology, and the deposit number is CCTCC NO: C2022300. The hybridoma cell line PLM1 secretes a monoclonal antibody for identifying porcine liver mesenchymal stem cells. The monoclonal antibody is used as a probe in flow cytometry, immunohistochemistry, immunofluorescence and immunoblotting, etc., for detecting and analyzing porcine liver mesenchymal stem cells. The present invention provides a specific method for the study of porcine liver mesenchymal stem cells, and a variety of detection techniques based on monoclonal antibodies of porcine liver mesenchymal stem cells can also be used in the study of porcine liver injury and regeneration.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody for identifying pig liver mesenchymal stem cells and a hybridoma cell line PLM1. Background Art

[0002] The liver has the functions of synthesis, detoxification, metabolism, secretion, biotransformation and immune defense. When affected by hepatotoxic substances, drugs, hepatitis viruses, etc., serious damage will occur, leading to abnormal liver function and even a large number of liver cell necrosis, thus causing liver failure. At present, liver transplantation is the best treatment for acute and chronic liver failure, but its application is limited by the shortage of donors and high costs. Mesenchymal stem cells (MSC) have self-renewal ability and multidirectional differentiation ability as well as low immunogenicity. It is considered to be an ideal cell source for research and treatment. At present, MSCs from various sources such as bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and umbilical cord blood mesenchymal stem cells have been studied and applied in various liver diseases. According to literature reports, Herrera et al. first isolated human liver mesenchymal stem cells from human liver tissue in 2006 and tested their biological characteristics. They found that their hematopoietic stem cell surface molecular markers such as CD34 and CD45 were negative, while markers such as CD29, CD73, and CD90 were positive, and they could secrete albumin, urea, etc.

[0003] In the previous study, the present invention successfully isolated and obtained porcine liver mesenchymal stem cells (PLMSC) from normal Chinese experimental miniature pig liver tissue. PLMSC has normal MSC cell functions and characteristics, and its CD29, CD73, CD90 and other markers are positively expressed, and CD34, CD45 and other hematopoietic stem cell surface molecules are negatively expressed. PLMSC cell glycogen staining is weakly positive, and it can also differentiate into adipocytes, osteoblasts and hepatocytes. It is a type of mesenchymal stem cell with application potential in various animal models of liver diseases. PLMSC can play an important physiological and repair function, especially in liver damage and regeneration, showing its unique value.

[0004] However, due to the lack of specific PLMSC cell surface markers, the identification, isolation and culture of PLMSC cells is a technical bottleneck in liver disease research. At present, the identification of PLMSC cells mainly relies on cell morphology observation and a series of combined determinations of non-specific cell surface antigens. These methods are often complicated to operate and difficult to achieve rapid and efficient cell detection, cell sorting and purification. In addition, due to the differences in PLMSC cell phenotypic characteristics in different animal individuals and treatment conditions, it is difficult to ensure the acquisition of high-purity PLMSC cells using traditional methods, which limits the research and application of PLMSC cells in pig liver injury and regeneration research.

[0005] Therefore, it is urgent to develop an efficient tool for identifying pig liver mesenchymal stem cells. Summary of the invention

[0006] The purpose of the present invention is to provide a monoclonal antibody and a hybridoma cell line PLM1 for identifying porcine liver mesenchymal stem cells in view of the deficiencies in the prior art.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: As a first aspect, a monoclonal antibody for recognizing porcine liver mesenchymal stem cells is provided, which is deposited in the China Center for Type Culture Collection and secreted by the anti-porcine liver mesenchymal stem cell monoclonal antibody hybridoma cell line PLM1 with the deposit number: CCTCC NO: C2022300.

[0008] Furthermore, the monoclonal antibody is a mouse IgG1 monoclonal antibody.

[0009] As a second aspect, the present invention provides a monoclonal antibody hybridoma cell line PLM1 against porcine liver mesenchymal stem cells, which was deposited in the China Center for Type Culture Collection and Wuhan University, Wuhan, China on October 19, 2022, with the deposit number: CCTCC NO: C2022300, and specifically secretes the above-mentioned monoclonal antibody.

[0010] As a third aspect, the present invention also provides an application of the monoclonal antibody for recognizing porcine liver mesenchymal stem cells in the detection of porcine liver mesenchymal stem cells, specifically: based on the monoclonal antibody as a primary antibody, or as a probe for application in flow cytometry, immunohistochemistry, immunofluorescence and immunoblotting.

[0011] As a fourth aspect, the present invention further provides a use of the monoclonal antibody for recognizing porcine liver mesenchymal stem cells in the culture, passage and expansion of porcine liver tissue or porcine liver mesenchymal stem cells.

[0012] The beneficial effects of the present invention are as follows:

[0013] The present invention has successfully developed a monoclonal antibody for identifying porcine liver mesenchymal stem cells and its hybridoma cell line PLM1, which has the advantages of high specificity and good repeatability, can specifically identify a surface antigen on porcine liver mesenchymal stem cells, and provides a specific method for the study of porcine liver mesenchymal stem cells, and provides an efficient tool for the rapid sorting, identification and purification of porcine liver mesenchymal stem cells. Various detection technologies based on monoclonal antibodies of porcine liver mesenchymal stem cells can also be applied to the study of acute injury and regeneration of porcine liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a morphological diagram of porcine liver mesenchymal stem cells obtained by using the collagenase four-step perfusion method and density gradient centrifugation combined with adherent culture method. Figure 1 A in the figure is the morphology of cultured primary pig liver mesenchymal stem cells; Figure 1 B is the morphology of the pig liver mesenchymal stem cells at the 5th passage of culture;

[0015] Figure 2 This is the growth curve of the fifth generation of pig liver mesenchymal stem cells;

[0016] Figure 3 This is a flow cytometry analysis of the fifth generation of pig liver mesenchymal stem cells, where: Figure 3 A in the figure is a flow cytometric analysis of porcine liver mesenchymal stem cells using CD45-PE monoclonal antibody as a probe; Figure 3 B is a flow cytometric analysis of porcine liver mesenchymal stem cells using CD34-FITC monoclonal antibody as a probe; Figure 3 C in the figure is a flow cytometric analysis of porcine liver mesenchymal stem cells using CD29-FITC monoclonal antibody as a probe; Figure 3 D in the figure is a flow cytometric analysis of porcine liver mesenchymal stem cells using CD90-FITC monoclonal antibody as a probe;

[0017] Figure 4 This is an analysis of the multidirectional differentiation ability of the fifth generation of pig liver mesenchymal stem cells, among which, Figure 4 A in the figure is the morphology of the fifth generation of cultured porcine liver mesenchymal stem cells; Figure 4 B is the Oil Red O staining image of pig liver mesenchymal stem cells directional differentiation into adipose; Figure 4 C in the figure is the Alizarin red staining image of porcine liver mesenchymal stem cells differentiating into osteoblasts;

[0018] Figure 5 This is the result of indirect immunofluorescence staining of the 5th generation porcine liver mesenchymal stem cells using PLM1 monoclonal antibody as the primary antibody;

[0019] Figure 6 This is the result of immunohistochemical staining to detect pig liver mesenchymal stem cells using PLM1 monoclonal antibody as the primary antibody. Figure 6 A in the figure is the immunohistochemical staining result of the negative control using mouse immunoglobulin as the primary antibody. Figure 6 Figure B shows the expression of the corresponding antigen of monoclonal antibody PLM1 in normal pig liver tissue. Figure 6 C in the figure is the expression result of the corresponding antigen of monoclonal antibody PLM1 in the liver of pigs with acute liver failure;

[0020] Figure 7 This is the result of flow cytometry detection of pig liver mesenchymal stem cells using PLM1 monoclonal antibody as the primary antibody; Figure 7 A is the analysis result of flow cytometry of negative control. Figure 7 B in the figure is the analysis result of flow cytometry using monoclonal antibody PLM1 as the primary antibody;

[0021] Figure 8 This is the result of Western blotting to detect the expression of corresponding antigens in porcine liver mesenchymal stem cells using PLM1 monoclonal antibody as the primary antibody. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below through specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. As mentioned throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present invention.

[0023] The monoclonal antibody specifically recognizing porcine liver mesenchymal stem cells of the present invention can specifically bind to the membrane surface protein of porcine liver mesenchymal stem cells, and has no cross reaction with other cells such as rat bone marrow mesenchymal stem cells, mouse bone marrow mesenchymal stem cells, porcine adipose mesenchymal stem cells, human liver cell lines huh751 and HepG2, human liver progenitor cells, human liver mesenchymal stem cells, and human hepatic stellate cell line HSC-Li.

[0024] In addition, the present invention also provides a mouse hybridoma cell line PLM1 that produces the above-mentioned monoclonal antibody. The spleen cells of Balb / c mice immunized with pig liver mesenchymal stem cells and mouse myeloma FO cells are obtained respectively, fused under the action of polyethylene glycol, selectively cultured in HAT and HT medium successively, screened positive cell clones by indirect immunofluorescence method, subcloned and screened three times in succession, selected positive cell clones, and finally obtained cells grown from single cell clones. After expanded culture, the mouse hybridoma cell line PLM1 is successfully constructed and preserved in the China Center for Type Culture Collection with the preservation number of CCTCCNO: C2022300. The cell line can stably secrete the above-mentioned monoclonal antibody, harvest the cell culture supernatant, or inoculate the cells into the peritoneal cavity of Balb / c mice to obtain ascites, and separate and purify the above-mentioned monoclonal antibody by existing technology.

[0025] Example 1: Isolation, culture, identification, cryopreservation and recovery of porcine liver mesenchymal stem cells.

[0026] (1) Isolation and culture of porcine liver mesenchymal stem cells:

[0027] Part of the liver tissue of a normal healthy pig was removed under sterile conditions, and the surface of the pig liver was rinsed with 1000 ml of phosphate buffer precooled to 4°C. The pig liver tissue was digested by a four-step in vitro perfusion method including split enzymes and collagenase to separate and obtain primary pig liver cells. Porcine liver mesenchymal stem cells were obtained by density gradient centrifugation combined with adherence screening. The primary cultured cells were cultured in a 37°C incubator containing 5% (v / v) CO2 using DMEM culture medium (v / v) containing 10% fetal bovine serum. After 48 hours, non-adherent cells were removed by replacing the DMEM culture medium, and long spindle-shaped adherent cells were visible. The culture medium was then replaced every 3 days. When the cells reached 70% to 80% fusion, they showed a colony distribution and attached to the bottom of the culture dish in a vortex-like form, with a morphology like Figure 1 As shown in A. 0.25% (w / v) trypsin was used for digestion, adherent cells were harvested, subcultured in T25 medium, and cultured continuously. The morphology of pig liver mesenchymal stem cells after subculture is as shown in Figure 1 As shown in B (unless otherwise specified, the fifth generation of porcine liver mesenchymal stem cells were used as the implementation object).

[0028] (2) Identification of the growth characteristics of porcine liver mesenchymal stem cells:

[0029] The 5th and 10th generation porcine liver mesenchymal stem cells were cultured and cultured at 1.0×10 4The initial density of cells / well was inoculated into a 6-well plate. The first cell count was performed 24 hours after culture. The cells were counted every 24 hours, and 3 wells were counted each time. The average value was obtained for kinetic analysis. The culture has the following common characteristics: after subculture, the cells are completely attached to the wall in 12 hours, and the cell morphology slowly changes back to a long spindle shape. The incubation period of subculture is about 24 to 48 hours; the logarithmic proliferation period of subculture is about 3 to 6 days; after the logarithmic proliferation period, it enters the plateau period. The proliferation ability of the 5th and 10th generation cells is basically maintained well. The growth and proliferation characteristics of porcine liver mesenchymal stem cells are as follows Figure 2 As shown, P5 and P10 are the growth curves of the 5th and 10th generations of pig liver mesenchymal stem cells, respectively.

[0030] (3) Identification of the phenotype of porcine liver mesenchymal stem cells:

[0031] The fifth generation cells were taken and the surface markers of porcine liver mesenchymal stem cells were analyzed by flow cytometry using CD29-FITC, CD34-FITC, CD90-FITC and CD45-PE monoclonal antibodies as probes. Figure 3 As shown in A~D, the horizontal axis is the fluorescence intensity, and the vertical axis is the number of cells. The surface molecules CD90 and CD29 of mesenchymal stem cells are positive, and the marker molecules CD34 and CD45 of hematopoietic stem cells are negative. The RT-PCR method (Reverse Transcription-Polymerase Chain Reaction, reverse transcription PCR) was used to further detect the cell surface molecules of pig liver mesenchymal stem cells. The results showed that the cells' CD29, CD44, CD71, CD73, and CD90 were all positively expressed.

[0032] (4) Identification of the multi-differentiation potential of porcine liver mesenchymal stem cells:

[0033] When the cells of the fifth generation were 80% to 90% confluent, they were digested with 0.25% (w / v) trypsin and plated at 5×10 4 The cells were seeded in 6-well culture plates at a density of 1.5 cells / well. The multipotential differentiation potential of PLDMSCs was evaluated under the conditions of adipogenic, osteogenic and hepatogenic differentiation, and the undifferentiated 5th generation PLDMSCs were used as the control group. Figure 4 A in the figure shows the morphology of the 5th generation of cultured porcine liver mesenchymal stem cells.

[0034] After 7 days of adipogenic induction, the cell volume increased, the morphology changed to oval, and small lipid droplets began to appear in the cytoplasm. As the induction continued, the circular characteristics of the cell outline became more obvious, and the number and volume of lipid droplets increased. At about 15-21 days, large lipid droplets almost occupied the entire cell, and the cell nucleus was biased to the edge to form a distinct feature. After 21 days of culture, the lipid droplets showed bright red after Oil Red O staining, confirming that PLDMSCs can successfully differentiate into adipocytes. Figure 4 As shown in B.

[0035] After 3-4 days of osteogenic induction, the cells adhered to the wall and gradually grew larger, forming tighter intercellular connections. Calcification spots were observed after 7 days, and polygonal and cubic cells increased significantly with the extension of induction time. Calcified nodule-like structures appeared in the cells around 14-21 days. After 21 days of induction, red-stained cells were found to aggregate in specific areas to form multi-layered nodular structures, indicating that the porcine liver mesenchymal stem cells successfully achieved osteogenic differentiation, while no nodular structures were observed in the control group. Figure 4 As shown in C.

[0036] (5) Cryopreservation and recovery of porcine liver mesenchymal stem cells:

[0037] Porcine liver mesenchymal stem cells were stored in DMEM culture medium containing 10% DMSO (v / v). After being frozen in liquid nitrogen for several months to several years, the MSC cell characteristics and functions were still maintained after resuscitation culture.

[0038] Example 2: Preparation and purification of monoclonal antibodies against porcine liver mesenchymal stem cells.

[0039] (1) Animal immunization:

[0040] PLMSCs of passage 5 were selected and the cell concentration was 1.0×10 6 Balb / c mice were intraperitoneally immunized once a week for three times. On the 20th day, blood was collected from the mice by eyeball blood sampling and serum was obtained by centrifugation. The antibody titer of the serum was detected by indirect immunofluorescence staining, and the mice corresponding to the serum samples with the highest titer were selected for booster immunization. The cell concentration for booster immunization was adjusted to 2.0×10 6 / , and cell fusion was performed 3 days later.

[0041] (2) Preparation of mouse hybridoma cell lines:

[0042] Peritoneal macrophages and spleen cells of normal mice without immunization were collected as feeder cells, spleen cells of mice immunized with PLMSC were obtained aseptically, mixed with myeloma cells FO at a ratio of 10:1, and fused with 50% (w / v) polyethylene glycol in a 38°C water bath. The fused cell clumps were inoculated into the 96-well plate prepared the day before and cultured in a 37°C, 5% (v / v) CO2 incubator. HAT culture medium was replaced every 3 days, and HT culture medium was used to continue culturing hybridoma cells after two weeks.

[0043] When the hybridoma cells grow to 1 / 3~1 / 2 of the bottom area of ​​the well, the indirect immunofluorescence staining method is used to screen positive clones. The method is as follows: select well-grown porcine liver mesenchymal stem cells for climbing and fixing, take the culture supernatant of hybridoma cells as the primary antibody, incubate at 37°C for 1h, wash 3 times with PBS, 1 minute each time, add FITC-labeled secondary antibody, incubate at 37°C for 30min, and wash with PBS. The positive clones screened out are inoculated into a new 96-well plate containing feeder cells by limiting dilution method for subcloning culture. Repeat the above steps until the positive rate of cloned cell antibody detection reaches 100% for 3 consecutive times. The hybridoma cells are continuously passaged in vitro, repeatedly frozen and revived, and regularly screened and tested until the hybridoma cell line can continuously and stably secrete monoclonal antibodies.

[0044] (3) Preparation and purification of monoclonal antibodies:

[0045] Balb / c mice were pretreated with paraffin oil and injected intraperitoneally with well-growing hybridoma cells PLM1. Each mouse was injected with 5×10 6 Cells. The abdomen of the mice was obviously swollen after about 2 weeks. The mice were killed by eye bleeding and ascites was collected. The ascites and saline were mixed at a ratio of 1:1 (v / v), and saturated ammonium sulfate was added while stirring until the final saturation of ammonium sulfate was 50%. The immunoglobulin was crudely extracted and purified by SPA (Staphylococcus aureus protein A method) to obtain the monoclonal antibody PLM1 against porcine liver mesenchymal stem cells.

[0046] (4) Anti-porcine liver mesenchymal stem cell monoclonal antibody PLM1 ascites titer and subtype identification:

[0047] The purified monoclonal antibody PLM1 was diluted in equal proportions and used as the primary antibody. The FITC-labeled goat anti-mouse immunoglobulin antibody was used as the secondary antibody. The titer of the detection was 2.0×10 4 The hybridoma cell culture supernatant was obtained, and the subtype of the monoclonal antibody was determined to be IgG1 using the ELISA method (Enzyme-Linked Immunosorbent Assays).

[0048] Example 3: Specific identification of monoclonal antibodies against porcine liver mesenchymal stem cells.

[0049] (1) Tissue-specific detection of pig liver:

[0050] Tissue materials: tendons, muscles, vascular walls, ligaments, nerves, liver, small intestine, skin, lungs, fat and bone marrow from normal pigs. The above tissue specimens are all from normal pig tissues or organs removed. Anti-pig liver mesenchymal stem cell monoclonal antibody PLM1 was used as an antibody to detect paraffin sections of normal pig tissues by immunohistochemical staining. The results showed that scattered positive cells were visible in normal pig liver tissue sections, and other normal pig tissues were negatively expressed, which shows that the monoclonal antibody has the characteristics of specific binding and recognition of pig liver mesenchymal stem cells.

[0051] (2) Species-specific detection:

[0052] Cell materials: normal human, pig, rabbit, rat, mouse liver tissues and cultured rat and pig fat mesenchymal stem cells, etc. By immunohistochemical staining, pig liver mesenchymal stem cells were used as positive control, and anti-pig liver mesenchymal stem cell monoclonal antibody PLM1 was used as antibody to detect paraffin sections and cells of various tissues. The results showed that all tissue sections and cells were negative, which shows that monoclonal antibody PLM1 has a strong specificity for pig liver mesenchymal stem cells.

[0053] Example 4: Application of monoclonal antibodies in detecting pig liver mesenchymal stem cells by immunofluorescence assay.

[0054] In this example, the monoclonal antibody against porcine liver mesenchymal stem cells is used as the primary antibody, and the fluorescent-labeled goat or sheep anti-mouse immunoglobulin antibody is used as the secondary antibody to establish an indirect immunofluorescence staining method to detect porcine liver mesenchymal stem cells, and observe and analyze the positive expression of porcine liver tissue or porcine liver mesenchymal stem cells in culture under a fluorescence microscope. Specifically:

[0055] Well-grown porcine liver mesenchymal stem cells were selected for slide fixation, and monoclonal antibody PLM1 was used as the primary antibody. The indirect immunofluorescence staining method (the specific method is the same as the preparation of hybridoma cell line described in step (2) of Example 2) was used to observe under a fluorescence microscope and calculate the positive rate. The results showed that porcine liver mesenchymal stem cells were 100% positive and were all expressed on the cell membrane surface. Figure 5 The experiment showed that the monoclonal antibody PLM1 can be used as a surface marker of pig liver mesenchymal stem cells and can be effectively applied to immunofluorescence staining, which is conducive to further exploring the biological characteristics of the corresponding antigens of pig liver mesenchymal stem cells.

[0056] Example 5: Application of monoclonal antibody PLM1 in detecting pig liver mesenchymal stem cells by immunohistochemistry.

[0057] In this example, the monoclonal antibody against pig liver mesenchymal stem cells is used as the primary antibody, and the HRP-labeled goat or sheep anti-mouse immunoglobulin antibody is used as the secondary antibody to establish an immunohistochemical staining or cytochemical staining method to detect pig liver or pig liver mesenchymal stem cells, and to detect and analyze the positive expression of pig liver tissue or cultured pig liver mesenchymal stem cells. Specifically:

[0058] Well-grown porcine liver mesenchymal stem cells were selected for cell slide fixation, blocked at 37°C for 30 minutes, and monoclonal antibody PLM1 was used as the primary antibody. The cells were incubated at 37°C for 2 hours, washed 3 times with PBS for 2 minutes each time, and then HRP-labeled anti-mouse immunoglobulin antibody was added as the secondary antibody. The cells were incubated at 37°C for 1 hour and washed with PBS. DAB color development was performed, and the color development time was controlled under a microscope. After washing with distilled water, hematoxylin was used for counterstaining for 2 minutes, and the distilled water washing method was the same as before. The positive expression of the corresponding antigens in porcine liver mesenchymal stem cells can be observed under a microscope, which is helpful for the localization and quantitative analysis of porcine liver mesenchymal stem cells. The same method was used to detect liver tissue sections of normal pigs and pigs with acute liver failure. Scattered positive cells could be seen in normal pig liver tissue under a microscope, such as Figure 6 As shown in B; more positive cells can be seen in the liver of pigs with acute liver failure, such as Figure 6 As shown in C; mouse immunoglobulin was used as the primary antibody as a negative control, and the results are shown in Figure 6 As shown in A. This shows that monoclonal antibodies have good specificity and sensitivity and can be effectively used in tissue immunohistochemistry.

[0059] Example 6: Application of monoclonal antibodies in flow cytometry detection of porcine liver mesenchymal stem cells.

[0060] In this example, a monoclonal antibody against porcine liver mesenchymal stem cells was used as the primary antibody, and a fluorescently labeled goat or sheep anti-mouse immunoglobulin antibody was used as the secondary antibody to establish a flow cytometer to detect porcine liver mesenchymal stem cells and to detect and analyze the positive expression of porcine liver mesenchymal stem cells. Specifically:

[0061] Well-growing porcine liver mesenchymal stem cells were selected, monoclonal antibody PLM1 was used as a probe, FITC-labeled anti-mouse immunoglobulin antibody was used as a secondary antibody, and the flow cytometry test results were as follows Figure 7 As shown in B, the horizontal axis is the fluorescence intensity and the vertical axis is the number of cells, showing that the PLMSC positive rate is 97.1%. At the same time, the flow cytometer test results using mouse immunoglobulin as the primary antibody and as a negative control are as follows Figure 7As shown in A, it shows that the monoclonal antibody PLM1 can efficiently bind to the surface protein of porcine liver mesenchymal stem cells and is suitable for the quantitative analysis of porcine liver mesenchymal stem cells.

[0062] Example 7: Application of monoclonal antibodies in detecting pig liver mesenchymal stem cells by western blot.

[0063] In this example, a monoclonal antibody against porcine liver mesenchymal stem cells was used as the primary antibody, and an HRP-labeled goat or sheep anti-mouse immunoglobulin antibody was used as the secondary antibody to establish an immunoblotting method to detect porcine liver or porcine liver mesenchymal stem cells, and analyze the positive expression of porcine liver tissue or porcine liver mesenchymal stem cells after culture. Specifically:

[0064] The in vitro expanded porcine liver mesenchymal stem cells were lysed with protein lysis buffer, and the total protein was extracted. SDS-PAGE electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, and the membrane was blocked with 5% skim milk powder at room temperature for 1 hour after electrotransfer. The monoclonal antibody PLM1 was added as the primary antibody, and the cells were incubated at 4°C overnight. The cells were washed with TBST, where TBST was prepared by adding 0.5% (v / v) Tween-20 to TBS (Tris buffered saline). After washing, the secondary antibody was added, and the cells were reacted at room temperature for 2 hours. After washing with TBST, ECL was added for 1 minute, and the expression of the corresponding antigen was detected by exposure in a dark room. The results showed that the monoclonal antibody PLM1 specifically bound to a protein band with a molecular weight of about 50 KD (kilodaltons), such as Figure 8 The results show that the monoclonal antibody PLM1 can be used in immunoblotting to qualitatively and semi-quantitatively analyze the corresponding antigens of porcine liver mesenchymal stem cells.

[0065] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody for recognizing porcine liver mesenchymal stem cells, characterized in that: It is secreted and produced by the hybridoma cell line PLM1 with the deposit number of CCTCC NO: C2022300.

2. The monoclonal antibody for recognizing porcine liver mesenchymal stem cells according to claim 1, characterized in that: The monoclonal antibody is a mouse IgG1 monoclonal antibody.

3. Use of the monoclonal antibody for recognizing porcine liver mesenchymal stem cells according to claim 1 or 2 in the detection of porcine liver mesenchymal stem cells, characterized in that: The application is specifically: the monoclonal antibody is used as a probe in flow cytometry, immunohistochemistry, immunofluorescence or immunoblotting.

4. Use of the monoclonal antibody for recognizing porcine liver mesenchymal stem cells according to claim 1 or 2 in the culture, passage and expansion of porcine liver tissue or porcine liver mesenchymal stem cells.

5. A hybridoma cell line PLM1, characterized in that: It was deposited in the China Center for Type Culture Collection on October 19, 2022, with the accession number: CCTCC NO: C2022300.

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