CN112062832A

CN112062832A - Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit

Info

Publication number: CN112062832A
Application number: CN202011017231.5A
Authority: CN
Inventors: 翟晋豫; 柴素真; 徐星星; 王亚杰; 张东辉; 常秋霜; 齐华
Original assignee: Henan Celnovtebio Biotechnology Inc
Current assignee: Henan Celnovtebio Biotechnology Inc
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2020-12-11
Anticipated expiration: 2040-09-24
Also published as: CN112062832B

Abstract

The invention relates to Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit. The invention takes NTKLDNNWGREERQS or NTKLDNNWGREERQSVFPF as epitope peptide, artificially synthesizes the epitope peptide and couples with carrier protein; and taking the monoclonal antibody as immunogen to immunize BALB/C mice, adopting cell fusion technology and indirect ELISA method to screen positive hybridoma cells, and finally obtaining a hybridoma cell strain C6C10 which can stably secrete mouse anti-human Galectin-3 monoclonal antibody, with the preservation number of CCTCC NO: C2020135; monoclonal antibodies were prepared by induction in animals and antibody specificity was identified by immunohistochemistry. The mouse anti-human Galectin-3 monoclonal antibody provided by the invention has better specificity and sensitivity, and can be used for preparing a Galectin-3 in-vitro immunoassay reagent or kit, so that the defects of low selectivity, high price and the like of the existing Galectin-3 antibody are overcome.

Description

Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit

Technical Field

The invention relates to Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit, and belongs to the technical field of immunology.

Background

The human Galectin 3(Galectin-3) gene is located on chromosome 14q 21-22 and consists of 6 exons and 5 introns, and Galectin-3 is mainly located in cytoplasm, is also expressed on cell nucleus, cell surface and outside cell and plays multiple effects through interaction with corresponding ligands. Galectin-3 binds glycosylated extracellular matrix proteins and membrane proteins in a lectin-sugar interaction manner.

Galectin-3 is one of beta-galactoside animal lectin family members, is also an endogenous lectin existing in various organisms, is mainly expressed in epithelial cells, immune cells and macrophages, not only participates in the processes of cell proliferation, adhesion, differentiation, migration, apoptosis, inflammatory reaction and the like, but also plays an important role in the processes of tumor generation, development and metastasis.

Galectin-3 is a powerful proinflammatory factor, and can promote the differentiation of monocytes to macrophages and the accumulation thereof at endangium damage parts, and stimulate the release of inflammatory factors such as interleukin-6, tumor necrosis factor alpha, monocyte chemotactic protein 1 and the like. During the cancer detection process, the polypeptide has high expression level in ovarian cancer, esophageal cancer and liver cancer, and accelerates the stimulation of the proliferation and invasion of tumor cells.

At present, a common in-vitro immune detection method for Galectin-3 is an Immunohistochemistry (IHC) method, a monoclonal antibody which is specifically combined with human Galectin-3 plays a key role in the process of detecting the expression condition of the Galectin-3 in isolated histiocytes by the IHC method, and the specificity and the affinity of the monoclonal antibody determine the sensitivity and the specificity of the detection method. The existing Galectin-3 in vitro immunodetection antibody has fewer types, the sensitivity needs to be improved and the price is high.

Disclosure of Invention

The invention aims to provide a Galectin-3 epitope peptide, and an antigen prepared by coupling the epitope peptide with a carrier protein can be used for immunizing animals to obtain monoclonal antibodies or polyclonal antibodies aiming at the epitope peptide.

Secondly, the invention provides a Galectin-3 monoclonal antibody or polyclonal antibody prepared by taking the antigen as an immunogen.

Meanwhile, the invention provides a hybridoma cell strain capable of stably secreting mouse anti-human Galectin-3 monoclonal antibody and a method for preparing the Galectin-3 monoclonal antibody by using the hybridoma cell strain.

The invention further provides application of the Galectin-3 antibody in preparation of a Galectin-3 in-vitro immunoassay reagent or kit.

Finally, the invention provides a Galectin-3 immunohistochemical detection reagent and a kit.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to the first aspect of the invention, a Galectin-3 epitope peptide is selected from the amino acid sequence 174-188 th site, namely the amino acid sequence SEQ ID NO:1 is epitope peptide, or four amino acids are added at the C end of the epitope peptide, and the four amino acids are sequentially as follows: VFPF, i.e. SEQ ID NO:2 is an epitope peptide, and the amino acid sequence of the epitope peptide is as follows: 1) NTKLDNNWGREERQS (SEQ ID NO: 1) or 2) NTKLDNNWGREERQSVFPF (SEQ ID NO: 2) as shown.

The second aspect of the invention provides a Galectin-3 antigen, which is prepared by coupling the Galectin-3 antigen epitope peptide with a carrier protein.

The antigen can be obtained by constructing an expression vector containing the epitope peptide and the nucleotide corresponding to the carrier protein, and then transfecting the expression vector into a host cell for expression; the above-mentioned SEQ ID NO:1 or SEQ ID NO:2, and coupling the Galectin-3 epitope peptide with a carrier protein.

Preferably, the carrier protein is any one of OVA (ovalbumin), BSA (bovine serum albumin), KLH (cyankey).

Preferably, the epitope peptide and the carrier protein are coupled by the SMCC method.

The Galectin-3 antigen can be used as an immunogen to prepare an anti-human Galectin-3 antibody.

The third aspect of the invention provides an anti-human Galectin-3 antibody, which is a monoclonal antibody or a polyclonal antibody prepared by taking the Galectin-3 antigen as an immunogen. The preparation method can adopt the conventional technology in the field.

The fourth aspect of the invention provides a hybridoma cell strain of a mouse anti-human Galectin-3 monoclonal antibody, wherein the hybridoma cell strain is as follows: the hybridoma cell line Galectin-3 (Clone: C6C10), deposited at: china center for type culture Collection, accession number: CCTCC NO: C2020135, preservation date: 29/7/2020. A female BALB/c mouse with 6-8 weeks of age is immunized by the Galectin-3 antigen, spleen cells of the mouse are taken to be fused with SP2/0 cells, monoclonal cells are obtained by a limiting dilution method, and positive hybridoma cells are screened by an indirect ELISA method to obtain a hybridoma cell line capable of stably secreting the anti-Galectin-3 specific antibody.

The fifth aspect of the invention provides a preparation method of a mouse anti-human Galectin-3 monoclonal antibody, which comprises the steps of inoculating the anti-human Galectin-3 monoclonal antibody hybridoma cell strain to an abdominal cavity of a mouse, collecting ascites, and purifying by a Protein A column to obtain the Galectin-3 monoclonal antibody.

The mouse anti-human Galectin-3 monoclonal antibody can be used for preparing a Galectin-3 in-vitro immunoassay reagent or kit.

The sixth aspect of the invention provides a Galectin-3 immunohistochemical detection reagent and a kit, wherein the reagent comprises a mouse anti-human Galectin-3 monoclonal antibody or a polyclonal antibody, and preferably also comprises BSA protein, PBS buffer solution and Proclin-300 preservative components. The kit comprises a hydrogen peroxide blocking agent, a primary antibody, a peroxidase-labeled goat anti-mouse and goat anti-rabbit secondary antibody, a DAB substrate, a DAB buffer solution and a hematoxylin staining solution, wherein the primary antibody comprises a mouse anti-human Galectin-3 monoclonal antibody or a polyclonal antibody. The reagent and the kit can be used for in vitro immunodetection of the expression condition of Galectin-3 in tissue cells.

The invention has the beneficial effects that:

1. the Galectin-3 epitope peptide provided by the invention takes a Galectin-3 in-vitro immunodetection antibody as a research and development target, experiments verify that the Galectin-3 epitope peptide can be used for preparing a Galectin-3 antigen at an IHC level, the Galectin-3 antigen has good antigenicity, and an animal immunized by the antigen (immunogen) prepared by the Galectin-3 epitope peptide can generate the Galectin-3 antibody with high specificity and sensitivity.

2. The invention provides a hybridoma cell strain capable of stably secreting a mouse anti-human Galectin-3 monoclonal antibody, which comprises the following steps: the hybridoma cell line Galectin-3 (Clone: C6C10), accession number: CCTCC NO: C2020135, preservation date: 29/7/2020, depository: china center for type culture Collection, collection address: the preservation center of Wuhan university in Wuchang Lodojia mountain, Hubei province, is prepared by using NTKLDNNWGREERQS, NTKLDNNWGREERQSVFPF coupled KLH protein as antigen through an immunological method; the hybridoma cell strain can secrete a mouse anti-human Galectin-3 monoclonal antibody with strong specificity and high sensitivity, and has high expression quantity and good expression stability.

3. The mouse anti-human Galectin-3 monoclonal antibody provided by the invention can be used for in vitro immunodetection of the Galectin-3 expression condition of isolated histiocytes, has the characteristics of strong specificity, high sensitivity and good accuracy, has stronger specificity than imported antibodies compared with expensive imported antibodies, is cheap, can reduce the burden of patients and reduce the diagnosis cost.

Drawings

FIG. 1 is a graph showing the results of IHC assay of mouse sera after immunization with Galectin-3 antigen;

FIG. 2 is a graph of the IHC results of control antibody EP 412;

FIG. 3 is a graph comparing the results of the chip of the C6C10 monoclonal antibody secreted by the Galectin-3 cell line with that of the control antibody EP 412;

FIG. 4 is a graph showing the expression of Galectin-3 in thyroid follicular gonadal cancer tissues detected by murine anti-human Galectin-3 monoclonal antibody as a primary antibody against immunohistochemistry;

FIG. 5 is a graph showing the detection of Galectin-3 expression in thyroid follicular gonadal cancer tissue using control antibody EP412 as an anti-immunohistochemical antibody;

FIG. 6 is a graph showing that a murine anti-human Galectin-3 monoclonal antibody is used as a primary antibody for immunohistochemical detection of Galectin-3 expression in colon cancer tissues;

FIG. 7 is a graph showing the detection of Galectin-3 expression in colon cancer tissues by immunohistochemistry using the control antibody EP412 as an anti-antibody;

FIG. 8 is a graph showing the results of immunohistochemical detection of Galectin-3 expression in lung cancer tissues using a mouse anti-human Galectin-3 monoclonal antibody as a primary antibody;

FIG. 9 is a graph showing the results of immunohistochemical detection of Galectin-3 expression in lung cancer tissues using the control antibody EP412 as an anti-antibody;

FIG. 10 is a graph showing the results of immunohistochemical detection of Galectin-3 expression in thyroid tissue using a mouse anti-human Galectin-3 monoclonal antibody as a primary antibody;

FIG. 11 is a graph showing the results of measuring Galectin-3 expression in thyroid tissue using the control antibody EP412 as an anti-immunohistochemical antibody.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto:

example 1

The Galectin-3 epitope peptide in this example has the amino acid sequence NTKLDNNWGREERQS (SEQ ID NO: 1), which is the core polypeptide sequence selected from the protein sequence P17931 published by Uniprot with the amino acid sequence position 174-188 as the epitope.

Example 2

The Galectin-3 epitope peptide in this example has the amino acid sequence NTKLDNNWGREERQSVFPF (SEQ ID NO: 2), which is the core polypeptide sequence with the amino acid sequence position 174-192 as the epitope selected according to the P17931 protein sequence published by Uniprot.

Example 3

The Galectin-3 antigen in the embodiment is prepared by artificially synthesizing the Galectin-3 epitope peptide in the embodiment 1 and coupling the synthesized Galectin-3 epitope peptide with a carrier protein KLH.

1. Artificial synthesis of epitope peptide: synthesized by the amino acid direct synthesis method by Nanjing Kinshire biology company.

2. Coupling of epitope peptide with carrier protein: KLH-polypeptide was synthesized in two steps by SMCC (succinimide-4- (N-maleimide) cyclohexane-1-carboxylate). The main reaction process comprises the following steps: 1) KLH with amino groups was reacted with several times the coupling reagent and after completion the unreacted SMCC was removed by desalting column or dialysis. 2) Then reacting with polypeptide protein containing sulfhydryl. KLH protein was from Nanjing Kinshire; SMCC, DMF was purchased from Sigma.

The method comprises the following steps:

1) 20mg of SMCC was dissolved in 2ml of DMF.

2) 0.8ml of KLH was added to a 25ml round bottom flask and supplemented with 1 XPBS (pH 7.2) to give a final protein concentration of 15 mg/ml.

3) The dissolved SMCC solution is slowly added into a 120mg KLH protein system in a dropwise manner, and the reaction is stirred at room temperature for 1 h.

4) Free SMCC was removed by dialysis against 1L of 1 XPBS (pH 7.4) at 4 ℃ for 6 hours.

5) The dialyzed KLH protein was poured into a 50ml centrifuge tube, the volume thereof was determined by the scale of the centrifuge tube, the concentration of the dialyzed protein was calculated from the amount of KLH protein added before the reaction, and then 2.5mg of KLH-SMCC solution was transferred into a 5ml centrifuge tube according to the concentration thereof.

6) The amount of KLH protein added was 120mg and the volume of KLH protein after dialysis was 20 ml.

7) 3.0mg of polypeptide was dissolved in 0.6ml of 1 XPBS (pH 7.2).

8) Thiol groups in polypeptides were detected with Ellman's reagent: adding 100 μ l Ellman reagent stock solution into 96-well plate, adding 10 μ l polypeptide solution, measuring its ultraviolet absorption value with Nano spectrophotometer at λ 412nm, and performing the next step if OD value is greater than 0.15; the OD value is less than 0.15 and more than 0.05, and the polypeptide is added until the requirement is met; and the OD value is less than 0.05, and the polypeptide is returned to the polypeptide synthesis step for quality control again.

9) The polypeptide is dripped into a KLH-SMCC tube and is mixed uniformly by a vertical mixer at room temperature for 4 hours.

10) Thiol groups in polypeptides were detected with Ellman's reagent: mu.l of Ellman reagent stock solution was added to a 96-well plate, 10. mu.l of the trained polypeptide solution was added, and the UV absorbance was measured at λ 412nm using a Nano spectrophotometer. The OD value is less than 0.03, which indicates that the crosslinking rate of the polypeptide and the KLH protein reaches more than 80 percent; OD >0.03 and then adding activated KLH protein of SMCC to continue crosslinking. If the Ellman reagent turns yellow, the coupling of the polypeptide to the KLH protein is incomplete; if the Ellman reagent does not appear yellow, it indicates that the polypeptide has been fully conjugated to the KLH protein.

Example 4

The Galectin-3 antigen in the embodiment is prepared by artificially synthesizing the Galectin-3 epitope peptide in the embodiment 2 and coupling the Galectin-3 epitope peptide with a carrier protein KLH, and the specific steps are the same as those in the embodiment 3.

Example 5

The Galectin-3 polyclonal antibody in the present example was prepared by a method comprising the steps of:

galectin-3 antigen was prepared according to example 3 or example 4, and the prepared antigen was diluted to a concentration of 2mg/mL by PBS, rapidly mixed with 50 μ L of water-soluble adjuvant at a dose of 100 μ g (i.e., 50 μ L), and then injected into the leg muscle of mice. Immunizing two needles on days 0 and 21 respectively, collecting tail blood of mice on day 35, centrifuging at room temperature at 8000rpm for 1min, and collecting supernatant at-20 deg.C for storage to obtain polyclonal antibody.

Example 6

the antigen prepared in example 3 and example 4 was diluted to a concentration of 2mg/mL with PBS, and mixed rapidly with 50. mu.L of water-soluble adjuvant at a dose of 50. mu.g + 50. mu.g protein (i.e., 25. mu.L + 25. mu.L), and injected into the leg muscle of mice. Immunizing two needles on days 0 and 21 respectively, collecting tail blood of mice on day 35, centrifuging at room temperature at 8000rpm for 1min, and collecting supernatant at-20 deg.C for storage to obtain polyclonal antibody.

Example 7

The hybridoma cell line Galcetin-3 (Clone: C6C10) in the embodiment is prepared by the method comprising the following steps:

1. animal immunization: the antigens prepared in example 3 and example 4 were diluted to a concentration of 2mg/mL with PBS, and 50. mu.g, i.e., 50. mu.g + 50. mu.g of each of the antigens prepared in example 3 and example 4 (i.e., 25. mu.L + 25. mu.L) were rapidly mixed with 50. mu.L of a water-soluble adjuvant to serve as immunogens. A BALB/c mouse of 6-8 weeks old is immunized by an intramuscular injection method, the immunization dose is 40 mu g/mouse, and the second immunization is carried out after three weeks, and the immunization dose is the same as the first immunization dose. After two times of immunization, tail blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer.

2. And (3) measuring the titer:

the detection method comprises the following steps:

1. antigen was coated by PB buffer to ELISA assay plates in an amount of 200 ng/well. Standing overnight at the temperature of 2-8 ℃;

2. discarding the coating solution, adding 150 μ L of sealing solution into each well, and sealing at 37 deg.C for 2 hr;

3. removing the blocking solution, adding 100 mu L of tail blood diluted in a gradient way, wherein the dilution ratio of the tail blood is respectively as follows: 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, 1: 128000, repeating 3 holes for each dilution ratio;

incubating the 96-well plate added with the diluted tail blood for 1h at the temperature of 37 ℃;

4. washing the plate for 5 times, adding 100 mu L of goat anti-mouse HRP labeled secondary antibody diluted by 1: 5000 into each hole, and incubating for 1h at 37 ℃;

5. the plate was washed 5 times, and 50. mu.L each of the developing solution A, B was added. Incubating at 37 deg.C for 10 min;

6. stop solution 50. mu.L was added and read under an enzyme plate.

The detection result is as follows:

	mouse 1	Mouse 1	Mouse 1	Mouse 2	Mouse 2	Mouse 2	Mouse 3	Mouse 3	Mouse 3
										1/2000	2.145	2.233	2.219	2.112	2.106	2.133	2.234	2.251	2.246
1/4000	1.83	1.863	1.852	1.644	1.606	1.639	1.904	1.885	1.894
										1/8000	1.223	1.246	1.206	1.121	1.195	1.173	1.333	1.362	1.394
1/16000	0.783	0.754	0.773	0.431	0.459	0.472	0.803	0.833	0.812
										1/32000	0.311	0.344	0.375	0.114	0.121	0.109	0.541	0.533	0.523
1/64000	0.104	0.098	0.085	0.032	0.033	0.031	0.334	0.352	0.341
										1/128000	0.028	0.026	0.026	0.026	0.027	0.026	0.121	0.126	0.119
Negative control	0.026	0.028	0.027	0.027	0.027	0.027	0.026	0.027	0.026

As can be seen from the above table, the titer level of mouse 3 reached 10⁶And performing impact immunization and cell fusion operation with the highest overall titer level.

2. Cell fusion: the myeloma cells adopt BALB/c-derived sp2/0, and are in logarithmic growth phase when fused; taking the spleen of an immunized mouse, and preparing a lymphocyte single cell suspension; mixing mouse spleen lymphocyte and myeloma cell at 1: 5, adding 1mL 50% PEG (PH8.0) at 37 deg.C, adding incomplete culture medium and the rest stop solution, centrifuging, removing supernatant, adding HAT culture medium, suspending, mixing, adding 50mL to desired volume, adding into 96-well cell culture plate, placing at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator.

3. Screening and cloning: cell clones were selected within 7-10 days of fusion and tested by indirect ELISA using Galectin-3 polypeptide, as follows:

1) performing overnight incubation at the temperature of 2-8 ℃ according to the concentration of 100 ng/hole by using Galectin-3 polypeptide antigen (coupled KLH) as an antigen for coating and TBS buffer solution as a coating solution, wherein the volume of supernatant in each hole is 100 mu L;

2) discarding the supernatant on the second day, adding 150 μ L of blocking solution into each well, and incubating for 2h in an incubator at 37 ℃;

3) discarding the supernatant, adding 100 μ L of cell fusion supernatant into each well, incubating at 37 deg.C for 30min, and washing the plate 5 times with a plate washing machine;

4) adding the components according to the proportion of 1: the goat anti-mouse secondary antibody is labeled with 5000-diluted horseradish peroxidase, each well is 100 mu L, the goat anti-mouse secondary antibody is incubated for 30min at the temperature of 37 ℃, and then a plate washing machine is used for washing the plate for 5 times each time;

5) sequentially adding 50 mu L of color development liquid A and 50 mu L of color development liquid B, and incubating for 3-5 min at 37 ℃. After color development, adding 50 mu L of stop solution for stopping;

6) reading by a microplate reader.

Limiting dilution is carried out on positive hole cells, ELISA values are measured 5-6 days after each limiting dilution, and OD is selected₄₅₀And (4) carrying out limited dilution on the monoclonal wells with higher positive values until the whole plate result of the 96-well plate is positive by ELISA (enzyme-Linked immunosorbent assay). And selecting a monoclonal antibody with a high positive value to determine a cell strain.

And finally selecting a cell strain C6C10 which has stable antibody secretion and better cell strain state through 3 times of subclone screening, and preserving the obtained Galectin-3 cell strain, wherein the hybridoma cell strain is as follows: the hybridoma cell line Galectin-3 (Clone: C6C10), deposited at: china center for type culture Collection, collection address: the preservation center of Wuhan university in Wuchang Lodojia mountain in Wuhan city, Hubei province has the preservation number: CCTCC NO: C2020135, preservation date: 29/7/2020.

Example 8

The mouse anti-human Galectin-3 monoclonal antibody in the embodiment is prepared by adopting a mouse abdominal cavity induction method, and comprises the following steps:

8-10 week old BALB/C mice were intraperitoneally injected with 0.5ml liquid paraffin, and one week later each mouse was intraperitoneally injected with 1ml syringe with Galectin-3 (Clone: C6C10) suspension resuspended by PBS washing, and the cell usage was 5X 10⁶One/only. Collecting ascites after ascites of the mouse accumulates, centrifuging to take supernatant, purifying the ascites by Protein A column chromatography, measuring the concentration of the purified monoclonal antibody, subpackaging, and freezing to store at-20 ℃. The concentration of the prepared mouse anti-human Galectin-3 monoclonal antibody is 3.4mg/mL by measuring with an ultraviolet spectrophotometer method.

Purification conditions of the ProteinA chromatographic column:

1. first, the column was equilibrated with PBS;

2. diluting ascites with PBS (phosphate buffer solution) in a volume ratio of 9: 1, and loading the diluted ascites into a chromatographic column;

3. after the sample loading is finished, the column is balanced by PBS;

4. washing the chromatographic column with citric acid eluent of pH3.0, collecting the antibody at the peak in Tris buffer solution containing pH8.0, and neutralizing the antibody damage under acid condition;

5. then, the antibody buffer system is replaced by a desalting column, and the obtained antibody is collected after centrifugal concentration.

Example 9

The embodiment is an immunohistochemical reagent for in vitro detection of human Galectin-3, the reagent comprises a mouse anti-human Galectin-3 monoclonal antibody or polyclonal antibody, and also comprises BSA protein, PBS buffer solution and Proclin-300 preservative components, and the reagent can be used for detecting the expression condition of the Galectin-3 in isolated tissue cells.

Example 10

The embodiment is an immunohistochemical kit for detecting human Galectin-3, which comprises a hydrogen peroxide blocking agent, a primary antibody, a peroxidase-labeled goat anti-mouse and goat anti-rabbit secondary antibody, a DAB substrate, a DAB buffer solution and a hematoxylin staining solution, wherein the primary antibody comprises a mouse anti-human Galectin-3 monoclonal antibody or a polyclonal antibody.

Test example 1

In this test example, the antigen specificity of the antigen epitope peptide was verified at the IHC level by the following steps:

1. the paraffin tissue is sliced, the thickness of the slice is 3 mu m, and the slice is baked for 2 hours at the temperature of 65 ℃.

2. Performing gradient dewaxing according to xylene 15min, anhydrous ethanol 5min, 95% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min, and 50% ethanol 5 min.

3. The dewaxed slices were soaked in purified water for 5 min.

4. The slices were placed in ETDA repair solution of pH9.0, and repaired under high pressure for 3 min.

5. After natural cooling, the mixture is soaked in PBST washing liquor for 5 min.

6. Adding hydrogen peroxide sealant, and sealing at room temperature for 5 min.

7. The primary antibodies were added, respectively, as a positive control with the control antibody EP412, at 37 ℃ for 1 h. The primary antibody is the Galectin-3 polyclonal antibody prepared in example 6 (obtained by immunizing a mouse with 50. mu.g + 50. mu.g of protein, which is a fusion protein obtained by coupling epitope peptides shown in SEQ ID NO:1 and SEQ ID NO:2 with a carrier protein KLH, respectively, as an immunogen).

8. PBST washing solution was washed 2 times, and each soaking time was 5 min.

9. Adding secondary antibody, and keeping at 37 deg.C for 30 min.

10. PBST washing solution was washed 2 times, and each soaking time was 5 min.

11. Adding DAB color development solution, and incubating at room temperature for 10 min.

12. Washing with pure water for 2 times, and soaking for 5min each time.

13. And adding hematoxylin for counterstaining for 5 min. And (5) washing with pure water.

14. Processing with 50% ethanol for 2min, 70% ethanol for 2min, 80% ethanol for 2min, 95% ethanol for 2min, anhydrous ethanol I, II for 2min, and xylene I, II for 2min, and sealing with neutral gum.

15. And (5) reading the film and taking a picture.

The fusion protein obtained by coupling the epitope peptides shown in SEQ ID NO. 1 and SEQ ID NO. 2 with the carrier protein KLH respectively is used as immunogen, the mice are immunized according to the total amount of 50 mug +50 mug protein, the serum IHC detection result of the immunized mice is shown in figure 1, and the IHC result of the control antibody EP412 is shown in figure 2. As can be seen from the figure, when a polyclonal antibody prepared by coupling the antigen epitope peptides shown by SEQ ID NO. 1 and SEQ ID NO. 2 with a carrier protein as an immunogen is used as an IHC primary antibody reagent, the specificity is stronger than that of a control antibody EP412, so that the Galectin-3 antigen epitope peptides (SEQ ID NO. 1 and SEQ ID NO. 2) provided by the application can be used for preparing the Galectin-3 antigen and have good antigenicity, and animals immunized by the antigen (immunogen) prepared by the polyclonal antibody can generate the Galectin-3 antibody with high specificity and sensitivity.

Test example 2

In this test example, the specificity of the mouse anti-human Galectin-3 monoclonal antibody secreted from the hybridoma cell line Galectin-3 (Clone: C6C10) of example 7 was identified on the IHC layer using a tissue chip, and an imported antibody was used as a control (FIG. 3).

The control clone number of Galectin-3 is EP412, and the manufacturer is: cellmarqe

The IHC procedure is the same as that of test example 1, wherein the primary antibody is a mouse anti-human Galectin-3 monoclonal antibody secreted by hybridoma cell line Galectin-3 (Clone: C6C10) or a control imported antibody EP 412.

The organisational chip layout is as follows:

the statistics of the IHC detection result of the chip are as follows:

	control antibody	C6C10
			Number of positive cases	45	45
Number of negative cases	29	29

At the level of both IHCs, the results of the specificity comparisons (using tissue chips) were as follows: the positive positioning consistency rate reaches 100 percent in 74 tissues in total, and the accuracy is excellent. As can be seen from fig. 4 and 5, during staining of papillary thyroid carcinoma tissue, the mass staining and nuclear staining of self-developed antibody C6C10 in follicular staining were more clear than those of control antibody EP412, and the nuclear staining was more intense than that of the control antibody, and the cells were confirmed to have no staining in the periphery. As can be seen from FIGS. 6 and 7, the staining effect of the self-developed antibody C6C10 in the colon cancer tissue was consistent with that of the control EP412 in the colon cancer tissue staining process. The result of comprehensive analysis on tissue chip staining shows that the specificity of the mouse-anti-human Galectin-3 monoclonal antibody secreted by the hybridoma cell strain Galectin-3 (Clone: C6C10) is higher than that of the imported antibody.

Test example 3

In the test example, the immunohistochemical detection of the mouse anti-human Galectin-3 monoclonal antibody secreted by the hybridoma cell strain Galectin-3 (Clone: C6C10) as a primary antibody reagent comprises the following steps:

1. respectively taking wax blocks of thyroid and lung cancer tissues, slicing by using a Leica tissue slicer, wherein the tissue thickness is 3-4 mu m, and drying and baking for 2h at 65 ℃.

2. The antibody of the invention is subjected to immunohistochemical staining test by using a Leica Bond Max immunohistochemical automatic staining machine, and dewaxing and hydration conditions carried by the machine are used, and the specific steps are as follows: incubate at 60 ℃ for 30min and wash 3 times with deparaffinized solution (Leica). Antigen retrieval was performed by incubating antigen retrieval solution 2(ER2, Leica) at 100 ℃ for 30 min. A mouse anti-human Galectin-3 monoclonal antibody secreted by hybridoma cell line Galectin-3 (Clone: C6C10) or a control antibody EP412 was diluted to a final concentration of 0.5. mu.g/ml and 150. mu.l with an antibody diluent (Leica). The antibody was incubated at room temperature for 30 min. A secondary antibody (Leica) was used in an amount of 150. mu.l and incubated at room temperature for 8 min. Mu.l of polymer detection solution (Leica) was used and incubated for 8min at room temperature. Endogenous peroxidase blocking solution was used and incubated at 150. mu.l for 5min at room temperature. 150 μ l of DAB staining solution (Leica) was used and incubated at room temperature for 10 min. Hematoxylin counterstain, incubate for 5min at room temperature.

3. Dehydration and transparency: cleaning with deionized water for 3 min; ethanol 85% for 1 min; 95% ethanol for 1 min; 100% ethanol for 2min,2 times; xylene for 2min,3 times; and (5) sealing the neutral gum.

4. And (6) microscopic examination.

FIGS. 8 and 10 are graphs showing the results of immunohistochemical detection of Galectin-3 expression in lung cancer tissue and thyroid tissue using a mouse anti-human Galectin-3 monoclonal antibody as a primary antibody, respectively; FIGS. 9 and 11 are graphs showing the results of immunohistochemical detection of Galectin-3 expression in lung cancer tissue and thyroid tissue using the control antibody EP412 as an anti-antibody, respectively, and FIGS. 8 to 11 show that intracellular Galectin-3 protein is localized in cell membrane and cytoplasm, exhibits specific expression in follicular thyroid carcinoma and papillary carcinoma, and has stronger specificity and sensitivity than those of the imported antibody.

SEQUENCE LISTING

<110> Henan Sainur Biotechnology Ltd

<120> Galectin-3 epitope peptide, antigen, antibody, hybridoma cell strain and kit

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<221> Galectin-3 epitope peptide 1

<400> 1

Asn Thr Lys Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln Ser

1 5 10 15

<210> 2

<211> 19

<212> PRT

<213> Artificial sequence

<221> Galectin-3 epitope peptide 2

<400> 2

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

1 5 10 15

Phe Pro Phe

Claims

1. A Galectin-3 epitope peptide is characterized in that the amino acid sequence of the epitope peptide is shown as SEQ ID NO:1 or SEQ ID NO:2, respectively.

2. A Galectin-3 antigen, which is obtained by coupling the Galectin-3 epitope peptide of claim 1 to a carrier protein.

3. The Galectin-3 antigen of claim 2, wherein the carrier protein is any one of OVA, BSA and KLH.

4. Use of the Galectin-3 antigen of claim 2 or 3 as an immunogen in the preparation of an anti-Galectin-3 antibody.

5. An anti-human Galectin-3 antibody, which is a monoclonal antibody or a polyclonal antibody produced from the Galectin-3 antigen of claim 2 or 3 as an immunogen.

6. A mouse anti-human Galectin-3 monoclonal antibody hybridoma cell strain is characterized in that: the hybridoma cell line Galectin-3 (Clone: C6C10), accession number: CCTCC NO: C2020135.

7. The anti-human Galectin-3 antibody of claim 5, wherein the antibody is a monoclonal antibody produced from the hybridoma cell line of claim 6.

8. The method of claim 7, comprising the steps of: the monoclonal antibody hybridoma cell line for human Galectin-3 according to claim 6, which is obtained by collecting ascites and purifying the collected ascites.

9. The use of the anti-human Galectin-3 antibody of claim 5 or 7 in the preparation of a Galectin-3 in vitro immunoassay reagent or kit.

10. A Galectin-3 immunohistochemical detection reagent, wherein the reagent comprises the anti-human Galectin-3 antibody according to claim 5 or 7.