CN108912229B

CN108912229B - anti-Bcl 6 protein monoclonal antibody, cell strain thereof, preparation method and application

Info

Publication number: CN108912229B
Application number: CN201810960674.4A
Authority: CN
Inventors: 杨清海; 张丹萍; 贾坤志; 陈惠玲; 谢成杰
Original assignee: Fuzhou Maixin Biotechnology Development Co ltd
Current assignee: FUZHOU MAIXIN BIOTECHNOLOGY DEVELOPMENT Co.,Ltd.
Priority date: 2018-08-22
Filing date: 2018-08-22
Publication date: 2021-07-16
Anticipated expiration: 2038-08-22
Also published as: CN108912229A

Abstract

The invention provides an anti-Bcl 6 antibody, a secretory cell strain thereof, a preparation method and application. The invention selects the protein coded by the nucleotide sequence shown in SEQID1 as immunogen, and the immunogen is recombined and expressed by escherichia coli. The recombinant protein is used for immunizing BALB/c mice for multiple times. Cell fusion, screening and subcloning are carried out to obtain a monoclonal cell line 1E6A4H10C 9. The antibody secreted by 1E6A4H10C9 is IgG_2aThe subtype is. The antibody can specifically detect Bcl6 protein in tissues.

Description

anti-Bcl 6 protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-Bcl 6 protein monoclonal antibody, a cell strain, a preparation method and application thereof.

Background

Bcl6, also known as LAZ-3, is a member of the POZ/zinc finger (POK) family, which contains an N-terminal POZ domain and 6C-terminal zinc finger motifs. Bcl6, also known as LAZ-3, is a member of the POZ/zinc finger (POK) family, which contains an N-terminal POZ domain and 6C-terminal zinc finger motifs, a zinc finger transcription factor encoded by a proto-oncogene located on chromosome III. Bcl6 is a transcription regulator that plays an important role in the survival and differentiation of lymphocytes, myocytes, male germ cells, and keratinocytes. Mainly expressed in normal germinal center B cells and associated lymphomas. Expression of Bcl6 was detectable in follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma.

In normal lymphoid tissues, Bcl6 is expressed predominantly in germinal center cells, and its expression is gradually down-regulated after B cells undergo germinal center maturation, not in memory B cells or plasma cells, nor in mantle zone cells, marginal zone cells, and myeloid precursor B cells. Expression of Bcl6 protein in tissues is considered to be one of the molecular markers of germinal center B cells, which is also expressed in lymphomas of germinal center B cell origin. Bcl6 is used as transcription inhibitor of various genes, and has important functions in the formation of germinal center, the regulation and the survival of different tumors. The reduction of the expression level of the protein by transcription and mutation has been confirmed during the formation of tumors. Relevant clinical studies indicate that the expression level of Bcl6 is closely related to the prognosis of diffuse large B-cell lymphoma DLBCL.

Diffuse large B-cell lymphoma DLBCL is the most common, and highly malignant non-Hodgkin's lymphoma (NHL) has found relief in only 40% -50% of patients on chemotherapy for malignancies. DLBCL has occult disease, most of the DLBCL is mainly clinically manifested by painless progressive enlargement of lymph nodes (most commonly, cervical lymph nodes, supraclavicular lymph node, axillary lymph nodes) and corresponding compression symptoms caused by the compression of the enlarged lymph nodes can also appear in lymph nodes of thoracic and abdominal organs (liver, spleen, gastrointestinal tract, thyroid gland, tonsil and the like). Therefore, the preparation of the Bcl6 antibody for IHC detection has important significance for the prediction of disease development and the formulation of individualized treatment strategies in the treatment process of patients.

Disclosure of Invention

Therefore, it is necessary to provide a specific and sensitive anti-Bcl 6 protein monoclonal antibody suitable for immunological detection, particularly immunohistochemical detection.

In order to achieve the aim, the inventor provides an anti-Bcl 6 protein monoclonal antibody which is produced by a hybridoma cell line with the preservation number of CGMCC NO. 16210. The cell strain is a mouse hybridoma cell line 1E6A4H10C9, and is classified and named as: a mouse hybridoma cell line which has been deposited at 9.1.2017 in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and which is located at the institute of microbiology, China academy of sciences, No. 3, West Lu 1 Hospital, North Kyoto Kogyo, Beijing, and the like.

Further, the monoclonal antibody specifically recognizes Bcl6 protein.

Further, the monoclonal antibody specifically recognizes the amino acid sequence encoded by seq id1 in the Bcl6 protein.

Further, the monoclonal antibody is mouse IgG_2aThe subtype is.

The invention also provides a preparation method of the anti-Bcl 6 protein monoclonal antibody, which is characterized in that the preparation method selects the protein coded by the nucleotide sequence shown by SEQID1 as immunogen, and the immunogen is recombined and expressed by escherichia coli.

The inventor also provides a hybridoma cell strain secreting anti-Bcl 6 protein molecules, wherein the cell strain is a mouse hybridoma cell line 1E6A4H10C9, and is classified and named as: mouse hybridoma cell line. The cell line is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms at 9.1.2017, and the accession number is CGMCC NO.16210, at the institute of microbiology, China academy of sciences, No. 3, West Lu 1, North Chen of the Korean district, Beijing.

The inventor further provides the application of the anti-Bcl 6 protein monoclonal antibody in the Bcl6 protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

Different from the prior art, the Bcl6 molecule is analyzed according to published sequences in the technical scheme, and regions which are suitable for soluble expression and have good immunogenicity are selected for recombinant expression according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure on cell membranes.

Meanwhile, in order to promote the soluble expression of the recombinant protein and optimize the expression behavior, the segments are subjected to codon optimization, are cloned into an expression vector pET-28a after gene synthesis for recombinant expression, and are used as immunogen after the target protein is purified, so that BALB/c mice are immunized for multiple times. Through cell fusion, screening and subcloning, a monoclonal cell line 1E6A4H10C9 capable of efficiently secreting the anti-Bcl 6 antibody and the Bcl6 antibody secreted by the cell line are obtained. The Bcl6 antibody has high specificity and sensitivity, and can specifically recognize cells expressing Bcl 6. The hybridoma cell line is used for preparing ascites by a mouse, and the ascites is purified by protein G column affinity chromatography to obtain a mouse monoclonal antibody. The subclass of the monoclonal antibody is IgG by ELISA technology_2aMonoclonal antibody with affinity constant of 5.12X 10¹⁰L/mol. Immunohistochemical experiments show that the antibody can specifically recognize tumor cells expressing Bcl6 molecules.

Drawings

FIG. 1 is a graph showing the result of electrophoresis of purified Bcl6 recombinant antigen protein; wherein M is a protein marker, 1 is an unloaded expression product, 2 is an unloaded IPTG expression product, 3 is target protein expression, 4 is a target protein and

IPTG expression product

5,6 is an expression product elution result of 200mM imidazole, the size of the expression product is about 40kDa, and 10% SDS PAGE gel is used. The purified recombinant Bcl6 protein was soluble and could partially retain its native conformation at a concentration of 0.34 mg/mL.

FIG. 2 is a graph showing the results of electrophoresis and immunoblotting of Bcl6-His target protein; m is a protein marker, 1 is purified Bcl6-His target protein, 2 is bovine serum albumin as a negative control, and 3 and 4 are: the result is the Western Blot immunoblotting result corresponding to 1, 2.

FIG. 3 is a graph showing the results of staining diffuse large B-cell lymphoma; the left side is 1E6A4H10C9 antibody (abbreviated as 1E6A4), and the right side is control antibody LN 22.

FIG. 4 is a graph showing the result of immunohistochemical staining of tonsil tissues; the left side is 1E6A4H10C9 antibody (abbreviated as 1E6A4), the middle is control antibody LN22, and the right side is negative control.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

Example 1 preparation of recombinant Bcl6 protein fragment

First, gene optimization and synthesis

Bcl6 was directly optimized into a gene fragment suitable for expression in E.coli BL21(DE3) by selecting a protein fragment of N-350 amino acids according to the protein sequence with accession number P41182 in the Uniprot database. EcoRI and HindIII sites were added to the 5 'and 3' ends of the gene during PCR, respectively.

And separating and recovering the PCR product through agarose gel electrophoresis, performing enzyme digestion on the recovered fusion protein gene and a plasmid vector pET-28a for expression by EcoRI and Hind III, performing electrophoresis recovery again, connecting by T4DNA ligase, transforming escherichia coli competent cells BL21(DE3) by the connecting product, selecting clone on a plate for inoculation, and performing bacteria liquid PCR identification. And selecting clones with positive PCR results for sequencing analysis, and using the clones with completely correct sequences.

The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. The Bcl6 molecules were analyzed according to published sequences, based on structure on cell membrane, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure, selecting regions suitable for soluble expression and good immunogenicity for recombinant expression, and N-350 amino acids of Bcl6 were selected for codon optimization with a molecular weight of about 40 kDa. The recombinant immunogen is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of antigenic Bcl6 protein fragments and protein tags for recombinant protein purification, and the protein tags consist of 5 to 7 histidines.

II, protein expression and purification

The overnight bacteria cultured in the single colony was transferred to 100mL LB medium at a ratio of 1:100, kanamycin was added to a final concentration of 10. mu.g/mL, and shaking culture was carried out at 37 ℃ until OD₆₀₀Adding IPTG (1 mmol/L) in the culture medium at 0.6-0.8, performing shake culture at 16 ℃ overnight, collecting bacteria, and performing ultrasonic disruption. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Eluting with 200mmol/L imidazole, and separating and detecting SDSPAGE, wherein FIG. 1 shows the purification result of histidine-tag fused recombinant Bcl6 protein, the protein concentration is 0.34mg/mL, and the method can be used for animal immunization and antibody screening and identification.

EXAMPLE 2 establishment of hybridoma cell line 1E6A4H10C9

Immunization

The fusion protein purified in example 1 and a rapid adjuvant (Beijing Boolong company) were mixed as required by the specification, and female BALB/c mice (purchased from Wu's laboratory animal center, Shanghai) of about 6 weeks old were immunized and injected into the hind leg muscle of the mice at a dose of 20. mu.g/mouse. Day 21, following the same protocol, immunization 1 needle was placed or boosted. On day 35, the titer of the polyclonal antibody against the immunogen in the mouse serum was measured by ELISA (wavelength 450nm), the highest titer of the mice was subjected to intraperitoneal injection for impact immunization, and the antigen was mixed with physiological saline at a dose of 50. mu.g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, and is mixed with mouse myeloma cell SP2/0 (Shanghai cell bank of Chinese academy of sciences) at a ratio of 5:1 and centrifuged at 1500r/min for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1mL of PEG1450 (Sigma) was added slowly over 1 minute, and the cells were agitated. Standing in warm water for 1min, adding 10mL serum-free RPMI-1640 mediumMixing, and centrifuging at 1000r/min for 5 min. Discarding supernatant, adding 10mL HAT culture medium, carefully beating cells, mixing well, spreading on 96-well plate, adding 5% CO at 37 deg.C₂Culturing in an incubator.

Third, ELISA screening positive hybridoma cell

After 10 days, 100. mu.L of the supernatant was screened by ELISA using the purified fusion protein. Selecting positive clones with high titer, small quantity and good state for subcloning. After 7 days a second ELISA screening was performed and the positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 mu L of supernatant is taken for the third ELISA screening, and positive clones are successively transferred into a 6-well plate and a cell culture bottle for amplification culture and frozen storage.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted 1X 10⁵0.5 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid was centrifuged at 12000r/min at 4 ℃ for 30 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid using Protein G (Nanjing Kinshire) affinity chromatography as described. The purity was assessed on SDS-PAGE gels (see FIG. 2) and the concentration was determined by BCA method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

Diluting 6 different subclass identification reagents (IgA, IgM, IgG) with 0.05M coating solution (pH 9.6) at a ratio of 1:1000₁、IgG_2a、IgG_2b、IgG₃Add 100. mu.L/well and incubate at 37 ℃ for 1h (Sigma). The wells were discarded, washed 3 times with PBS, and 200. mu.L of PBSM blocking solution was added to each well and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS. mu.L of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBST and 3 times with PBS. Use the sealThe blocking solution 1 is HRP-IgG enzyme-labeled secondary antibody diluted at 8000, each well is 100 mu L, and the incubation is carried out for 1h at 37 ℃. The liquid was decanted, washed 3 times with PBST, 3 times with PBS, and patted dry. Adding 100 μ L of color development solution into each well, incubating at 37 deg.C for 10-20min, adding 50 μ L of stop solution, and measuring OD value at 450nm wavelength.

The results show that the monoclonal antibody of the invention is IgG_2aType murine monoclonal antibodies.

Second, determination of affinity constant

Recombinant Bcl6 protein was coated at 425.00ng/mL, 106.25ng/mL and 26.56ng/mL, 100. mu.L/well, coated overnight at 4 ℃ and washed 3 times with PBS. Add 200. mu.L of blocking solution per well and incubate 2h at 37 ℃ and wash 3 times with PBS. The monoclonal antibody purified in example 4 was diluted in a 1: 2000-fold gradient, incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T and 3 times with PBS. HRP-IgG enzyme-labeled secondary antibody was diluted with blocking solution 1:8000, and incubated at 37 ℃ for 1h with 100. mu.L of blocking solution per well, washed 3 times with PBST and 3 times with PBS. Add 100. mu.L of chromogenic solution to each well, incubate at 37 ℃ for 10-20min, and add 50. mu.L of stop solution. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. The fitted curves were drawn using origin8.0 software, IC from each curve₅₀The affinity constant was calculated to be 5.12X 10 according to the following formula¹⁰L/mol。

In the formula: [ Ab ]]Indicates the antigen concentration is [ Ag ]]Time IC₅₀The corresponding antibody concentration;

[Ab]_tindicates the antigen concentration is [ Ag ]]_tTime IC₅₀The corresponding antibody concentration;

n＝[Ab]/[Ab]_t

III, monoclonal antibody reaction specificity and application effect

Recombinant Bcl6 protein is selected, and the recognition specificity of the monoclonal antibody of the invention is detected by an immunoblotting method, wherein the immunoblotting experiment process is as follows: each protein was loaded at about 5-10ng and subjected to 10% polyacrylamide gel electrophoresis. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibody 1E6A4H10C9(1:100000 dilution) was added and incubated at 37 ℃ for 1H. After washing the membrane with TBST, goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotechnology Co., Ltd.) diluted at 1:8000 was added and incubated at 37 ℃ for 1 h. Washing the membrane with TBST again, adding ECL (Ecl hypersensitive color development liquid) (Wuhan Sanying biotechnology, Inc.), and collecting chemiluminescence image with fluorescence imaging system (Bio-Rad).

FIG. 2 is a graph showing the results of SDS electrophoresis and immunoblotting of Bcl6-His target protein in FIG. 2; m is a protein marker, 1 is purified Bcl6-His target protein, 2 is bovine serum albumin as a negative control, and 3 and 4 are: the result is the Western Blot immunoblotting result corresponding to 1, 2.

Example 5 tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Dewaxing by conventional xylene for 3 times, 6min each time, hydrating in 100%, 95%, 85% gradient ethanol for 3min each time, and finally washing with tap water. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3X 3min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3X 3 min. Throwing off PBS, dropwise adding and sealingThe solution (1% BSA solution) was incubated at room temperature for 10 min. Spin-drying the slices, dripping primary antibody (the dilution ratio of the antibody is designed according to the concentration of the antibody in the first dilution) diluted in a proper proportion, incubating at room temperature (25 ℃) for 1h, washing with PBS for 3 x 3min, dripping secondary antibody, incubating at room temperature for 20-30min, washing with PBS for 3 x 3min, throwing off the PBS, and developing with freshly prepared DAB developing solution for 3-10 min. Hematoxylin counterstain for 25s, and PBS rewet for 30 s. Dehydrating according to an alcohol gradient of 85% (3min) -95% (3min) -100% (3min) -100% (3min), finally, clearing xylene for 3min, and sealing with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody (1E6A4H10C9) and the commercial antibody (LN22) were tested simultaneously on different human tumor tissue chips (including 32 cases of diffuse large B-cell lymphoma, 24 cases of follicular lymphoma, 20 cases of mantle cell lymphoma, and 22 cases of T-cell lymphoma) and the results were compared.

Immunohistochemical results for Bcl6 were counted. The whole experimental process adopts a double-blind design, and the statistical results are shown in the following table.

Compared with the commercial antibody (LN22), the positive rate and the sample staining intensity of the antibody on the tumor cells are obviously higher than those of the commercial antibody (1E6A4H10C9), which shows that the specificity, the sensitivity and the affinity of the antibody are higher than those of the commercial antibody on the diagnosis of the tumor cells.

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (1E6A4H10C9) and the commercial antibody (murine monoclonal antibody LN22) were tested simultaneously on normal tissue chips, and the negative and positive results were consistent, indicating that the specificity of the antibody in normal tissues was equivalent to that of the commercial antibody.

FIG. 3 is a graph showing the results of staining diffuse large B-cell lymphoma; left is 1E6A4H10C9 antibody, right is control antibody LN 22;

FIG. 4 is a graph showing the result of immunohistochemical staining of tonsil tissues; the left is the 1E6A4H10C9 antibody, the middle is the control antibody LN22, and the right is the negative control.

The staining results in FIG. 3 and FIG. 4 show that 1E6A4H10C9 can specifically recognize Bcl6 protein in tissue cells, has good specificity, has higher sensitivity compared with the commercial antibody, and does not show any non-specific staining.

The technical scheme is that Bcl6 molecules are analyzed according to published sequences, and regions which are suitable for soluble expression and have good immunogenicity are selected for recombinant expression according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure on cell membranes. Meanwhile, in order to promote the soluble expression of the recombinant protein and optimize the expression behavior, the segments are subjected to codon optimization, are cloned into an expression vector pET-28a after gene synthesis for recombinant expression, and are used as immunogen after the target protein is purified, so that BALB/c mice are immunized for multiple times. Through cell fusion, screening and subcloning, a monoclonal cell line 1E6A4H10C9 capable of efficiently secreting the anti-Bcl 6 antibody and the Bcl6 antibody secreted by the cell line are obtained. The Bcl6 antibody has high specificity and sensitivity, and can specifically recognize cells expressing Bcl 6.

The hybridoma cell line is used for preparing ascites by a mouse, and the ascites is purified by protein G column affinity chromatography to obtain a mouse monoclonal antibody. The subclass of the monoclonal antibody is IgG2a type monoclonal antibody determined by ELISA technique, and the affinity constant is 5.12 × 10¹⁰L/mol. Immunohistochemical experiments showed that the antibody specifically recognized cells expressing the Bcl6 molecule.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

Sequence listing

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Claims

1. An anti-Bcl 6 protein monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO. 16210.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes a Bcl6 protein.

3. The monoclonal antibody of claim 2, wherein the monoclonal antibody specifically recognizes the amino acid sequence encoded by SEQ ID No.1 of the Bcl6 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG_2aThe subtype is.

5. A hybridoma cell strain secreting anti-Bcl 6 protein is a mouse hybridoma cell line 1E6A4H10C9, and is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation number is as follows: CGMCC NO. 16210.