CN115044560B

CN115044560B - Hybridoma cell strain, anti-human ERG protein monoclonal antibody and application thereof

Info

Publication number: CN115044560B
Application number: CN202210727663.8A
Authority: CN
Inventors: 范磊
Original assignee: Beijing Sino Fir Jinqiao Biological Technology Co ltd
Current assignee: Beijing Sino Fir Jinqiao Biological Technology Co ltd
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2023-07-28
Anticipated expiration: 2042-06-23
Also published as: CN115044560A

Abstract

The invention provides a hybridoma cell strain, an anti-human ERG protein monoclonal antibody and application thereof, wherein the hybridoma cell strain is preserved in the China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) with the preservation number of 45154 in the 5-month-9 of 2022, and the anti-human ERG protein monoclonal antibody is secreted by the hybridoma cell strain. The hybridoma cell strain can stably secrete monoclonal antibodies, is specifically combined with ERG proteins, remarkably improves the specificity, accuracy and reliability of ERG protein immunodetection, and is widely applicable to the labeling of ERG proteins in various tumors.

Description

Hybridoma cell strain, anti-human ERG protein monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain, an anti-human ERG protein monoclonal antibody and application thereof.

Background

ERG is also called erythrocyte transformation-specific (ETS) related gene, is located on chromosome 21q22 and belongs to members of ETS transcription factor family. All members of this family are key regulators of embryonic development, cell proliferation, differentiation, angiogenesis, inflammation and apoptosis. The protein encoded by the gene is mainly expressed in the nucleus. It comprises a DNA binding domain and a PNT domain, which is involved in self-binding of chimeric oncoproteins. This protein adheres to the subendothelium in platelets and is necessary to induce vascular cell remodeling. It also regulates differentiation and maturation of hematopoietic cells and megakaryocytes.

The gene is involved in chromosomal translocation, resulting in different fusion gene products. For example, ERG can be fused with androgen regulated transmembrane serine protease 2 (TMPRSS 2) to form a TMPRSS2-ERG fusion gene, resulting in high expression of ERG protein in prostate cancer. ERG proteins have been reported to be also expressed in vascular endothelial tumors, epithelial-derived tumors and other non-epithelial tumors, positive expression in vascular endothelial-derived tumors, 96% vascular sarcoma, 97.7% epithelial-like vascular endothelial tumors and positive expression of ERG in all kaposi's sarcomas. Simultaneously, ERG can regulate endothelial cell apoptosis and angiogenesis. In addition, ERG is also expressed in lymphatic endothelial cells and bone marrow stem cells. Positive expression of ERG is also seen in meningiomas, epithelioid sarcomas, malignant rhabdoid tumours, acute myelogenous leukemias and extramedullary myelosarcomas. ERG gene rearrangements are found in Ewing sarcoma. Markers of current hemangiosarcomas include potential markers such as CD31 (major markers), CD34, D2-40, and Vascular Endothelial Growth Factor Receptors (VEGFRs), but none of these markers have the desired sensitivity and specificity. The strong expression of ERG in vascular endothelial cells makes it one of the highly sensitive and specific markers of vascular-derived tumors.

Currently, the expression of ERG in tumor tissues is mainly detected clinically by Immunohistochemistry (IHC) pathology experiments. The core of IHC experiments is a primary antibody which can be specifically combined, and the performance of the primary antibody directly determines the sensitivity and the specificity of the whole experiment. Therefore, the development of a monoclonal antibody which specifically binds to ERG protein has important significance.

Disclosure of Invention

In view of the above, the present invention aims to provide a hybridoma cell strain, an anti-human ERG protein monoclonal antibody and application thereof, so as to provide an anti-human ERG protein mouse monoclonal antibody with good specificity and high affinity.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

a hybridoma cell strain is named OTI4H7, and is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 45154 in the 5 th month and 9 th year of 2022.

An anti-human ERG protein monoclonal antibody is secreted by the hybridoma cell strain.

The screening method of the hybridoma cell strain and the preparation method of the anti-human ERG protein monoclonal antibody are as follows:

(1) Construction of recombinant expression vectors: the ORF nucleotide sequence of the ERG gene is shown as SEQ ID No.1, the ORF length is 1437bp, the corresponding amino acid sequence is shown as SEQ ID No.2, and the amino acid length is 479aa. Designing a primer according to the ORF complete sequence of the ERG gene, obtaining a target fragment through PCR amplification, and connecting the target fragment with an expression vector pCMV6-Entry to construct an ERG recombinant expression plasmid pCMV6-rERG.

(2) Expression and purification of ERG recombinant proteins: and (3) transfecting the pCMV6-rERG recombinant expression plasmid into HEK293T cells, performing lysis and centrifugation to obtain supernatant, and purifying by using a DDK affinity chromatography column to obtain the purified ERG recombinant protein.

(3) Screening and preparation of monoclonal antibodies: immunizing a BALB/c mouse by using the recombinant ERG protein, fusing spleen cells of the mouse with SP2/0 cells, obtaining monoclonal cells by a limiting dilution method, and screening positive hybridoma cells by an ELISA method to obtain a hybridoma cell strain capable of secreting an anti-ERG specific antibody; antibodies were prepared by serum-free medium and ERG monoclonal antibodies were purified by affinity chromatography column. The sensitivity and the specificity of the monoclonal antibody are verified by Western-blot and immunohistochemical experiments.

Further, the anti-human ERG protein monoclonal antibody specifically binds to ERG protein.

Further, the light chain variable region of the anti-human ERG protein monoclonal antibody contains 105aa and comprises an amino acid sequence shown as SEQ ID NO. 3.

Further, the light chain variable region of the anti-human ERG protein monoclonal antibody comprises 3 antigenic determinants, namely CDR1 shown as SEQ ID No.5, CDR2 shown as SEQ ID No.6 and CDR3 shown as SEQ ID No.7, and the amino acid residue sequences of the light chain variable region are QNINVW, KVS, QQGQSY respectively.

Further, the heavy chain variable region of the anti-human ERG protein monoclonal antibody contains 106aa and comprises an amino acid sequence shown as SEQ ID NO. 4.

Further, the heavy chain variable region of the anti-human ERG protein monoclonal antibody comprises 3 antigenic determinants, namely CDR1 shown as SEQ ID No.8, CDR2 shown as SEQ ID No.9 and CDR3 shown as SEQ ID No.10, and the amino acid residue sequences of the heavy chain variable region are GYTFTEYL, INPGSGGT, ARLGGNFPN respectively.

The application of the anti-human ERG protein monoclonal antibody in preparing an immunodetection tool;

further, the immunodetection tool is used for detecting ERG proteins;

further, the immunoassay tool is a kit, a chip or test paper.

The application of the anti-human ERG protein monoclonal antibody in preparing a kit for marking tumor tissues;

further, the tumor tissue comprises one or more of prostate cancer, angiosarcoma, epithelioid vascular endothelial tumor, and all Kaposi's sarcoma.

An immunohistochemical detection kit comprises the anti-human ERG protein monoclonal antibody, and can detect the expression condition of ERG protein in tissue cells.

Compared with the prior art, the hybridoma cell strain, the anti-human ERG protein monoclonal antibody and the application thereof have the following advantages:

the hybridoma cell strain can stably secrete monoclonal antibodies, is specifically combined with ERG proteins, remarkably improves the specificity, accuracy and reliability of ERG protein immunodetection, and is widely applicable to the labeling of ERG proteins in various tumors.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is a graph showing the result of Western blot detection of ERG recombinant plasmid expression in HEK293T cells according to example 1 of the present invention; lane L is the detection result of HEK293T cell lysate transfected with empty vector pCMV6-Entry plasmid; lane R is the detection result of HEK293T cell lysate transfected with pCMV6-rERG recombinant plasmid, and the primary antibody used for hybridization is a DDK tag antibody;

FIG. 2 is a chart showing the result of coomassie brilliant blue staining for detecting pCMV6-rERG recombinant proteins by SDS-PAGE electrophoresis as described in example 1 of the present invention, purifying ERG proteins by using a DDK affinity chromatography column, and staining the purified proteins by SDS-PAGE electrophoresis and coomassie brilliant blue;

FIG. 3 is a graph showing the result of detecting the expression of ERG protein in hemangioma tissue by immunohistochemical method according to example 3 of the present invention, wherein the primary antibody used for hybridization is anti-human ERG protein monoclonal antibody OTI4H7 of the present invention;

FIG. 4 is a graph showing the results of immunohistochemical detection of ERG protein expression in prostate cancer tissue according to example 3 of the present invention, wherein the primary antibody used for hybridization is the anti-human ERG protein monoclonal antibody OTI4H7 of the present invention.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to the following examples and drawings.

Example 1: preparation of anti-ERG murine monoclonal antibodies

1. Construction of ERG recombinant expression plasmid

Primers are designed according to the sequence of the ERG gene (NM_ 182918) in NCBI, and PCR is utilized to amplify and connect with an expression vector pCMV6-Entry to construct a recombinant expression plasmid pCMV6-rERG of ERG.

2. Expression and purification of recombinant ERG proteins

1. Transfection of HEK293T cells

HEK293T cells were passaged 1:3 and cultured continuously in petri dishes; adding serum-free and antibiotic-free DMEM culture medium into a 50ml tube, adding PEI MegaTran1.0, mixing, adding the ERG recombinant plasmid DNA, mixing, and standing for 30 min; the mixture was added to HEK293T cell culture dishes and incubated at 37℃in a 5% CO2 incubator. 24 hours after transfection, 2M sodium butyrate was added.

2. Lysing cells

After 48 hours of transfection, the medium was aspirated, rinsed with PBS and the PBS was aspirated. Lysis buffer was added and protease inhibitors PI and PMSF (ready to use) were added prior to use. Placing in ice box, shaking on shaking table, collecting lysate, centrifuging at 4deg.C, and collecting supernatant.

3. DDK affinity chromatography column purification

Filtering the supernatant of the centrifuged lysate by using a PVDF membrane filter, transferring the supernatant into a 15ml tube, adding 1ml of mixed Sepharose Beads, sealing the mixture, putting the mixture into a 360-degree mixer, and combining the mixture for 2 hours at 4 ℃; pouring the lysate into BIO-RAD chromatographic column, and collecting the permeate, sampling WB for detection after dripping, as shown in FIG. 1 (FIG. 1 shows normal expression of ERG recombinant plasmid in HEK293T cells), washing column with lysis Buffer 1-2 times, washing Beads 3 times after dripping, eluting with 0.1M Glycine Buffer (pH3.5), eluting with 200. Mu.l of the first time, collecting no supernatant, 500. Mu.l of each of the second and third times, 250. Mu.l of the fourth time, collecting into 1.5ml centrifuge tube, and rapidly adding NaH ₂ PO ₄ Buffer (pH 11.0) was neutralized to about pH7.0, and glycerol was added to a final concentration of 10% and Tween-80 to a final concentration of 0.1% per tube. After purification, the ERG recombinant protein was identified by SDS-PAGE, and the results are shown in FIG. 2.

3. Preparation and screening of OTI4H7 secretion anti-ERG protein monoclonal antibody

1. Immunization of animals

The purified ERG antigen is emulsified by complete Freund's adjuvant, and BALB/c mice with 6-8 weeks of age are immunized by subcutaneous or intraperitoneal injection, wherein the immunization dose is 50 mug/mouse, and the immunization is carried out for the second time after two weeks, and the immunization dose is 30 mug/mouse by incomplete Freund's adjuvant emulsification. Tail blood is taken after two times of immunization, and serum titer is measured by ELISA method gradient dilution; and determining whether to boost according to the result, and selecting the mice with the highest antibody titers for cell fusion.

2. Cell fusion

Myeloma cells adopt BALB/c sp2/0 and are in logarithmic growth phase during fusion; taking spleen of the immunized mice to prepare lymphocyte single-cell suspension; mixing spleen lymphocytes of mice with myeloma cells at a ratio of 1:5-1:10, dripping 1mL of 50% PEG (pH 8.0) at 37 ℃, adding incomplete culture medium and stop solution thereof, centrifuging, removing supernatant, adding HAT culture medium, suspending, mixing uniformly, MC constant volume to 50mL, subpackaging into 3.5cm culture dishes, placing in a wet box, and adding 5% CO at 37 DEG C ₂ In a constant temperature incubatorCulturing.

3. Screening and cloning

Hybridoma clones were selected within 7-10 days of fusion, ELISA testing was performed using purified recombinant ERG protein, and corresponding cell line numbers were labeled. Limiting dilution is carried out on positive hole cells, ELISA values are measured 5-6 days after each limiting dilution, and OD is selected ₂₈₀ The monoclonal well with higher positive value is subjected to limiting dilution until the result of ELISA measurement of the whole 96-well plate is positive. And selecting monoclonal fixed strains with high positive values.

4. Preparation and purification of monoclonal antibodies

And (3) culturing hybridoma cells in a serum-free culture medium in a suspension manner, collecting cell suspension after about 1 week when the volume of cell supernatant is expanded to 60% -70% of the workload and the cell death rate, centrifuging to obtain supernatant, performing antibody purification by using an affinity chromatography method, and selecting corresponding column materials for purification according to antibody subtypes. The concentration of the purified monoclonal antibody is measured, split charging and freezing storage are carried out at-20 ℃.

Example 2: monoclonal antibody OTI4H7 variable region gene and amino acid sequence analysis

By means ofRACE 5'/3' kit (Takara Bio USA Co.) the light and heavy chain variable region gene sequences of the hybridoma cell functional antibodies were amplified using the 5' RACE (RapidAmplification of cDNA Ends ) technique. For a specific experimental procedure see->RACE 5'/3' kit user manual.

1. Recombinant hybridoma cell OTI4H7 variable region gene and sequencing

According to the antibody OTI4H7 is of an IgG1 subtype, specific gene primers pRace-H-GSP and pRace-K-GSP aiming at the 3' -end of Ig and Kappa constant regions of the antibody are designed, and the primer sequences are as follows:

pRace-H-GSP:CATCDGTCTATCCACTGGCCCCTG

pRace-K-GSP:CTTCCCACCATCCAGTGAGCAGTT

mRNA is extracted from hybridoma cell OTI4H7, reverse transcription is carried out to obtain cDNA, RACE amplifies DNA fragments of antibody light chain and heavy chain, the amplified light chain and heavy chain are respectively connected with a cloning vector PUC119, positive clones are selected by utilizing blue white screening, a ABI 3730 sequencer is utilized to sequence the purified positive plasmids, and sequencing primers are universal primers M13f and M13r.

2. Amino acid sequence analysis of monoclonal antibody OTI4H7 variable region gene

And (3) respectively carrying out data analysis on the variable region nucleotide sequences of the light chain and the heavy chain obtained by sequencing by using IMGT/V-QUEST online analysis software (http:// www.imgt.org), so as to obtain the light chain variable region amino acid sequence of the mouse monoclonal antibody OTI4H7, wherein the light chain variable region amino acid sequence is shown as SEQ ID NO.3, and the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 4. The VL has a total length of 105 amino acids, the FR has 4 domains of 26, 17, 36 and 11 amino acids, the CDR has 3 domains of 6, 3 and 6 amino acids, and the CDR1, CDR2 and CDR3 have 27aa-32aa,50aa-52aa and 89aa-94aa, respectively, and their amino acid sequences are QNINVW, KVS, QQGQSY, respectively.

By analysis, the murine monoclonal antibody OTI4H7 VH has a total length of 116 amino acids, the FR 4 domains thereof have amino acid numbers of 25, 17, 38 and 11, the CDR3 domains thereof have amino acid numbers of 8, 8 and 9, respectively, the CDR1, CDR2 and CDR3 have amino acid numbers of 26aa-33aa,51aa-58aa and 97aa-105aa, respectively, and the amino acid sequences thereof are GYTFTEYL, INPGSGGT, ARLGGNFPN, respectively.

Example 3: immunohistochemical experiments Using the monoclonal antibody of the present invention as the primary antibody

Monoclonal antibody OTI4H7 is used as a primary antibody, and the specificity of hemangioma and prostatic cancer tissues is verified by detecting the hemangioma and prostatic cancer tissues through IHC experimental technology.

1. Formalin-fixed hemangioma and prostate cancer tissues were paraffin-embedded and sectioned using a Leica tissue microtome at a tissue thickness of 4 μm.

2. Dewaxing and hydration: analytical grade xylene 10min×3 times, absolute ethanol 10min×3 times, 95% ethanol 5min,85% ethanol 5min,75% ethanol 5min, deionized water 3min×3 times.

3. Repairing: adding antigen retrieval liquid into the autoclave for autoclave retrieval for 3min, opening the autoclave when the temperature of the autoclave is reduced to about 90 ℃, taking out the specimen, and naturally cooling to room temperature. The deionized water is soaked for 3min multiplied by 3 times.

4. And (3) inactivation: tissue endogenous peroxidase was inactivated with 3% hydrogen peroxide, and left to stand at room temperature for 10min. The deionized water is soaked for 5min multiplied by 3 times.

5. Incubation resistance: the monoclonal antibody secreted by the OTI4H7 of the invention was added at a 1:300 dilution, placed in a wet box and incubated for 90min at room temperature. Wash 5min×3 times with PBST (0.1% Tween-20).

6. Secondary antibody incubation: each piece was incubated with 200. Mu.l of PV-8000 at 37℃for 30min. Wash 5min x 3 times using PBST.

7. Color development: the DAB solution is used for color development for 3 to 10 minutes. Washing with distilled water.

8. Counterstaining: hematoxylin counterstains the nuclei for 2min, rinsing with distilled water, and differentiating with 1% hydrochloric acid. Rinsing with distilled water for 3 times, and standing at room temperature for 1min.

9. Dehydration and transparency: 75% ethanol 5min,85% ethanol 5min,95% ethanol 5min,100% ethanol 3X 5min; xylene 3×5min, neutral resin seals.

10. And (5) microscopic examination: as shown in fig. 3-4.

2. Experimental results

The result shows that the anti-ERG protein monoclonal antibody secreted by the hybridoma cell OTI4H7 has good specificity on the detected tissue, and is concretely expressed as follows,

(1) As can be seen from the results of fig. 3, the expression of the anti-ERG protein monoclonal antibody secreted by the hybridoma cell OTI4H7 on the hemangioma tissue, and the dotted arrow indicates that the tumor cell exhibits positive expression, i.e., EGR protein expression; solid arrows indicate that the mesenchymal cells are negative.

(2) As can be seen from the results of fig. 4, the expression of the anti-ERG protein monoclonal antibody secreted by the hybridoma cell OTI4H7 in the prostate cancer tissue, the dashed arrow indicates positive expression on vascular endothelium, and the solid arrow indicates non-expression on cancer.

The above results demonstrate that the monoclonal antibody secreted by OTI4H7 can specifically bind to ERG protein in vascular and vascular-derived tumor tissue, making it one of the highly sensitive and specific markers of vascular-derived tumors.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

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Claims

1. A hybridoma cell line, characterized in that: the hybridoma cell strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45154 in the year 5 and the month 9 of 2022.

2. An anti-human ERG protein monoclonal antibody, characterized in that: is secreted by the hybridoma cell line of claim 1.

3. The anti-human ERG protein monoclonal antibody of claim 2, wherein: the anti-human ERG protein monoclonal antibody specifically binds to ERG protein.

4. The anti-human ERG protein monoclonal antibody of claim 2, wherein: the light chain variable region of the anti-human ERG protein monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 3.

5. The anti-human ERG protein monoclonal antibody of claim 2, wherein: the light chain variable region of the anti-human ERG protein monoclonal antibody comprises a CDR1 shown as SEQ ID No.5, a CDR2 shown as SEQ ID No.6 and a CDR3 shown as SEQ ID No. 7.

6. The anti-human ERG protein monoclonal antibody of claim 2, wherein: the heavy chain variable region of the anti-human ERG protein monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 4.

7. The anti-human ERG protein monoclonal antibody of claim 2, wherein: the heavy chain variable region of the anti-human ERG protein monoclonal antibody comprises a CDR1 shown as SEQ ID No.8, a CDR2 shown as SEQ ID No.9 and a CDR3 shown as SEQ ID No. 10.

8. Use of an anti-human ERG protein monoclonal antibody according to any one of claims 2-7 for the preparation of an immunoassay kit.

9. The use according to claim 8, characterized in that: the immunoassay tool is used for detecting ERG proteins.

10. The use according to claim 9, characterized in that: the immunodetection tool is a kit, a chip or test paper.

11. Use of an anti-human ERG protein monoclonal antibody according to any one of claims 2-7 for the preparation of a kit for labelling tumor tissue.

12. The use according to claim 11, characterized in that: the tumor tissue includes one or more of prostate cancer, angiosarcoma, epithelioid vascular endothelial tumor, and kaposi's sarcoma.

13. An immunohistochemical detection kit, characterized in that: comprising an anti-human ERG protein monoclonal antibody according to any one of claims 2-7.