CN112480260B

CN112480260B - anti-PSMA protein monoclonal antibody, cell line, preparation method and application thereof

Info

Publication number: CN112480260B
Application number: CN202011449337.2A
Authority: CN
Inventors: 李玲玲; 陈惠玲; 王小亚; 吴茂; 邓晓; 李恢波
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2022-03-08
Anticipated expiration: 2040-12-09
Also published as: CN112480260A

Abstract

The invention relates to the field of biological detection, and provides a PSMA protein resisting monoclonal antibody, wherein the amino acid sequences of the heavy chain and light chain variable regions are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3. The antigen for immunizing mice is recombinant protein, the recombinant protein is coded by a nucleotide sequence shown in SEQ ID No.1, and is expressed through escherichia coli recombination. The inventor also provides a hybridoma cell line secreting the PSMA-resistant protein, wherein the cell line is a mouse hybridoma cell line 11D1E5E10 with the preservation number of: CGMCC NO. 19683. The PSMA protein resisting monoclonal antibody has high specificity and sensitivity, can specifically identify PSMA protein expressing cells, and is suitable for immunological detection, especially immunohistochemical detection.

Description

anti-PSMA protein monoclonal antibody, cell line, preparation method and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to a PSMA protein-resistant monoclonal antibody, a cell line, a preparation method and application thereof.

Background

Prostate cancer is the most common malignancy in men, and the third mortality associated with cancer in men worldwide. Statistically, about 1-2% of men currently die from prostate cancer. In recent years, with the rapid development of society, the quality of life of people is improved, the average life of people is prolonged, and diagnosis and treatment technologies are gradually improved, but the incidence rate of prostate cancer is still gradually increased, and the incidence rate of prostate cancer is about 11.2 percent at present. Early diagnosis of prostate cancer is also receiving increasing attention from scholars.

Prostate Specific Membrane Antigen (PSMA) is uniquely overexpressed on the surface of prostate cancer cells and in the neovasculature of a variety of solid tumors. Thus, PSMA has attracted attention as a clinical biomarker for the detection and treatment of prostate cancer. The prostate specific membrane antigen, abbreviated as PSMA, was first discovered in 1987. It is a type II transmembrane glycoprotein, which is composed of 750 amino acids and comprises an intracellular region with 19 amino acids, a transmembrane region with 24 amino acids and an extracellular region with 707 amino acids, and the molecular weight of the protein is 84KDa, and after glycosylation, the molecular weight is about 100 KDa. The gene for PSMA maps to the arm of chromosome 11, 60Kb in full length, and includes 19 exons and 18 introns.

PMSA is highly expressed in the epithelial cell membrane of the prostate cancer, and the expression level of the PMSA is positively correlated with the number and the invasiveness of tumor cells. PSMA is predominantly expressed on the membrane of the prostate epithelium, with a very high proportion of prostate cancer, and is further increased in more malignant, metastatic, and hormone refractory prostate cancers. Therefore, PSMA has high prostate specificity, is closely related to malignant tumor disease process, and is an ideal tumor marker and a prostate cancer biological treatment target. Immunohistochemistry shows that PSMA is expressed in normal tissue, benign hyperplastic tissue, intraepithelial hyperplasia and cancer tissue of prostate, the expression intensity is increased in sequence, and the expression intensity is highest in prostate cancer; positive expression was found in prostate cancer lymph node metastases, bone metastases. Meanwhile, PSMA has certain expression in various non-prostate malignant tumors, which mainly comprise renal clear cell carcinoma, transitional cell carcinoma of bladder, colon adenocarcinoma, pancreatic duct cancer, non-small cell lung cancer, such as histiosarcoma, breast cancer and the like. PSMA has high specificity and the characteristic of being located in the epithelial cell membrane of prostate, has high expression in prostate cancer and metastasis thereof, and has important significance in the targeted treatment of the prostate cancer.

Disclosure of Invention

The invention provides a monoclonal antibody for resisting PSMA protein, wherein the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

Furthermore, the heavy chain and light chain variable region amino acid sequences of the monoclonal antibody are respectively encoded by the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.

Further, the monoclonal antibody specifically recognizes the PSMA protein.

Further, the monoclonal antibody specifically recognizes the amino acid sequence shown as SEQ ID NO. 1.

Further, the monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO. 19683. The cell strain is a mouse hybridoma cell line 11D1E5E10, and is classified and named as: a mouse hybridoma cell line which has been deposited at 24.04.2020 by the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at the institute of microbiology, China academy of sciences, No.3, West Lu 1 Hospital, North Cheng, Chaoyang, Beijing.

Further, the anti-PSMA protein is a mouse IgG1 subtype monoclonal antibody.

Further, the anti-PSMA protein monoclonal antibody takes a recombinant protein as an immunogen to prepare the recombinant protein, and the recombinant protein comprises an antigenic PSMA fragment and a histidine protein tag, wherein the PSMA fragment is an amino acid fragment from 307 th to 750 th positions of human PSMA protein.

The inventor also provides a hybridoma cell line secreting PSMA-resistant protein, the cell line is a mouse hybridoma cell line 11D1E5E10, the cell line is preserved in China general microbiological culture Collection center, and the preservation unit address is as follows: china Beijing, the preservation date is 2020, 4 months and 24 days, and the preservation number is: CGMCC NO. 19683.

The inventor also provides the application of the anti-PSMA protein monoclonal antibody in the immunodetection of the PSMA protein.

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

In contrast to the prior art, the above-described protocol selects for recombinant expression the region from amino acid 307 to amino acid 750 of PSMA, which is suitable for soluble expression and which is also highly immunogenic. The histidine tag of 6 His can be used as a purification tag of the fusion protein. Immunizing a mouse, and obtaining a monoclonal cell line 11D1E5E10 which efficiently secretes the anti-PSMA protein monoclonal antibody and the anti-PSMA protein monoclonal antibody secreted by the cell line through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing PSMA protein, is suitable for immunological detection, particularly immunohistochemical detection, can reduce misreading and improve the accuracy of diagnosis.

Drawings

FIG. 1: polyacrylamide gel electrophoresis image of purified PSMA monoclonal antibody.

FIG. 2: a prostate cancer immunohistochemical staining result graph; left is (11D1E5E10) PSMA, right is commercially available PSMA (1D 6).

Detailed Description

Example 1 preparation of recombinant PSMA protein fragments

First, Gene cloning

The PSMA protein sequence with number Q04609 was selected as the standard sequence from the Uniprot database (http:// www.uniprot.org). According to the structure of binding with DNA, antigenicity, hydrophilicity and hydrophobicity of amino acid and secondary structure, the region with proper length and special antigenicity is selected as antigenic peptide. The PSMA amino acid sequence from 307 th to 750 th bit is selected, and the corresponding nucleotide sequence is SEQ ID No. 1. The PSMA recombinant protein plasmid is synthesized by connecting a target protein fragment to a plasmid vector pet beta 2M (pet beta 2M vector is purchased from Kelly bioengineering, Inc., of Wuhan gold, and contains a beta 2M protein sequence and a His tag). Transforming into colon bacillus competent cell TOP10, selecting clone on the plate, inoculating, expanding, extracting plasmid DNA, and performing PCR identification. And (4) sequencing and analyzing the clone with positive target gene shown by PCR, and using the clone with completely correct sequence for next experiment.

β 2M is a component of the Major Histocompatibility Complex (MHC) class I, and is involved in presentation of antigenic peptides to the immune system, facilitating antibody production. The histidine tag of 6-His can be used as a purification tag for the fusion protein.

Secondly, recombinant protein expression and purification

And (3) transforming the recombinant plasmid into an escherichia coli competent cell Rosetta, selecting a clone on a plate, inoculating, expanding and culturing, and preserving bacteria. The resulting suspension was inoculated into 4mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and cultured overnight at 37 ℃ under shaking at 270 rpm. Then, the cells were transferred to 200mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and cultured at 37 ℃ and 270rpm for 8H with shaking, and then the cells were collected. The cells expressing the recombinant protein were subjected to ultrasonication. Centrifuging 8000g of the bacterial liquid for 10min, removing a supernatant culture medium, keeping the bacterial liquid, resuspending and washing the bacterial liquid by using a proper amount of PBS buffer solution, and adding a nickel column balance buffer solution into the bacterial liquid for ultrasonic crushing. And (4) carrying out ultrasonic crushing until bacterial liquid is clarified, centrifuging, and respectively collecting precipitate and supernatant. And (4) carrying out SDS-PAGE electrophoresis on the crushed supernatant, the precipitate and the thalli subjected to induced expression, and analyzing the existence position of the recombinant protein.

Purifying the recombinant protein by adopting a nickel column affinity chromatography. The main purification steps were carried out according to the instructions of Changzhou Tiandi and Biotech Co., Ltd. The purified target protein is dialyzed to remove salt, and is concentrated by an ultrafiltration tube. And finally, determining the protein concentration by an ultraviolet micro spectrophotometer, detecting the protein purity by SDS-PAGE electrophoresis, and determining the specificity by ELISA detection analysis. Table 1 shows the results of PSMA antigen-specific detection. FIG. 1: polyacrylamide gel electrophoresis image of purified PSMA monoclonal antibody.

Table 1: ELISA for detecting PSMA antigen specificity

Note: over indicates that the measurement range of the instrument is exceeded, i.e. the response is strong.

EXAMPLE 211 establishment of the hybridoma cell line D1E5E10

Immunization

The PSMA protein obtained in example 1 was diluted to 1mg/mL, mixed and emulsified with an equal volume of complete Freund's adjuvant (CFA, Sigma Co.), and 18-20g of Balb/c mice (purchased from Wu's laboratory animal, Fuzhou) were immunized by abdominal injection at a dose of 50. mu.g/mouse. Thereafter, the booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (IFA, Sigma Co.) at a dose of 25. mu.g/mouse. 2 nd boost

The multi-antibody titer of the anti-immunogen in the serum of the mouse is detected by indirect ELISA (wavelength of 450nm) 14 days later, the mouse with the highest titer is injected by tail vein for impact immunization, and the antigen is mixed with PBS solution uniformly, and the dosage is 50 mu g/mouse.

Second, cell fusion

Aseptically preparing mouse spleen cell suspension with qualified immunity, mixing with mouse myeloma cell sp2/0(ATCC) at a ratio of 5:1, centrifuging at 1000rpm for 10min, discarding supernatant, adding 1mL of PEG (Sigma) solution preheated to 37 ℃ from slow to fast within 1 min, and slightly rotating the centrifuge tube during the addition process to make the cells fully contact with PEG. Standing at room temperature for 90s, adding 4mL of serum-free DMEM (Hyclone company) culture medium preheated to 37 ℃ from slow to fast within 2min, then adding 10mL of preheated serum-free DMEM culture medium within 2min, finally adding the rest preheated serum-free DMEM culture medium within 2min, and fixing the volume to 50mL, wherein the centrifugal tube needs to be slowly shaken in the whole adding process to ensure uniform mixing and reduce the damage to cells. Standing at room temperature for 10min, centrifuging (1000rpm, 5min), discarding supernatant, resuspending cells in 10-20 mLHAT (Sigma) medium, and diluting with HAT medium to final concentration of 0.5X 10⁶cells/mL, all solutions were transferred to 96-well plates at 200. mu.L/well and labeled. The 96-well plates were carefully transferred to a 37 ℃ 5% CO2 incubator for incubation. The growth state and potential pollution of cells are regularly checked, and the incubator is opened and closed as little as possible to ensure the stability of the culture environment. On day 5 post-fusion, plates were supplemented with HAT medium at 50. mu.L/well.

Third, ELISA screening positive hybridoma cell

When the fused cell diameter is about 1-2mm, 50-200. mu.L of culture supernatant is aspirated for the first cell selection (ELISA, IHC-P and other methods of detection), and HAT medium is added to the culture wells to 200. mu.L. And (3) detecting the culture solution supernatant by ELISA, transferring all cell culture solution in the culture hole with the positive result obtained by detection to a 24-hole culture plate, supplementing HT medium, culturing for 3 days at a concentration of 2 mL/hole.

And repeatedly screening each cell strain in the 24-pore plate, and removing the culture well cells which are not positive results to obtain the culture well cells with better positive results. Performing subclone screening on the positive well cells obtained from the 24-well culture plate by limiting dilution method, namely adding cell sap obtained by limiting dilution method into 96-well culture plate, and transferring to CO₂Culturing in an incubator for 11 days, and repeating cell screening when the diameter of the cloned cell is 1-2 mm. According to the detection result, 4 well-grown monoclonal positive culture wells are selected from each subcloned cell strain, and transferred to a 24-well plate for continuous culture. And after a period of time, screening the positive clone cell strain cloned in the 24-pore plate again, namely the hybridoma cell strain 11D1E5E10 secreting the specific monoclonal antibody. The cell strain is transferred into a T-75 culture flask to be amplified to a logarithmic growth phase for seed preservation or subsequent experiments are carried out.

EXAMPLE 3 preparation of monoclonal antibodies by in vitro culture

First, in vitro culture

After obtaining the stable hybridoma cell line, the monoclonal antibody is obtained mainly by an in vitro culture method.

The hybridoma cells were cultured in DMEM complete medium containing 10% fetal bovine serum, and then centrifuged at low speed to collect culture supernatant, which was stored at 4 ℃ for further use.

Secondly, purification of monoclonal antibody

Antibody purification using rProtein A sepharose Fast Flow (GE) affinity column: filling a proper amount of purchased ProteinA filler into a gravity chromatography column, and washing the gravity chromatography column with an equilibrium buffer solution (0.1M Tris solution, pH7.0) until the balance is achieved; secondly, loading the sample, adding the ascites filtered by the filter membrane of 0.22 mu m into the packed chromatographic column, and controlling the flow rate to be 1 drop/second; thirdly, balancing, and washing the sample solution to be balanced by using a balancing buffer solution after the sample solution is loaded; eluting, adding an elution buffer solution (0.1M citric acid solution, pH3.0) to wash the column and collecting the eluent; fifthly, regenerating, adding an equilibrium buffer solution to wash the column to be balanced after the elution is finished, washing the column with 2 times of column volume of 20 percent ethanol, and storing the column at 4 ℃. Finally, the purity of the antibody is identified by an SDS-PAGE method (as shown in figure 2), the purity reaches more than 95%, and the concentration of the antibody is determined by an ultraviolet micro-spectrophotometer method and reaches more than 3.0 mg/mL.

EXAMPLE 4 characterization of monoclonal antibodies

Identification of one, two subtypes

The cell supernatants were diluted to 1. mu.g/mL coated ELISA plates, 100. mu.L per well, coated overnight at 4 ℃, the solution was decanted, the plates were washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.L of blocking solution (PBS-T solution containing 2% BSA) was added per well, and incubated for 1h at 37 ℃. The liquid was decanted and washed 3 times with PBS-T. 0.1mL of culture supernatant of hybridoma cell line diluted 5 times was added to each well, and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Using a confining liquid 1: HRP-labeled goat anti-mouse (kappa, lambda, IgM, IgG1, IgG2a, IgG2b, IgG) diluted 400₃IgA) antibody (Southern Biotech) was added in an amount of 0.1mL per well and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. 100 mu of LTMB (Hiroshi Biotech, Inc., Huzhou) substrate (A, B mixed solution with equal volume) is added into each well for color development, the reaction is carried out for 15min at room temperature, 50 mu L of 1N HCl solution is added into each well to stop the color development reaction, and then the OD value at the wavelength of 450nm is measured by a microplate reader. The results show that the monoclonal antibody of the present invention is a murine monoclonal antibody of the IgG1 type.

Second, determination of affinity constant

PSMA protein was coated at 100. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 1h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was diluted to the following concentrations (unit: ng/mL): 2000. 500, 125, 62.5, 31.25, 15.625, 3.125, 0.625, incubation at 37 ℃ for 1h, PBS-T wash 3 times. HRP-labeled goat anti-mouse secondary antibody 1: diluted at 5000, 100. mu.l/well, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. Mu.l of TMB (Hiroshi Biotech, Inc., England, Huzhou) color developing solution was added to each well, and the reaction was stopped by adding 100. mu.l of 1.0N salt solution for color development for 13 min. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. The OD value is plottedThe antibody concentration a corresponding to the "plateau OD value" of 1/2 was found from the curve corresponding to the dilution factor of the antibody. The affinity constant was calculated to be 4.96X 10 using the following formula⁹L×mol^-1。

Example 6 tissue chip staining and characterization

First, tissue wax block preparation process

HE section staining was performed on the sample tissue to determine the tumor lesion site. And (5) circling the lesion site and preparing to punch. When the receptor wax block is manufactured, a plastic frame is placed on a mold, melted paraffin (the melting point is 56-58 ℃) is poured into the mold, the tissue block is placed into wax liquid in the mold, then a proper amount of wax liquid is added to enable the tissue block to be completely embedded in the wax liquid, the mold is placed into a refrigerator with the temperature of-20 ℃ for 6min after being cooled to the room temperature, the wax block is taken out of the mold, and the section is sliced or placed into the refrigerator with the temperature of 4 ℃ for storage. After trimming, continuous slicing is carried out, the thickness is determined to be 4 mu m, the continuous slicing is rinsed in 40% alcohol, the slices are naturally unfolded, then the separated slices are transferred to warm water at 45 ℃ for 30 seconds, a glass slide treated by 2% APES acetone solution is used for mounting the slices, the prepared tissue chip is placed into an oven at 60 ℃ for baking for 2 hours, the slices are taken out for cooling at room temperature, and the tissue chip is placed into a refrigerator at-4 ℃ for storage.

Second, IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each time with 100%, 95%, 85% gradient ethanol hydration for 3 minutes each time and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. The PBS was spun off and the peroxidase blocker was added dropwise and incubated for 10 minutes at room temperature. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution.Hematoxylin counterstain for 20 seconds, PBS turns blue. Dehydration was carried out in a gradient of 85% (3 min), 95% (3 min), 100% (3 min) and 100% (3 min) in order, and finally two times xylene was cleared for 10min, followed by 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 "+ + + +";

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

Thirdly, sample detection results:

the results of simultaneous detection of 33 prostate carcinomas using the anti-PSMA protein monoclonal antibody (11D1E5E10) and the control antibody, commercially available PSMA antibody 1D6, are shown in the table below.

The result shows that the monoclonal antibody (11D1E5E10) resisting the PSMA protein has accurate staining location, clear staining, no non-specific staining and clean background, which indicates that the monoclonal antibody (11D1E5E10) resisting the PSMA protein has strong specificity. And in 33 cases of prostate cancer, the number of positive cells and the positive intensity using the anti-PSMA protein monoclonal antibody (11D1E5E10) were comparable to those using the control reagent, indicating that the sensitivity and affinity of the anti-PSMA protein monoclonal antibody (11D1E5E10) were comparable to those of the commercial antibody.

And the anti-PSMA protein monoclonal antibody (11D1E5E10) and the control antibody-commercially available 1D6 are used for synchronous detection on a normal tissue chip, and the positive and negative results of the sample are consistent, which indicates that the specificity of the antibody in normal tissues is equivalent to that of the commercially available antibody. FIG. 2 is a graph showing an example of immunohistochemical staining for prostate cancer; left is (11D1E5E10) PSMA, right is commercially available PSMA (1D 6).

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Claims

1. The PSMA protein resisting monoclonal antibody is characterized in that the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are encoded by the nucleotide sequences set forth in SEQ ID No.4 and SEQ ID No.5, respectively.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes a PSMA protein.

4. The monoclonal antibody of claim 3, wherein the monoclonal antibody specifically recognizes the amino acid sequence of PSMA protein shown as SEQ ID No. 1.

5. The monoclonal antibody according to claim 1, which is produced by a hybridoma cell line having a collection number of CGMCC No. 19683.

6. The monoclonal antibody of claim 1, wherein the anti-PSMA protein monoclonal antibody is a mouse IgG1 subtype monoclonal antibody.

7. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is prepared from an immunogen which is a recombinant protein consisting of an antigenic fragment of PSMA from amino acid 307 to amino acid 750 of human PSMA and a histidine tag.

8. A hybridoma cell line for secreting monoclonal antibodies against PSMA protein is a mouse hybridoma cell line 11D1E5E10, and is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO. 19683.