CN114507286B

CN114507286B - anti-PD-L1 recombinant rabbit monoclonal antibody and application thereof

Info

Publication number: CN114507286B
Application number: CN202210406174.2A
Authority: CN
Inventors: 张东旭; 李静; 孟凡华; 程育苗; 刘杨
Original assignee: Suzhou Baidao Medical Technology Co ltd
Current assignee: Suzhou Baidao Medical Technology Co ltd
Priority date: 2022-04-18
Filing date: 2022-04-18
Publication date: 2022-07-05
Anticipated expiration: 2042-04-18
Also published as: CN114507286A

Abstract

The invention relates to an anti-PD-L1 recombinant rabbit monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5. Compared with the commercially available anti-PD-L1 recombinant rabbit monoclonal antibody, the anti-PD-L1 recombinant rabbit monoclonal antibody provided by the invention has higher affinity with PD-L1 protein, can identify and detect the expression of PD-L1 protein on tumor cells or immune cells with high specificity and high sensitivity, can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like, is favorable for obtaining more accurate detection and evaluation results, and reduces the detection cost and the interference of background signals.

Description

anti-PD-L1 recombinant rabbit monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-PD-L1 antibody and application thereof, in particular to application in immunohistochemical detection.

Background

PD-L1(programmed cell death ligand 1) is called programmed death receptor ligand 1, and is the ligand of PD-1 (programmed cell death receptor 1). PD-L1 belongs to a transmembrane protein on a cell membrane, is expressed on immune cells such as T cells, B cells and the like and tumor cells, and researches show that when PD-L1 on the tumor cell membrane is combined with PD-1 on the immune cells such as T cells and the like, the tumor cells send inhibitory signals, and then the T cells can not recognize the tumor cells and have killing effect on the tumor cells, so that the immune function of an organism is inhibited.

How to screen patients who may benefit from PD-1/PD-L1 inhibitor therapy is the most clinically interesting issue. The PD-L1 immunohistochemical detection is a simple and effective method for predicting the curative effect of the PD-1/PD-L1 inhibitor. Currently, PD-L1 immunohistochemical detection as a predictive marker of efficacy of PD-1/PD-L1 immune checkpoint inhibitor drugs has been approved by the FDA as a companion or complementary diagnosis for immunotherapy. And an anti-PD-L1 antibody with high sensitivity and strong specificity plays an important role in identifying and detecting the expression of PD-L1 protein on tumor cells or immune cells.

Disclosure of Invention

Technical problem to be solved

In view of the defects and shortcomings of the prior art, the invention provides the anti-PD-L1 recombinant rabbit monoclonal antibody which is wide in application and can accurately identify the expression of PD-L1, and the antibody can well detect the expression of PD-L1 protein on tumor cells or immune cells through immunohistochemical detection of various different tissues, and can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like. The invention also relates to a nucleotide sequence for coding the anti-PD-L1 recombinant rabbit monoclonal antibody, a recombinant plasmid, a preparation method and application of the anti-PD-L1 recombinant rabbit monoclonal antibody in a PD-L1 protein detection method or device, and the like.

(II) technical scheme

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

in a first aspect, the present invention provides an anti-PD-L1 recombinant rabbit monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.

The anti-PD-L1 recombinant rabbit monoclonal antibody (PD-L1 rabbit-derived antibody) can be used for immunohistochemical detection, and can identify and detect the expression of PD-L1 protein on tumor cells or immune cells with high specificity and high sensitivity.

The anti-PD-L1 monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-PD-L1 recombinant rabbit monoclonal antibody provided by the invention is generated by fusion screening of rabbit hybridomas and 293 cell eukaryotic expression. When the anti-PD-L1 monoclonal antibody is prepared, an antigen for an immune rabbit (New Zealand white rabbit) is a synthetic polypeptide, and the amino acid sequence of the synthetic polypeptide is shown as SEQ ID No.1 and is obtained by artificial chemical synthesis. Obtaining a positive hybridoma cell line capable of efficiently secreting the monoclonal antibody by cell fusion and clone screening, obtaining a nucleotide sequence for coding a heavy chain amino acid sequence and a light chain amino acid sequence of the antibody by using a molecular cloning technology, constructing the nucleotide sequence on a eukaryotic expression vector, transfecting a 293 cell line by using a transfection reagent, collecting cell supernatant, and purifying the cell supernatant by Protein A column affinity chromatography to obtain the rabbit monoclonal antibody. Immunohistochemical detection shows that the antibody can specifically recognize PD-L1 protein.

The anti-PD-L1 monoclonal antibody can recognize recombinant PD-L1 antigen protein and PD-L1 molecules on tumor cells and immune cells; the anti-PD-L1 monoclonal antibody can also be applied to immunohistochemical pathological diagnosis agents.

In a second aspect, the present invention provides a coding gene for encoding the above recombinant rabbit monoclonal antibody against PD-L1.

Preferably, the coding gene comprises a DNA sequence shown as SEQ ID No.2 and is used for coding a heavy chain variable region of the anti-PD-L1 recombinant rabbit monoclonal antibody; and the DNA sequence shown as SEQ ID No.3 is used for coding the light chain variable region of the anti-PD-L1 recombinant rabbit monoclonal antibody.

In a third aspect, the present invention relates to a nucleic acid molecule comprising a coding gene for encoding said recombinant anti-PD-L1 rabbit monoclonal antibody.

In a fourth aspect, the invention provides an expression vector comprising a nucleic acid molecule as described above.

In a fifth aspect, the invention provides a preparation method of the anti-PD-L1 recombinant rabbit monoclonal antibody, which comprises the steps of transfecting cells by using the expression vector, culturing the transfected cells, collecting cell supernatant and purifying to obtain the anti-PD-L1 recombinant rabbit monoclonal antibody.

More preferably, the preparation method comprises the following steps:

(1) immunizing a rabbit: firstly, analyzing a PD-L1 protein molecule sequence, selecting and using a proper polypeptide sequence as an immunogen according to the structure, antigenicity, hydrophilicity and hydrophobicity of amino acids and a secondary structure of PD-L1 on a cell membrane, coupling by KLH or OVA to serve as the immunogen, and immunizing a rabbit; the polypeptide is artificially synthesized polypeptide shown in SEQ ID No. 1;

(2) preparation of hybridoma cell lines: obtaining a positive hybridoma stable cell line capable of efficiently secreting antibodies through cell fusion and cloning screening, and separating total RNA from the hybridoma cell line;

(3) obtaining the antibody sequence: obtaining the nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody by using a specific primer and a PCR amplification technology;

(4) antibody expression and purification: cloning the nucleotide sequence into an expression vector, transiently transfecting cultured cells by using a transfection method, collecting supernatant after culturing, and purifying the supernatant by using Protein A to obtain the antibody with the purity of more than 95%.

In a sixth aspect, the anti-PD-L1 recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule and the expression vector are applied to the preparation of a PD-L1 protein molecule detection device. The detection device includes but is not limited to a kit, an antibody chip, and the like.

In a seventh aspect, the invention further provides a PD-L1 detection kit, which comprises the above anti-PD-L1 recombinant rabbit monoclonal antibody and an immunohistochemical detection reagent.

Preferably, the PD-L1 detection kit comprises: the kit comprises an anti-PD-L1 recombinant rabbit monoclonal antibody, an HRP enzyme-labeled secondary antibody, an EDTA repair solution, a catalase blocking solution, a DAB concentrated solution, a DAB buffer solution, hematoxylin and a blue returning solution.

When the immune tissue detection is carried out, the detection steps comprise dewaxing, antigen retrieval, inactivation of endogenous peroxidase, sealing, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration, mounting, microscopic examination and the like.

(III) advantageous effects

The anti-PD-L1 recombinant rabbit monoclonal antibody provided by the invention has high specificity and high sensitivity in combination with a PD-L1 protein molecule, can specifically recognize and detect the expression of PD-L1 protein on tumor cells or immune cells, and shows positive high expression when detecting the PD-L1 protein, so that the antibody can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like, and is favorable for obtaining accurate evaluation and detection results. Compared with the commercially available anti-PD-L1 monoclonal antibody, the PD-L1 recombinant rabbit monoclonal antibody cloned by the AC37 has the characteristics of good specificity, strong positive signal and the like, so that the evaluation is easier in IHC staining, and the detection and the differentiation of cancers are more accurate.

Drawings

FIG. 1 is a comparison graph of the results of immunohistochemical detection of the anti-PD-L1 monoclonal antibody prepared by the present invention and a commercially available anti-PD-L1 monoclonal antibody in human tonsil, placenta and lung cancer tissues, wherein the primary antibody is used at a concentration of 0.6. mu.g/mL.

FIG. 2 is a statistical chart of titer measurements of the AC37 anti-PD-L1 monoclonal antibody and the commercially available anti-PD-L1 monoclonal antibody at 7 gradient concentrations.

FIG. 3 is a graph comparing fluorescence signal intensities (MFI) of a blank control, a commercial anti-PD-L1 antibody sample, and an AC37 anti-PD-L1 monoclonal antibody sample obtained by adding equal amounts of a commercial anti-PD-L1 antibody and an AC37 anti-PD-L1 monoclonal antibody to cells expressing PD-L1 and performing flow cytometry staining analysis.

FIG. 4 shows the detection result of Western blotting of the anti-PD-L1 recombinant rabbit monoclonal antibody AC37 as a primary antibody in example 5, which proves that the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody can specifically recognize the PD-L1 protein.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer. The human tissue samples are formalin-fixed paraffin-embedded human tissue samples, are pathologically verified, and are informed consent of patients.

Example 1

This example is the preparation and screening of recombinant rabbit monoclonal antibodies against PD-L1, comprising the steps of:

(1) antigen preparation

The specific sequence of the PD-L1 antigen is shown as SEQ ID NO. 1.

SEQ ID No. 1：GIQDTNSKKQSDTHLEET。

The polypeptide sequence is selected by analyzing the PD-L1 molecular sequence according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure of PD-L1 protein molecules on cell membranes. The polypeptide with the sequence shown in SEQ ID NO.1 is artificially synthesized, and the synthesized polypeptide is used as the antigen for immunizing rabbits. When in immunization, the polypeptide with the sequence shown in SEQ ID NO.1

Rabbits were immunized as immunogens after coupling via KLH or OVA.

(2) Immunization

The polypeptide sequence (PD-L1 antigen) of SEQ ID NO.1 is respectively mixed and emulsified with complete Freund's adjuvant (1: 1), a plurality of New Zealand white rabbits are respectively immunized by adopting a subcutaneous injection method, and the PD-L1 antigen containing the sequence (polypeptide shown in SEQ ID NO. 1) is emulsified with the complete Freund's adjuvant (1: 1) after two weeks for the second and third immunization. After three times of immunization, blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; selecting the rabbit with the highest titer of the antigen-antibody with SEQ ID NO.1 to perform the next cell fusion.

(3) Cell fusion

Murine sp2/0 myeloma cells were prepared in advance so that the sp2/0 myeloma cells were in logarithmic growth phase at the time of fusion. Taking the spleen of the immunized rabbit, and preparing lymphocyte single cell suspension; mixing rabbit spleen lymphocytes and the myeloma cells, dropwise adding 50% PEG1500, adding IMDM culture medium, centrifuging, removing supernatant, adding HAT culture medium, suspending, mixing, diluting to 800mL, subpackaging in 96-well plate, standing at 37 deg.C with 5% CO₂Culturing in a constant temperature incubator. Observing the state of fused cells in a 96-well plate after fusion for 6-9 days, and continuously placing the fusion medium in a temperature range of 37 ℃ and 5% CO by using HT for liquid exchange₂Culturing in a constant temperature incubator.

(4) Screening and cloning

Cloned cells were screened 7-10 days after fusion by ELISA assay using PD-L1 antigen (SEQ ID NO: 1). Marking the corresponding cell strain number, and performing limited dilution on the positive well cells until the whole plate result of the 96-well plate is positive by ELISA determination. The stable monoclonal antibody with high positive value was selected to obtain a hybridoma cell line secreting a specific monoclonal antibody, which was designated as AC 37.

(5) Performing antibody sequencing on the screened hybridoma cell strain

Total RNA was isolated from AC37 hybridoma cells according to the instructions of the reagent TriZol, reverse-transcribed into cDNA according to the instructions of the TIANCcript first strand cDNA Synthesis kit, amplified using specific primers (heavy chain variable region primer, VH-F: AGACTGGGCTGCGCTGGCTTC, VH-RGTGAGGGTGCCCGAG; light chain variable region primer: VK-F ATGGACAYGAGGGCCCCCACTC, VK-R: GGTGGGAAGATGAGGACAGTAGG) to obtain the nucleotide sequences of the antibody heavy chain variable region and the antibody light chain variable region, and then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk 1) to prepare for cell transfection.

(6) Cell transfection and selection

293 cells to be transfected were prepared in advance, centrifuged to replace fresh medium and placed in 24-well plates at a density of 3 x 10 in a desired amount of 1.5ml per well⁶One per ml.

Will be describedThe eukaryotic expression vector and PEI are mixed according to the proportion of 1:6 and then added into prepared 293 cells, and the mixture is placed at 37 ℃ and 5% CO₂The shaking table of (4) was cultured. After culturing for 3-5 days, performing ELISA detection on the transfected cell supernatant and the corresponding antigen to screen positive holes, and then performing immunohistochemical detection on the cell supernatant of the positive holes, wherein if the immunohistochemical detection is positive, the detected antibody sequence is correct.

(7) Preparation and purification of cell supernatant monoclonal antibody

And (3) performing mass cell transfection on the positive expression vector, continuously culturing for 3-5 days, collecting cell suspension, centrifuging, taking supernatant, and purifying by using an affinity chromatography method. And (4) measuring the concentration of the purified monoclonal antibody, subpackaging and storing in a refrigerator at 4-8 ℃.

Finally, the amino acid sequence of the heavy chain variable region of the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2, and the amino acid sequence of the light chain variable region of the anti-PD-L1 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No. 3.

The specific sequence of SEQ ID Nos. 2-3 is as follows:

SEQ ID No.2：

cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggattctccctcagtagctatagaatgagctgggtccgccaggctccagggaaggggctggaatacatcggaatcattagtgttactgctaacacatactacgcgaactgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaaaattaccagtccgacaaccgaggacacggccacctatttccgtgccagatatggtggtagtggtgattataacatctggggcccaggcaccctggtcaccgtctcctcaa

SEQ ID No.3：

caaattgtgatgacccagactccaccctcggtgtctggagctgtgggaggcacagttaccatcaattgccaggcaagtgagagcattagcaactggttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgctgcatccactctggcatctggggtctcatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcggcgtggagtgtgccgatgctgccacttactactgtcaacagggttggagtgaaagtaatattgagaatactttcggcggagggaccgaggtggtggtcaaag。

translating the obtained base sequence into an amino acid sequence, and analyzing to obtain the heavy chain variable region amino acid sequence of the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody shown as SEQ ID No.4, and the light chain variable region amino acid sequence of the anti-PD-L1 recombinant rabbit monoclonal antibody shown as SEQ ID No. 5.

The specific sequences of SEQ ID Nos. 4 to 5 are as follows:

SEQ ID No.4：

QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYRMSWVRQAPGKGLEYIGIISVTANTYYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFRARYGGSGDYNIWGPGTLVTVSS

SEQ ID No.5:

QIVMTQTPPSVSGAVGGTVTINCQASESISNWLAWYQQKPGQPPKLLIYAASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQGWSESNIENTFGGGTEVVVK。

example 2

This example is an immunohistochemical assay of anti-PD-L1 recombinant rabbit monoclonal antibody as a primary antibody, the method was as follows:

(1) sample section preparation: and (3) baking the formalin-fixed paraffin-embedded human tonsil, placenta and lung cancer tissue sections in a thermostat at 60 ℃ for 1-2h, and storing for later use.

(2) Slicing and dewaxing: paraffin slices are firstly placed in fresh dimethylbenzene for dewaxing, and are soaked for 2 times, and each time lasts for 10 min.

(3) Hydration of the slices: soaking in anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5min for hydration, washing with purified water for 2 times, each for 3 min.

(4) Antigen retrieval: repairing with high temperature heat repairing method for 3min (98 deg.C for 20 min if using automatic repairing instrument), cooling the slices to room temperature, looping the tissue with immunohistochemical pen, washing with purified water for 2 times, 3min each time.

(5) Inactivation of endogenous peroxidase: dropping proper amount of endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 10min, washing with purified water for 2 times (3 min each time), and washing with PBST once.

(6) Primary and commercial control antibody incubations: the tissues were completely covered by the addition of 100. mu.L of AC37 anti-PD-L1 recombinant rabbit monoclonal antibody and commercial PD-L1 control antibody, respectively, incubated for 1h at 37 ℃ in an incubator and washed 3 times with PBST, 5min each.

(7) And (3) secondary antibody incubation: and (4) performing secondary antibody incubation according to the instruction of the DAB staining solution kit of the secondary antibody staining system, after the incubation is finished, washing the PBST by using the washing sheet for 3 times, 5min each time, and washing by using purified water for 1 time.

(8) DAB color development: preparing DAB color developing solution according to the DAB staining solution kit instruction, dripping proper amount of the prepared DAB color developing solution until the tissue is completely covered, stopping staining when the color is not deepened, and washing with purified water for 3 times.

(9) Hematoxylin counterstaining: the sections were counterstained according to the protocol and recommendations of the hematoxylin manufacturer's instructions, washed back to blue with PBST or tap water.

(10) And (3) dehydrating and transparency: soaking in 70%, 85%, 95%, 100%, 100% gradient alcohol for 3min each time; 2 times xylene clear, 5min each time.

(11) And (3) sealing: the samples were mounted with neutral gum.

As can be seen from the results in FIG. 1, the PD-L1 protein showed specific cell membrane staining in human tonsil, placenta and lung cancer tissues, and the PD-L1 signal of the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody sample set was stronger in all three tissues than that of the commercial PD-L1 antibody set at the same antibody concentration (0.6. mu.g/ml). The PD-L1 group which is sold in the market has weak staining positive signals, and the problem of low detection sensitivity exists in some early cancer tissues, while the PD-L1 recombinant rabbit monoclonal antibody cloned by AC37 has the characteristics of good specificity, strong positive signals and the like, so that the evaluation is easier in IHC staining, and the detection and the cancer differentiation are more accurate.

Example 3

This example is a determination of the affinity of AC37 for recombinant rabbit monoclonal antibody PD-L1 as follows:

(1) the purified PD-L1 protein was removed from the column at 4 ℃ and returned to room temperature. Diluted to a concentration of 1. mu.g/ml, added to a 96-well plate at 100. mu.L/well and incubated overnight at 4 ℃ followed by blocking overnight at 4 ℃ with 2% BSA.

(2) The PD-L1 recombinant rabbit monoclonal antibody cloned from AC37 and the commercial anti-PD-L1 antibody were diluted to an initial concentration of 0.5. mu.g/mL, and were serially diluted in 2-fold gradients, setting up 7 concentration gradients for comparison.

(3) Respectively adding the diluted anti-PD-L1 recombinant rabbit monoclonal antibody to a 96-well enzyme label plate with polypeptide according to 100 mu L/well, covering a sealing plate membrane, and incubating at the constant temperature of 37 ℃ for 1h to balance the reaction.

(4) And (3) taking out the enzyme label plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and patting to dry the water.

(5) The HRP-labeled goat anti-rabbit IgG was diluted according to the instructions for the use of the secondary antibody, added to the ELISA plate at 100. mu.L/well, and incubated at 37 ℃ for 1h to allow the reaction to reach equilibrium.

(6) And (3) taking out the enzyme label plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and patting to dry the water.

(7) TMB developing solution was added to the reaction vessel at a rate of 100. mu.L/well, and the reaction was carried out at room temperature for 6 minutes.

(8) After the reaction was complete, 2M H was added at 50. mu.L/well₂SO₄The color development was terminated.

(9) OD values were read at 450nm on a microplate reader, and the data were collated and analyzed as shown in FIG. 2. The results show that in 7 concentration gradient comparison tests, the anti-PD-L1 recombinant rabbit monoclonal antibody cloned by AC37 has stronger affinity to PD-L1 protein molecules and higher sensitivity compared with the commercial antibody under each gradient concentration, can still reach higher OD value under the condition of lower antibody concentration, and can save the test and detection cost.

Example 4

This example performs flow cytometry staining analysis of AC37 secreted anti-PD-L1 antibody and a commercially available anti-PD-L1 antibody, according to the following embodiment:

(1) 1 x 10^6 cells expressing the PD-L1 protein were collected, washed once with 500. mu.L PBS, and resuspended in 100. mu.L PBS.

(2) 0.5. mu.g of a commercially available anti-PD-L1 antibody and the AC37 cloned anti-PD-L1 recombinant rabbit monoclonal antibody were added to the cells, mixed well by gentle blowing and incubated for 15min at room temperature.

(3) Cells were incubated for 2 washes with 500 μ L PBS and resuspended in 100 μ L PBS.

(4) 0.5 mu g of Alexa fluor 488 fluorescence-labeled goat anti-rabbit fluorescent secondary antibody is added into the cells, and the cells are evenly mixed by light blowing and incubated for 15min at room temperature in a dark place.

(5) The cells were washed 2 times with 500. mu.L PBS to wash the residual fluorescent secondary antibody and resuspended in 500. mu.L PBS for detection on the flow machine, and 20000 cells were sampled for analysis of the intensity of the fluorescence signals of the two antibodies. The fluorescence signal intensity (MFI) results are shown in Table 1, and the flow assay results are shown in FIG. 3.

Table 1:

the experimental results show that the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody has better antibody affinity, and higher fluorescence signal intensity can be obtained under the condition of the same antibody dosage.

Example 5

This example is an immunoblotting (Western blotting) assay of the recombinant rabbit monoclonal antibody AC37 against PD-L1 as a primary antibody, the method is as follows:

(1) selecting PVDF membranes of 293-PD-L1 cell lysate and 293 cell lysate for activation, activating with methanol for 1min, washing the membrane with pure water for 2 times, and then washing with TBST for 3 times;

(2) and (3) sealing: placing the membrane in a blocking solution prepared from 5% BSA, and mixing and shaking at room temperature for 2 h;

(3) primary antibody incubation: diluting the AC37 antibody to 0.5 mu g/mL concentration, putting the sealed membrane into the corresponding diluted antibody, and incubating overnight at 4 ℃ with mixed shaking;

(4) taking out the membrane, and washing in TBST solution for 3 times (2 × 5min +1 × 10 min);

(5) and (3) secondary antibody incubation: diluting HRP-anti-rabbit IgG with FG solution at a ratio of 1:5000, mixing uniformly, adding a membrane strip, and mixing and shaking at room temperature for 1 h;

(6) taking out the membrane strip, and washing the membrane strip in TBST solution for 4 times (3 × 5min +1 × 8 min);

(7) substrate: mixing the same amount of Luminol/enhancer solution and Peroxide solution diluted by 5 times with pure water in the same container, adding membrane strip, and incubating for 2 min;

(8) exposure: placing the negative film in a cassette, and respectively exposing the X-ray film for different time periods according to the fluorescence intensity; then carrying out the operations according to the sequence of developing, cleaning and fixing for 1min, and finally cleaning and drying. The results are shown in FIG. 4.

As can be seen from the results of fig. 4, wherein lane 1 (left): HEK 293 whole cell lysate transfected with PD-L1, lane 2 (right): 293 whole cell lysate, band size: 45 kDa. In lane 1 (left), the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody can specifically recognize 293 cell over-expressed PD-L1 protein with a molecular weight of about 45kd, while in lane 2 (right), the AC37 anti-PD-L1 recombinant rabbit monoclonal antibody does not recognize other proteins, which shows that the PD-L1 recombinant rabbit monoclonal antibody cloned by AC37 of the present invention can specifically recognize PD-L1 protein.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Suzhou Baidao medical science and technology Co., Ltd

<120> anti-PD-L1 recombinant rabbit monoclonal antibody and application thereof

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<141> 2022-02-15

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Claims

1. An anti-PD-L1 recombinant rabbit monoclonal antibody, which is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.

2. A gene encoding the recombinant rabbit monoclonal antibody against PD-L1 of claim 1.

3. The coding gene according to claim 2, characterized in that it comprises: the DNA sequence shown in SEQ ID No.2 for encoding the heavy chain variable region of the anti-PD-L1 recombinant rabbit monoclonal antibody, and the DNA sequence shown in SEQ ID No.3 for encoding the light chain variable region of the anti-PD-L1 recombinant rabbit monoclonal antibody.

4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A preparation method of an anti-PD-L1 recombinant rabbit monoclonal antibody is characterized in that the expression vector of claim 5 is adopted to transfect cells, the cells are continuously cultured after the transfection, cell supernatants are collected and purified, and the anti-PD-L1 recombinant rabbit monoclonal antibody is obtained.

7. Use of the anti-PD-L1 recombinant rabbit monoclonal antibody of claim 1, the coding gene of claim 2 or 3, the nucleic acid molecule of claim 4, the expression vector of claim 5 for the preparation of a PD-L1 detection device.

8. A PD-L1 detection kit, characterized in that it comprises the anti-PD-L1 recombinant rabbit monoclonal antibody of claim 1 and immunohistochemical detection reagents.

9. The PD-L1 test kit according to claim 8, characterized in that it comprises: the kit comprises an anti-PD-L1 recombinant rabbit monoclonal antibody, an HRP enzyme-labeled secondary antibody, an EDTA repair liquid, a catalase blocking liquid, a DAB concentrated solution, a DAB buffer solution, hematoxylin and a blue returning liquid.