CN115073602A - anti-CD 3 recombinant rabbit monoclonal antibody and application thereof - Google Patents
anti-CD 3 recombinant rabbit monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to an anti-CD 3 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. The anti-CD 3 recombinant rabbit monoclonal antibody provided by the invention has very high affinity with CD3 protein, can identify and detect the expression of CD3 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
Technical Field
The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-CD 3 antibody and application thereof, in particular to application in immunohistochemical detection.
Background
The CD3 molecule is a composite molecule composed of peptide chains with non-covalent bonds, is expressed on the surface of mature T cells, and plays a main role in blocking acute allograft rejection. The CD3 molecule and T cell antigen recognition receptor TCR form a composite receptor molecule, and activate tyrosine kinase to promote tyrosine (Y) phosphorylation in an immunoreceptor tyrosine activation motif of the CD3 molecule. The phosphorylated tyrosine (pY) further phosphorylates downstream tyrosine-containing proteins, thereby causing a cascade of kinase activation, regulating the target genes for T cell proliferation and activation, causing gene expression and transcription, and transforming T cells from a resting state to a proliferative and activated state. The CD3 molecule has the functions of stabilizing the TCR structure and transmitting an activation signal. The monoclonal antibody aiming at the CD3 molecule can stimulate or block T cell activation signal transduction, eliminate effector T cells or induce the generation of regulatory T cells, and provides a new method for treating organ transplant rejection and autoimmune diseases.
Since the 90 s of the 20 th century, the CD3 monoclonal antibody is applied to CIK cell therapy to activate T cell proliferation and activation, and under the combined action of other cytokines IL2, IL1a and the like, the CIK cell with the characteristics of high proliferation speed, high tumor killing activity, wide tumor killing spectrum and non-MHC-restricted tumor killing is generated, so that the CIK cell has obvious curative effects on various diseases such as cancer, chronic leukemia, liver diseases, neurological diseases and the like. The antibody can be used as a whole T cell marker of normal T cells, NK cells and tumors derived from the NK cells, and is helpful for diagnosis of T cell lymphoma and NK cell lymphoma. The specificity of CD3 for T cells, coupled with its ability to appear in all stages of T cell development, makes it an ideal marker for the detection of healthy T cells and T cell cancers (e.g., leukemia, lymphoma). In immunohistochemistry studies, CD3 is an immunohistochemical marker of T cells in tissue sections.
Disclosure of Invention
Technical problem to be solved
In view of the defects and shortcomings of the prior art, the invention provides the anti-CD 3 recombinant rabbit monoclonal antibody which is wide in application and can accurately identify CD3 expression, and the antibody can well detect the expression of CD3 protein on tumor cells or immune cells through immunohistochemical detection of various tissues, and can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, Western blotting (Western blotting), antibody chip preparation, flow cytometry and the like. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-CD 3 recombinant rabbit monoclonal antibody, a preparation method and application of the anti-CD 3 recombinant rabbit monoclonal antibody in a CD3 protein detection method or device.
(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-CD 3 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-CD 3 recombinant rabbit monoclonal antibody (CD3 rabbit source antibody) can be used for immunohistochemical detection, and can identify and detect the expression of CD3 protein on tumor cells or immune cells with high specificity and high sensitivity.
The anti-CD 3 monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-CD 3 recombinant rabbit monoclonal antibody provided by the invention is generated by fusion screening of rabbit hybridomas and 293 cell eukaryotic expression. When the anti-CD 3 monoclonal antibody is prepared, an antigen for an immune rabbit (New Zealand white rabbit) is a synthetic polypeptide, the amino acid sequence of the synthetic polypeptide is shown as SEQ ID No.1, and the synthetic polypeptide is obtained by artificial chemical synthesis. After immunization, cell fusion and clone screening are carried out to obtain a positive hybridoma cell line capable of efficiently secreting the monoclonal antibody, a molecular cloning technology is used for obtaining a nucleotide sequence for coding a heavy chain amino acid sequence and a light chain amino acid sequence of the antibody, the nucleotide sequence is constructed on a eukaryotic expression vector, the eukaryotic expression vector is transfected into a 293 cell line through a transfection reagent, cell supernatant is collected, and the cell supernatant is purified through Protein A column affinity chromatography to obtain the rabbit monoclonal antibody. Immunohistochemical detection showed that the antibody specifically recognized the CD3 protein.
The anti-CD 3 monoclonal antibody can recognize a recombinant CD3 antigen protein and a CD3 molecule on tumor cells and immune cells; the anti-CD 3 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 recombinant rabbit monoclonal antibody against CD3 described above.
Preferably, the coding gene comprises a DNA sequence shown as SEQ ID No.2, and is used for coding the heavy chain variable region of the anti-CD 3 recombinant rabbit monoclonal antibody; and the DNA sequence shown in SEQ ID No.3 is used for coding the light chain variable region of the anti-CD 3 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 rabbit monoclonal antibody against CD 3.
In a fourth aspect, the invention provides an expression vector or recombinant plasmid comprising a nucleic acid molecule as described above.
In a fifth aspect, the invention provides a preparation method of the anti-CD 3 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-CD 3 recombinant rabbit monoclonal antibody.
More preferably, the preparation method comprises the following steps:
(1) immunizing a rabbit: analyzing a CD3 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 CD3 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-CD 3 recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule, the expression vector or the recombinant plasmid are applied to the preparation of a CD3 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 present invention further provides a CD3 detection kit, which includes the above-mentioned anti-CD 3 recombinant rabbit monoclonal antibody and an immunohistochemical detection reagent.
Preferably, the CD3 test kit comprises: the kit comprises an anti-CD 3 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-CD 3 recombinant rabbit monoclonal antibody provided by the invention can be combined with an epsilon chain of a CD3/T cell antigen receptor complex, and is used for flow cell and immunofluorescence applications to identify immune cell subsets expressing CD 3. Cells expressing the surface marker of CD3 are known to include about 60-85% normal peripheral blood lymphocytes, about 65-85% thymocytes, and Purkinje cells of cerebellum and parietal cells. Therefore, the antibody can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, Western blotting, antibody chip preparation, flow cytometry and the like, and is beneficial to obtaining accurate evaluation and detection results. In immunohistochemical research, the CD3 recombinant rabbit monoclonal antibody cloned in 230B2F6 has the characteristics of good specificity and high sensitivity.
Drawings
FIG. 1 is the result of immunohistochemical detection of the monoclonal antibody against CD3 in human appendices, breast cancers, colon cancers and lung adenocarcinoma tissues, and the primary antibody concentration is 0.5. mu.g/mL.
FIG. 2 is a statistical chart of titer detection of the 230B2F6 anti-CD 3 monoclonal antibody at 7 gradient concentrations.
FIG. 3 is a graph comparing fluorescence signal intensity (MFI) of a blank control and a 230B2F6 anti-CD 3 monoclonal antibody sample obtained by adding a quantitative 230B2F6 anti-CD 3 monoclonal antibody to cells expressing CD3 protein and then performing flow cytometry staining analysis.
FIG. 4 shows the result of Western blotting (Western blotting) of the 230B2F6 anti-CD 3 monoclonal antibody of the invention as a primary antibody to verify its recognition ability to CD3 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 that no limitation of the invention is intended.
The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. 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 by consent of patients.
Example 1
This example is the preparation and screening of recombinant rabbit monoclonal antibodies against CD3, comprising the steps of:
(1) antigen preparation
The specific sequence of the CD3 antigen is shown as SEQ ID NO. 1.
SEQ ID No.1：KGQRDLYSGLNQRRI。
The polypeptide sequence is selected by analyzing the sequence of the CD3 molecule according to the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure of the CD3 protein molecule on the cell membrane. 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. In the immunization, the polypeptide with the sequence shown in SEQ ID NO.1 is coupled through KLH or OVA and then used as immunogen to immunize rabbits.
(2) Immunization
The polypeptide sequence (CD3 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 CD3 antigen containing the sequence (the polypeptide shown in SEQ ID NO:1) is emulsified with the complete Freund's adjuvant (1:1) after two weeks to carry out the second immunization and the 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, and the sp2/0 myeloma cells were fusedIn the logarithmic growth phase. 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% CO 2 Culturing 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 exchange 2 Culturing in a constant temperature incubator.
(4) Screening and cloning
Cloned cells were screened 7-10 days after fusion by ELISA assay using CD3 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 strains with high positive values were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies, which were recorded as 230B2F 6.
(5) Performing antibody sequencing on the screened hybridoma cell strain
Total RNA was isolated from 451I3V0 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-R GTGAGGGTGCCCGAG; 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 a eukaryotic expression vector (InvivoGen, pfuse-rchg, pfuse2-rclk1) to prepare for cell transfection.
(6) Cell transfection and selection
293 cells to be transfected are prepared in advance, centrifuged to replace fresh medium and placed in 24-well plates with a density of 3X 10 and a required amount of 1.5ml per well 6 One per ml.
Mixing the eukaryotic expression vector and PEI according to the proportion of 1:6, adding the mixture into prepared 293 cells, and placing the cells at 37 ℃ and 5% CO 2 The shaking table of (4) was cultured. Culture No.3After 5 days, screening positive wells by ELISA detection of the transfected cell supernatants and the corresponding antigens, and further performing immunohistochemical detection on the cell supernatants of the positive wells, and confirming that the detected antibody sequences are correct if the immunohistochemical detection is positive.
(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. 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 230B2F6 anti-CD 3 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-CD 3 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No. 3.
The specific sequence of SEQ ID No.2-3 is as follows:
SEQ ID No.2：
cagtcgttggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcaatgactactggatgagttgggtccgccaggctccagggaaggggctggaatggatcgggattattattcgtgggggtaccacatggtacgcgaattgggtgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaagatgaccagcccgacaaccgaggacacggccacctatttctgtgcccgaattggtggtggtaatactggttttaatttgtggggcccaggcaccctggtcaccgtctcctcaa。
SEQ ID No.3：
gcccaagtgctgacccagactccatcctccgtgtctgaacctgtgggaggcacagtcaccatcaattgccaggccagtgagaacatttacatctctttagcctggtatcagcagaaaccagggcagcctcccaatctcctgatctatgatgcatccgatctggcatctggggtcccatcgcgcttcagcggcagtggatctgggacagagttcactctcaccatcagcggcgtgcagtgtgaagatgctgccacttactactgtcaaagctataattatagtagtgttactttcggcggagggaccgaggtggtggtcaaag。
the obtained base sequence is translated into an amino acid sequence, and the amino acid sequence of the heavy chain variable region of the 230B2F6 anti-CD 3 recombinant rabbit monoclonal antibody is shown as SEQ ID No.4, and the amino acid sequence of the light chain variable region of the anti-CD 3 recombinant rabbit monoclonal antibody is shown as SEQ ID No. 5.
The specific sequences of SEQ ID Nos. 4 to 5 are as follows:
SEQ ID No.4：
QSLEESGGRLVTPGTPLTLTCTASGFSLNDYWMSWVRQAPGKGL EWIGIIIRGGTTWYANWVKGRFTISKTSTTVDLKMTSPTTEDTATYFCA RIGGGNTGFNLWGPGTLVTVSS。
SEQ ID No.5:
AQVLTQTPSSVSEPVGGTVTINCQASENIYISLAWYQQKPGQPPNL LIYDASDLASGVPSRFSGSGSGTEFTLTISGVQCEDAATYYCQSYNYSS VTFGGGTEVVVK。
example 2
This example is an immunohistochemical assay of anti-CD 3 recombinant rabbit monoclonal antibody as a primary antibody, the method was as follows:
(1) preparation of a sample section: and baking the formalin-fixed paraffin-embedded appendix, breast cancer, colon cancer and lung adenocarcinoma 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 20min 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 antibody incubation: the tissue was completely covered with 100. mu.L of 0.5. mu.g/mL 230B2F6 recombinant rabbit monoclonal antibody to CD3, incubated at 37 ℃ for 1h, and washed 3 times with PBST for 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 specification, dripping a 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, 5min each time, xylene was clear.
(11) Sealing: the samples were mounted with neutral gum.
As can be seen from the results in FIG. 1, the CD3 protein shows specific cell membrane staining in the tissues covered by the 230B2F6 anti-CD 3 antibody, such as human appendix, breast cancer, colon cancer, lung adenocarcinoma, and the like. Therefore, the CD3 recombinant rabbit monoclonal antibody cloned from 230B2F6 is characterized by good specificity, strong positive signal and the like, so that the scoring in IHC staining is easier, and the detection and the differentiation of cancers are more accurate.
Example 3
This example is a determination of the affinity of 230B2F6 for recombinant rabbit monoclonal antibody against CD3 by the following method:
(1) the labeled CD3 polypeptide was removed from 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 CD3 recombinant rabbit monoclonal antibody cloned at 230B2F6 was diluted to an initial concentration of 0.5. mu.g/mL and serially diluted in 2-fold gradients for a total of 7 concentration gradients for comparison.
(3) And adding the diluted anti-CD 3 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/well 2 SO 4 The 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 result shows that in 7 concentration gradient tests, the anti-CD 3 recombinant rabbit monoclonal antibody cloned in 230B2F6 has strong affinity and high sensitivity to CD3 protein molecules, can still reach a 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 230B2F6 secreted anti-CD 3 antibody, the embodiment is as follows:
(1) 1X 10^6 cells expressing the CD3 protein were collected, washed once with 500. mu.L PBS, and resuspended in 100. mu.L PBS.
(2) To the cells, 0.5. mu.g of the anti-CD 3 recombinant rabbit monoclonal antibody cloned in 230B2F6 was added, mixed well with gentle blowing and incubated at room temperature for 15 min.
(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 out residual fluorescent secondary antibody and resuspended in 500. mu.L PBS for flow-on-machine detection, and 20000 cells were sampled to analyze the intensity of the fluorescence signal in both cases (no antibody and addition of 230B2F6 antibody). The results of fluorescence signal intensity relative values (MFI) are shown in Table 1, and the results of flow assay are shown in FIG. 3.
Table 1:
| sample (I) | MFI |
| Blank control | 1.48 |
| 230B2F6 antibody | 1585 |
The above experimental results show that the 230B2F6 anti-CD 3 recombinant rabbit monoclonal antibody of the invention has higher antibody affinity, compared with the blank control group, under the condition of the same cell number, the MFI signal intensity of the 230B2F6 added antibody group is increased by 1070 times, which indicates that the 230B2F6 anti-CD 3 recombinant rabbit monoclonal antibody of the invention can obtain very high fluorescence signal intensity.
Example 5
This example is an immunoblotting (Western blotting) assay of the anti-CD 3 recombinant rabbit monoclonal antibody 230B2F6 as a primary antibody, as follows:
(1) activating PVDF membrane of cell lysate of rabbit thymus tissue lysate, activating with methanol for 1min, washing with pure water for 2 times, and washing with TBST for 3 times; and (3) sealing: placing the membrane in a blocking solution prepared from 5% BSA, and mixing and shaking at room temperature for 2 h;
(2) primary antibody incubation: diluting 230B2F6 antibody to 0.5 μ g/mL, placing the sealed membrane into the corresponding diluted antibody, and incubating overnight at 4 deg.C with shaking;
(3) taking out the membrane, and washing in TBST solution for 3 times (2 × 5min +1 × 10 min);
(4) 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;
(5) taking out the membrane strip, and washing the membrane strip in TBST solution for 4 times (3 × 5min +1 × 8 min);
(6) 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;
(7) exposure: the negative film is put in a cassette, and the X-ray film is respectively exposed 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. Wherein r.thymus represents rabbit thymus tissue lysate; band size: 19 kDa.
As can be seen from the results in fig. 4, in the lane, the 230B2F6 anti-CD 3 recombinant rabbit monoclonal antibody can specifically recognize CD3 protein in rabbit thymus, and the molecular weight is about 19kDa, which indicates that the CD3 recombinant rabbit monoclonal antibody cloned in 230B2F6 of the present invention can recognize CD3 protein with high specificity.
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-CD 3 recombinant rabbit monoclonal antibody and application thereof
<130> EJS220570I
<141> 2022-05-24
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Claims (10)
1. An anti-CD 3 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 as 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 to CD3 of claim 1.
3. The encoding gene of claim 2, comprising: a DNA sequence shown as SEQ ID No.2 for encoding the heavy chain variable region of the anti-CD 3 recombinant rabbit monoclonal antibody, and a DNA sequence shown as SEQ ID No.3 for encoding the light chain variable region of the anti-CD 3 recombinant rabbit monoclonal antibody.
4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.
5. An expression vector or recombinant plasmid comprising the nucleic acid molecule of claim 4.
6. A preparation method of an anti-CD 3 recombinant rabbit monoclonal antibody, which is characterized in that the expression vector of claim 5 is adopted to transfect cells, the cells are continuously cultured after the transfection, cell supernatant is collected and purified, and the anti-CD 3 recombinant rabbit monoclonal antibody is obtained.
7. The method of claim 6, comprising the steps of:
(1) immunizing a rabbit: firstly analyzing the sequence of CD3 molecule, selecting and using proper polypeptide sequence as immunogen according to the structure, antigenicity, hydrophilicity and hydrophobicity of amino acid and secondary structure of CD3 molecule on cell membrane, and taking the immunogen as an immunogen to immunize rabbit after coupling by KLH or OVA; the polypeptide sequence 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 a eukaryotic expression vector, transiently transfecting the cultured 293 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%.
8. Use of the recombinant rabbit monoclonal antibody against CD3 of claim 1, the coding gene of claim 2 or 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant plasmid for the preparation of a CD3 detection device.
9. A CD3 test kit comprising the anti-CD 3 recombinant rabbit monoclonal antibody of claim 1 and immunohistochemical detection reagents.
10. The CD3 detection kit according to claim 9, characterized in that it comprises: the kit comprises an anti-CD 3 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.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116355093A (en) * | 2023-06-02 | 2023-06-30 | 苏州百道医疗科技有限公司 | anti-CK 7 recombinant rabbit monoclonal antibody and application thereof |
| CN117700544A (en) * | 2024-02-05 | 2024-03-15 | 苏州百道医疗科技有限公司 | anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9021679D0 (en) * | 1990-10-05 | 1990-11-21 | Gorman Scott David | Antibody preparation |
| US5968509A (en) * | 1990-10-05 | 1999-10-19 | Btp International Limited | Antibodies with binding affinity for the CD3 antigen |
| CN113234161A (en) * | 2021-06-24 | 2021-08-10 | 福州迈新生物技术开发有限公司 | anti-CD 3 protein monoclonal antibody and cell strain, preparation method and application thereof |
-
2022
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9021679D0 (en) * | 1990-10-05 | 1990-11-21 | Gorman Scott David | Antibody preparation |
| WO1992006193A1 (en) * | 1990-10-05 | 1992-04-16 | Scott David Gorman | Antibodies directed against cd3 |
| US5968509A (en) * | 1990-10-05 | 1999-10-19 | Btp International Limited | Antibodies with binding affinity for the CD3 antigen |
| CN113234161A (en) * | 2021-06-24 | 2021-08-10 | 福州迈新生物技术开发有限公司 | anti-CD 3 protein monoclonal antibody and cell strain, preparation method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116355093A (en) * | 2023-06-02 | 2023-06-30 | 苏州百道医疗科技有限公司 | anti-CK 7 recombinant rabbit monoclonal antibody and application thereof |
| CN116355093B (en) * | 2023-06-02 | 2023-08-18 | 苏州百道医疗科技有限公司 | anti-CK 7 recombinant rabbit monoclonal antibody and application thereof |
| CN117700544A (en) * | 2024-02-05 | 2024-03-15 | 苏州百道医疗科技有限公司 | anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof |
| CN117700544B (en) * | 2024-02-05 | 2024-04-16 | 苏州百道医疗科技有限公司 | anti-MSH 2 recombinant rabbit monoclonal antibody and application thereof |
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