CN108084263B - Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof - Google Patents
Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-human CD25 chimeric monoclonal antibody and a preparation method thereof, which comprises an amino acid sequence for coding a variable region of the antibody, a preparation method and antibody function identification. The invention extracts heavy chain variable region (mVH) and light chain variable region (mVL) from hybridoma secreting anti-human CD25 monoclonal antibody, connects with cloning vector (pJET cloning vector), transforms competent bacterium DH5a, reserves candidate heavy chain and light chain variable region sequence according to sequencing result, PCR amplifies heavy chain and light chain variable region sequence matched with expression vector again, connects PCR product with linear expression vector pretreated by double enzyme digestion, transforms the connection product into competent bacterium DH5a, and extracts plasmid after amplification culture. The expression vector connected with the target monoclonal antibody heavy chain and light chain variable region genes cotransfects the eukaryotic expression cell strain 293. The supernatant obtained by culture contains the target antibody, the flow detection expression monoclonal antibody is well combined with PBMC, the positive rate reaches more than 95 percent, and the CD25 mouse/human chimeric antibody is obtained.
Description
Technical Field
The invention relates to a preparation method of an antibody, in particular to an anti-human CD25 chimeric monoclonal antibody, a preparation method and application thereof, belonging to the technical field of biology.
Background
CD25, the alpha chain of the interleukin 2 (IL-2) heterotrimeric receptor complex, forms an IL-2 receptor complex with the beta (CD 122) and gamma (CD 132) chains of the IL-2 receptor. IL-2 is interleukin-2 (IL-2), also known as T cell growth factor (TCRF). Mainly from activated CD4+Th1 cells produce cytokines with a wide range of biological activities. Th0 and CTL are promoted to proliferate, so that they are important factors for regulating immune response and are also involved in antibody response, hematopoiesis and tumor surveillance.
IL-2 receptor alpha chain cytoplasmic region is short, and IL-2R beta and IL-2R gamma dimer form is an essential structure of IL-2 signal channel, but only with IL-2R beta and IL-2R gamma dimer form, IL-2 can not effectively through IL-2 receptor complex signal channel regulation of T cell proliferation, so IL-2 receptor alpha, namely CD25 has the function of improving the affinity of receptor and IL-2, and is mainly expressed in CD4+T cell membrane surface involved in regulatory CD4+T cells inDifferentiation and proliferation proceed at physiologically low levels of IL-2.
The interleukin 2 receptor complex does not have the capacity of activating a signal path, and activates a downstream signal conduction path by coupling with a non-receptor type protein tyrosine kinase, and the downstream signal conduction path is mainly conducted through 3 signal paths: in the Jak-STAT approach, Jak kinase is activated, the Jak protein binding part in the intracellular section of the IL-2R is phosphorylated, and STAT factors are started to be transferred into cell nucleus, so that target genes play a role in regulating cell proliferation and apoptosis; MAP kinase path, after Jak kinase and Syk kinase are activated, IL-2R intracellular segment is phosphorylated to trigger Shc protein and Grb-2 protein cascade activation, Raf-1 protein with serine/threonine protein kinase activity starts MAP kinase path, in addition, Pyk2 kinase can be excited after Jak kinase is activated, thus starting MAP kinase path, finally regulating cyclin dependent kinase to act on specific substrate, and promoting cell proliferation; the phosphoinositide-3-kinase pathway needs the participation of Jak kinase and Pyk2 kinase, lck kinase is used as a promoter, and the phosphoinositide-3-kinase is combined with Shc protein to stimulate the expression of oncogenes Akt, p70 and S6k and participate in apoptosis and proliferation activities.
CD4+CD25+Foxp3+Detection phenotype of cells being regulatory T cells (Tregs), CD4+CD25+T cells were also identified as tregs in some studies. CD4+CD25+T cells are in different proportions in normal tissues or peripheral blood, but are obviously increased in peripheral blood and tumor local tissue cells of patients with various malignant solid tumors such as lung cancer, ovarian cancer, gastrointestinal tumors, breast cancer and the like. Regulatory T cells secrete cytostatic factors, such as IL-10, which exert immunosuppressive functions, and also exert inhibitory effects through cell contact, both of which may be present together and synergistically contribute to the immunosuppressive response in the tumor milieu. The regulatory T cells mainly secrete IL-10 and TGF-beta to produce immunosuppressive action, wherein the IL-10 has the function of mediating contact immunosuppressive between the regulatory T cells and various immune cells. When the tumor metastasizes, lymph node tumor infiltration is formed in lymph nodesThe up-regulated tregs cooperate with tumor cells to block the anti-tumor immune effect. Thus, CD4 in tumor patients+CD25+Foxp3+Elevated levels of regulatory T cells are a poor factor in the prognosis of neoplastic disease.
Regulatory T cell expression is elevated in chronic lymphocytic leukemia patients, particularly in patients prior to chemotherapy, and decreased after fludarabine administration, despite fludarabine's reduced CD4+The side effect of the T cell is mainly achieved by reducing precursor T cells, so that the immunosuppression effect of the T cell of a chronic stranguria patient is reduced, the activity of an immune system inhibition signal conduction pathway is reduced, an organism is promoted to play an anti-tumor immunity effect, and the tumor immunity tolerance is broken. It has been suggested that anti-CD 25 monoclonal antibody drugs can induce apoptosis of tumor cells after administration to leukemia and fibrosarcoma patients. Thus, CD25 may also be a target for tumor therapy.
Disclosure of Invention
The invention provides an anti-human CD25 chimeric monoclonal antibody; the invention extracts heavy chain variable region (mVH) and light chain variable region (mVL) from hybridoma secreting anti-human CD25 monoclonal antibody, reserves candidate heavy chain and light chain variable region sequence according to sequencing result, PCR amplifies heavy chain and light chain variable region sequence matched with expression vector, connects PCR product with linear expression vector pretreated by double enzyme digestion, and the connection product transforms competent bacterium DH5 a. The expression vector connected with the target monoclonal antibody heavy chain and light chain variable region genes co-transfects a eukaryotic expression cell strain 293, the supernatant obtained by culturing contains the target antibody, and the CD25 mouse/human chimeric antibody is obtained by screening and purifying.
The invention clones the anti-human CD25 monoclonal variable region gene to a eukaryotic expression vector, transfects 293 cells to obtain the anti-human CD25 chimeric monoclonal antibody, and then compares the affinity and biological functions with other similar antibodies.
In order to achieve the purpose, the technical scheme of the invention is as follows:
as a first aspect of the present invention, a protein which is a heavy chain variable region (mVH) of a chimeric monoclonal antibody against human CD25 and which has an amino acid sequence identical to that of SEQ ID NO.1,
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYAISWVRQPPGKGLEWLGVMWTGGGTNYNSALKSRLNISKDNSKSQVFLKMNSLQTEDTARYYCARPYYYDGSWFAYWGQGTLVTVSA。
wherein the heavy chain variable region (mVH) has three hypervariable regions: GFSLTSYA, MWTGGGT, ARPYYYDGSWFAY.
As a second aspect of the present invention, a protein, which is a light chain variable region (mVL) of a chimeric monoclonal antibody against human CD25, and which has the same amino acid sequence as that of SEQ ID NO.2,
DIVLSQSPAILSASPGEKVTMTCRASSSVGYMHWYQQKPGSSPKPWIYATSNLASGVPVRFSGSESGTSYSLTISRVEAEDAATYYCQQWSSNPPTFGGGTKLEIK。
wherein the light chain variable region (mVL) has three hypervariable regions: SSVGY, ATS, QQWSNPPT.
As a third aspect of the present invention, a method for producing a chimeric monoclonal antibody against human CD25, comprising the steps of:
firstly, preparing a hybridoma of a CD25 monoclonal antibody;
immunizing a mouse;
(II) cell culture;
(III) fusing and screening;
secondly, preparing a CD25 mouse/human chimeric antibody;
(1) extraction of cDNA from hybridoma cells: extracting RNA from the hybridoma cell strain, and performing reverse transcription on the obtained RNA into cDNA by using an RT-PCR technology;
cloning the heavy chain variable region (mVH) and the light chain variable region (mVL) of the hybridoma cells by using an upstream primer and a downstream primer which are specially designed;
(1) the heavy chain variable region (mVH) and the light chain variable region (mVL) are respectively connected with a cloning vector (pJET cloning vector), a connecting product is transformed into a competent bacterium DH5a, and as the pJET vector carries an ampicillin (Amp +) resistance gene, a transformation bacterium solution can be coated on an Amp resistant LB solid culture medium and cultured at 37 ℃ overnight;
(2) the method comprises the following steps of (1) growing dispersed colonies by bacteria to be plated, selecting colonies with clear edges and good growth, and further sequencing and identifying;
(3) reserving candidate sequences of a heavy chain variable region (mVH) and a light chain variable region (mVL) according to a sequencing result, performing PCR amplification again to obtain sequences of a heavy chain variable region (mVH) and a light chain variable region (mVL) which are matched with an expression vector, connecting a PCR product with a double-enzyme digestion pre-treated linear expression vector, and transforming a competent bacterium DH5a by using a connecting product, wherein the expression vector is provided with a kanamycin (Kana +) resistance gene, and a transforming bacterium solution can be coated on a Kana-resistant LB solid culture medium and cultured at 37 ℃ overnight;
(4) comparing the two sequencing results according to the step (4), selecting the transformation bacteria with the correct sequence, and performing plasmid extraction after amplification culture;
(5) the expression vector connected with the target single-antibody heavy chain variable region (mVH) and light chain variable region (mVL) genes co-transfects a eukaryotic expression cell strain 293;
wherein, in the step (5), the eukaryotic Expression cell line 293 is suspension cultured, SFM4Transfx-293 without L-glutamine (liquid) serum-free Medium is subjected to passage amplification, and is replaced by Gibco FreeStyle ® 293 Expression Medium serum-free Medium during transfection; harvesting supernatant after 7 days of continuous culture, centrifuging for 30min at 4000g, removing impurities such as cells in the supernatant, and filtering and sterilizing by using a 0.46um filter;
(6) the harvested supernatant contains the target antibody, the flow detection expression monoclonal antibody is well combined with PBMC, the positive rate reaches more than 95%, and then the CD25 mouse/human chimeric antibody is obtained.
Wherein the protein is a heavy chain variable region (mVH) of an anti-human CD25 chimeric monoclonal antibody, the amino acid sequence of the protein is the same as that of SEQ ID NO.1,
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYAISWVRQPPGKGLEWLGVMWTGGGTNYNSALKSRLNISKDNSKSQVFLKMNSLQTEDTARYYCARPYYYDGSWFAYWGQGTLVTVSA。
further, the heavy chain variable region (mVH) has three hypervariable regions: GFSLTSYA, MWTGGGT, ARPYYYDGSWFAY.
Wherein the protein is a light chain variable region (mVL) of an anti-human CD25 chimeric monoclonal antibody, the amino acid sequence of the protein is the same as that of SEQ ID NO.2,
DIVLSQSPAILSASPGEKVTMTCRASSSVGYMHWYQQKPGSSPKPWIYATSNLASGVPVRFSGSESGTSYSLTISRVEAEDAATYYCQQWSSNPPTFGGGTKLEIK。
further, the light chain variable region (mVL) has three hypervariable regions: SSVGY, ATS, QQWSNPPT.
As a fourth aspect of the present invention, an application of the anti-human CD25 chimeric monoclonal antibody is characterized in that the anti-human CD25 chimeric monoclonal antibody is used for preparing a kit for inducing tumor cell apoptosis.
As a fifth aspect of the present invention, a kit comprising a chimeric monoclonal antibody against human CD 25.
The invention has the beneficial effects that:
the CD25 mouse/human chimeric antibody provided by the invention can be used for detecting T cell phenotype, in particular to CD4+CD25+Foxp3+A detection phenotype of regulatory T cells (tregs); the combined IL-2 is used for activating T cells in vitro, promoting the proliferation of the T cells and activating the biological functions of the T cells.
Drawings
FIG. 1 flow cytometry analysis of anti-human CD25 chimeric monoclonal antibody XGMHC26.1 recognition of PBMC surface CD25 molecule with grey peak as negative control.
A is positive control (positive control), and the primary antibody is anti-human CD25 monoclonal antibody marked by commercial PE.
B is an experimental group, the primary antibody is an anti-human CD25 chimeric antibody XGMHC26.1, and the secondary antibody is goat anti-human IgG labeled by fluorescein PE.
FIG. 2 IL-2 in combination with CD 25-elicited antibodies efficiently amplified TIL
A. Single IL-2 group (500U)
B. Single IL-2 group (100U)
C. IL-2 (100U) in combination with CD25 monoclonal antibody (5 μ g/ml) group
The proportion of CD8+ T cells of the IL-2 group with 100U dose combined with the mABCD25 monoclonal antibody group with 5 mug/ml is higher than that of the IL-2 group with 500U dose; the combined effect group can expand more CD8+ T cells under the effect of the same dose of IL-2.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Unless otherwise specified, the examples are all routine experimental techniques in the art.
The sources of biomaterials used in the examples were as follows:
EXAMPLE 1 acquisition of hybridoma producing CD25 monoclonal antibody
(I) immunizing mice
The fusion protein or the transgenic cells are used for immunizing a mouse for four times, the interval is 21 days every time, the orbital blood titer of the mouse is measured after the fourth immunization is carried out for 7-10, and the boosting immunization is carried out when the titer is measured.
(II) cell culture
1. 1 mouse of BALB/c 6-7 weeks old is taken and placed in 75% ethanol solution for 2min before fusion 1 d.
And 2, taking out the spleen of the mouse aseptically, placing the spleen in a stainless steel screen with 200 meshes, and grinding to obtain a single cell suspension. Wash twice (1400 rpm, 5 min) with 1640 basic medium for use. Adjusting the cell concentration to 2X 10 by using 1640 culture medium of 15% FBS5/ml, added dropwise to 96-well culture plates at a concentration of 100. mu.L/well, 37 ℃ and 5% CO2Culturing in an incubator.
3. The culture was overnight and observed under low power microscope the next day. And 150ul of subcloned cells were plated.
(III) fusion and screening
Preparing:
1. equipment: a mouse soaking cup; a simple dissecting table; a hybridoma package; a heat-preservation water bath cup; a thermometer; 96-well culture plates.
2. Reagent: 75% ethanol solution; preheating 1640 basic culture medium at 37 ℃; preheating PEG1ml at 37 ℃; preheating 1640 basic culture medium 14ml (PEG stop solution) at 37 ℃; medium was selected by pre-heating 1640 at 37 deg.C (15% FBS and the amount of HAT added was calculated so that the final concentration of HAT in the medium in the final 96-well plate was 1%).
The method comprises the following steps:
1. the immunized mice were washed with running water and placed in 75% ethanol solution for 2 min.
2. The spleens of the mice were aseptically removed, placed in a 200 mesh stainless steel screen, and ground to obtain a single cell suspension. Washed twice (1400 rpm, 5 min) with pre-heated 1640 base medium for use.
3. Well-grown SP2/0 cells in logarithmic growth phase were collected and washed twice (1400 rpm, 5 min) with pre-heated 1640 basal medium for future use.
4. SP2/0 cells or Ag8 cells and spleen cells were mixed in a 50ml clear plastic centrifuge tube, the ratio of spleen cells to myeloma cells was typically 5:1, the cell was washed once (1400 rpm, 5 min) with pre-heated 1640 basic medium, the supernatant was discarded (to avoid unnecessary dilution of PEG) and the tube bottom was rubbed with the palm (or flicked with the fingers) to mix the two cells well into suspension.
5. Placing the centrifuge tube in a 37 ℃ heat-preservation water bath cup for preheating, sucking 1ml of 50% PEG solution preheated at 37 ℃, completing adding at a constant speed within 1min, slightly shaking the centrifuge tube while adding, and slightly shaking in the 37 ℃ water bath for 60s after adding. (one drop for 3 seconds)
6. Gently add 14ml of 1640 basal medium pre-warmed at 37 ℃ along the tube wall to stop (1 ml for 1min, 3ml for 3min, and finally 10 ml slowly). (Uniform dropping)
After standing at 7.37 ℃ for 5min, the mixture was centrifuged (800 rpm, 5 min), and the supernatant was discarded (the tube was tilted, and the supernatant was aspirated).
8. And (3) lightly resuspending the precipitated cells with 1640 selection culture medium preheated at 37 ℃ (the cells cannot be blown to be beaten), adding the cells into the preheated culture medium prepared in advance, uniformly mixing, dropwise adding the cells into the 96-well culture plate containing the trophoblasts, culturing the cells in a culture box containing 5% CO2 at 37 ℃ in 100 mul/well, changing the culture solution after 3-4 days, culturing the cells in HT culture medium after 10 days, and culturing the cells in the culture medium containing 10% FBS 1640 after 2 weeks.
9. During the period, the clone growth condition in a 96-well plate is observed every day, and generally when hybridoma cells are fully distributed in the area of the bottom 1/10 of the well, the specific antibody can be detected, and the required hybridoma cell line can be screened. For cells with specific secretion antibodies, the cells should be cloned and frozen in time. It usually takes 3-5 subclones to obtain cells of stable genotype and stable secretory phenotype, and after a period of culture it needs to be subcloned again.
EXAMPLE 2 CD25 murine/human chimeric antibody obtaining method
1. Extraction of cDNA from hybridoma cells: extracting RNA from the hybridoma cell strain, and performing reverse transcription on the obtained RNA into cDNA by using an RT-PCR technology; cloning the heavy chain variable region (mVH) and the light chain variable region (mVL) of the hybridoma cell by using an upstream primer and a downstream primer which are specially designed;
2. mVH and mVL are respectively connected with a cloning vector (pJET cloning vector), the connection product is transformed into a competent bacterium DH5a, and the pJET vector carries an ampicillin (Amp +) resistance gene, so that a transformation bacterium solution can be coated on an Amp-resistant LB solid culture medium and cultured at 37 ℃ overnight;
3. the method comprises the following steps of (1) growing dispersed colonies by bacteria to be plated, selecting colonies with clear edges and good growth, and further sequencing and identifying;
4. reserving a candidate heavy-light chain variable region sequence according to a sequencing result, performing PCR amplification again to obtain a heavy chain variable region sequence and a light chain variable region sequence which are matched with an expression vector, connecting a PCR product with a linear expression vector subjected to double enzyme digestion pretreatment, and connecting a product to transform competent bacteria DH5a, wherein the expression vector is provided with a kanamycin (Kana +) resistance gene, so that a transformation bacterium solution can be coated on a Kana resistant LB solid medium and cultured overnight at 37 ℃;
5. comparing the two sequencing results according to the sequencing method 4, selecting the transformation bacteria with the correct sequence, carrying out amplification culture, and then carrying out plasmid extraction;
6. the expression vector connected with the target monoclonal antibody heavy chain and light chain variable region genes cotransfects the eukaryotic expression cell strain 293. 293 cells were suspension cultured, SFM4Transfx-293 with L-glutamine (liquid) serum-free Medium was passage-expanded, and replaced with Gibco FreeStyle 293 Expression Medium serum-free Medium during transfection. Harvesting supernatant after 7 days of continuous culture, centrifuging for 30min at 4000g, removing impurities such as cells in the supernatant, and filtering and sterilizing by using a 0.46um filter;
7. the harvested supernatant contains the target antibody, the flow detection expression monoclonal antibody is well combined with PBMC, the positive rate reaches more than 95%, and then the CD25 mouse/human chimeric antibody is obtained.
The CD25 murine/human chimeric antibody obtained by the above method, antibody heavy chain variable region: QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYAISWVRQPPGKGLEWLGVMWTGGGTNYNSALKSRLNISKDNSKSQVFLKMNSLQTEDTARYYCARPYYYDGSWFAYWGQGTLVTVSA
Three hypervariable regions: GFSLTSYA, MWTGGGT, ARPYYYDGSWFAY.
Antibody light chain variable region:
DIVLSQSPAILSASPGEKVTMTCRASSSVGYMHWYQQKPGSSPKPWIYATSNLASGVPVRFSGSESGTSYSLTISRVEAEDAATYYCQQWSSNPPTFGGGTKLEIK
three hypervariable regions: SSVGY, ATS, QQWSNPPT.
Example 3
1. Separation and extraction of PBMC
Aseptically extracting blood in a single blood-sampling platelet leukocyte filter, hereinafter referred to as filter blood, and diluting the blood with PBS (phosphate buffer solution) 1: 20; taking normal heparin anticoagulation peripheral blood of the same individual, hereinafter referred to as peripheral blood for short, and diluting the peripheral blood by PBS (phosphate buffer solution) 1: 2; separating the diluted filter blood and peripheral blood respectively by using conventional Ficoll to obtain PBMC, washing for 2 times by PBS, counting, and adjusting the cell density to 1x106/ml by 10% FBS-containing RPMI-1640 for later use, wherein the PBMC are whole blood PBMC cells and filter blood PBMC cells respectively.
2. CFSE-labeled cells
The method for detecting the in-vitro expansion of the human peripheral blood T cells by using CFSE (circulating fluid infusion) markers comprises the steps of taking a proper amount of separated human Peripheral Blood Mononuclear Cells (PBMC) in a proper amount of CFSE working solution, gently mixing the PBMC uniformly, then carrying out water bath at 37 ℃ for 10 min, removing a supernatant after centrifugation, adding 5 ml of a solution 1640 of 10% calf serum, removing the supernatant after centrifugation, repeating the operation for 1 time, and adding a proper culture medium to prepare a cell suspension.
3. PBMC amplified in vitro
PBMC cells were prepared into cell suspension by using RMPI1640 complete medium (containing 10% calf serum), then transferred into 24-well plates (1.0X 106/well), and then corresponding cytokines were added to the cell culture fluid: anti-CD 3mAb (50 ng/ml), IL-2 (500U/ml)) or anti-CD 3mAb (50 ng/ml), IL-2 (100U/ml)), CD25mAb (5. mu.g/ml) was added, and the plates were incubated in a CO2 incubator at 37 ℃ and 5% CO2 at 95% saturation humidity. Then, the culture medium containing the cytokine IL-2 (200U/ml) is supplemented every 2-3 days, namely every 3 days according to the cell state, and the culture is carried out for 10-14 days and observed every day.
4. Detection by CFSE experiment
On the basis of the above experiment, the adoptive immunotherapy cells marked by CFSE fluorescence were collected on the 10 th day of culture, and anti-human CD3-PECY5 antibody was added to detect the proliferation of CD3+ T.
FIG. 1 flow cytometry analysis of anti-human CD25 chimeric monoclonal antibody XGMHC26.1 recognition of PBMC surface CD25 molecule with grey peak as negative control.
A is positive control (positive control), and the primary antibody is anti-human CD25 monoclonal antibody marked by commercial PE.
B is an experimental group, the primary antibody is an anti-human CD25 chimeric antibody XGMHC26.1, and the secondary antibody is goat anti-human IgG labeled by fluorescein PE.
FIG. 2 IL-2 in combination with CD 25-elicited antibodies efficiently amplified TIL
A. Single IL-2 group (500U)
B. Single IL-2 group (100U)
C. IL-2 (100U) in combination with CD25 monoclonal antibody (5 μ g/ml) group
The proportion of CD8+ T cells of the IL-2 group with 100U dose combined with the mABCD25 monoclonal antibody group with 5 mug/ml is higher than that of the IL-2 group with 500U dose; the combined effect group can expand more CD8+ T cells under the effect of the same dose of IL-2.
SEQUENCE LISTING
<110> Suzhou Xuguangx Biotech Co., Ltd
<120> anti-human CD25 chimeric monoclonal antibody, and preparation method and application thereof
<130> 20161023
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 119
<212> PRT
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> High variable region
<222> (26)..(33)
<220>
<221> High variable region
<222> (51)..(57)
<220>
<221> High variable region
<222> (96)..(108)
<400> 1
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Met Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Asn Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Ala
85 90 95
Arg Pro Tyr Tyr Tyr Asp Gly Ser Trp Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 2
<211> 106
<212> PRT
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> High variable region
<222> (27)..(31)
<220>
<221> High variable region
<222> (49)..(51)
<220>
<221> High variable region
<222> (88)..(96)
<400> 2
Asp Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Gly Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Glu Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
Claims (5)
1. The anti-human CD25 chimeric monoclonal antibody is characterized in that the amino acid sequence of a heavy chain variable region (mVH) of the anti-human CD25 chimeric monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region (mVL) of the anti-human CD25 chimeric monoclonal antibody is shown as SEQ ID No. 2.
2. The chimeric anti-human CD25 monoclonal antibody according to claim 1, wherein the heavy chain variable region (mVH) has three hypervariable regions: GFSLTSYA, MWTGGGT, ARPYYYDGSWFAY.
3. The chimeric anti-human CD25 monoclonal antibody according to claim 1, wherein the light chain variable region (mVL) has three hypervariable regions: SSVGY, ATS, QQWSNPPT.
4. The application of the anti-human CD25 chimeric monoclonal antibody is characterized in that the anti-human CD25 chimeric monoclonal antibody is used for preparing a kit.
5. A kit comprising a chimeric monoclonal antibody against human CD 25.
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| CN201611167733.XA CN108084263B (en) | 2016-12-16 | 2016-12-16 | Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611167733.XA CN108084263B (en) | 2016-12-16 | 2016-12-16 | Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108084263A CN108084263A (en) | 2018-05-29 |
| CN108084263B true CN108084263B (en) | 2021-07-13 |
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| CN114957473B (en) * | 2022-04-25 | 2023-05-16 | 苏州旭光科星抗体生物科技有限公司 | ELISA detection kit for detecting content of soluble CD25 protein |
| CN116693687B (en) * | 2023-08-03 | 2023-10-03 | 天津旷博同生生物技术有限公司 | Anti-human CD25 engineering antibody and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101160137A (en) * | 2005-04-15 | 2008-04-09 | 诺瓦提斯公司 | Use of CD 25antibody in immunity treatment |
| CN101385742A (en) * | 2001-03-12 | 2009-03-18 | 梅里克斯生物科学公司 | CD4+CD25+regulatory T cells from human blood |
| CN102585000A (en) * | 2012-02-22 | 2012-07-18 | 尉军 | Tumor marker CD25 autoantibody and application thereof |
| CN102964449A (en) * | 2012-10-29 | 2013-03-13 | 武汉生物制品研究所有限责任公司 | Recombinant anti-human CD25 chimeric antibody |
| WO2016109410A2 (en) * | 2014-12-29 | 2016-07-07 | Novartis Ag | Methods of making chimeric antigen receptor-expressing cells |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101385742A (en) * | 2001-03-12 | 2009-03-18 | 梅里克斯生物科学公司 | CD4+CD25+regulatory T cells from human blood |
| CN101160137A (en) * | 2005-04-15 | 2008-04-09 | 诺瓦提斯公司 | Use of CD 25antibody in immunity treatment |
| CN102585000A (en) * | 2012-02-22 | 2012-07-18 | 尉军 | Tumor marker CD25 autoantibody and application thereof |
| CN102964449A (en) * | 2012-10-29 | 2013-03-13 | 武汉生物制品研究所有限责任公司 | Recombinant anti-human CD25 chimeric antibody |
| WO2016109410A2 (en) * | 2014-12-29 | 2016-07-07 | Novartis Ag | Methods of making chimeric antigen receptor-expressing cells |
Non-Patent Citations (1)
| Title |
|---|
| Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance;Li W, et al.;《Transplantation proceedings》;20061231;第38卷(第10期);第3207-3208页 * |
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