CN115772219B - Binding protein capable of specifically binding MRP1 protein, kit and application thereof - Google Patents
Binding protein capable of specifically binding MRP1 protein, kit and application thereof Download PDFInfo
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- CN115772219B CN115772219B CN202210904588.8A CN202210904588A CN115772219B CN 115772219 B CN115772219 B CN 115772219B CN 202210904588 A CN202210904588 A CN 202210904588A CN 115772219 B CN115772219 B CN 115772219B
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Abstract
The invention discloses a binding protein specifically binding MRP1 protein, a kit and application thereof, and relates to the technical field of biology. The binding protein has higher activity and higher affinity with MRP1 protein, can be used for developing corresponding detection reagents or kits, is used for detecting the expression level of the MRP1 protein in relevant tumor tissues, and can improve the detection sensitivity and specificity. The binding protein can be used for diagnosing diseases taking MRP1 as a marker, provides more protein choices for the detection of MRP1 and the diagnosis of diseases taking multi-drug resistance related protein MRP1 as a marker, and enriches the types of monoclonal antibodies aiming at MRP1 proteins.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a binding protein specifically binding MRP1 protein, a kit and application thereof.
Background
The phenomenon of multidrug resistance is a major obstacle in tumor chemotherapy, which leads to the development of resistance of tumor cells to various antitumor agents through the overexpression of various related proteins. The multi-drug resistance related protein MRP1 is one of the main proteins. MRP molecules are an intact membrane glycoprotein, belonging to the same ATP-binding cassette transmembrane transporter superfamily as P-glycoproteins, and are overexpressed in many P-glycoprotein-negative, multi-drug resistant cell lines and tumors. The main function of MRP in drug resistance is plasma membrane drug efflux pump, and intracellular isolation of drug affects drug redistribution, making drug unable to bind to target site and indirectly generate drug resistance.
MRP1 (multidrug resistance-associated protein 1) is a transmembrane transporter that is believed to be closely related to the occurrence of multidrug resistance in patients with breast cancer, lung adenocarcinoma, and the like. Compared with normal tissues, the MRP1 has obviously increased content in tumor tissues such as breast cancer, lung adenocarcinoma and the like, and is highly expressed in cancer tissues with recurrent metastasis. It can be seen that enhancing the detection of MRP1 is critical for the detection of multi-drug resistance of the relevant cancers.
Clinical detection of protein expression in tumor cells is often performed by Immunohistochemical (IHC) pathology experiments, and the accuracy and sensitivity of the IHC experiments are determined by the merits of monoclonal antibodies that specifically bind the protein. Therefore, developing a monoclonal antibody with higher binding specificity to MRP1 protein is of great importance to IHC to detect MRP1 expression level.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a binding protein specifically binding to MRP1 protein, a kit and application thereof, so as to solve the technical problems.
The inventor discovers that the binding protein provided by the invention has higher activity and higher affinity with MRP1 protein, can be used for developing corresponding detection reagents or kits for detecting MRP1 protein, and can improve the sensitivity and specificity of detection. The binding protein can be used for diagnosing diseases taking MRP1 as a marker, provides more protein choices for detecting MRP1 and diagnosing diseases taking MRP1 as the marker, and enriches the types of monoclonal antibodies aiming at MRP1 proteins.
Noun definition
The term "binding protein" refers broadly to all proteins/protein fragments comprising CDR regions, in particular antibodies or antibody functional fragments. "antibody functional fragments" include antigen compound binding fragments of these antibodies, including Fab, fab ', F (ab') 2, fd, fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody may be selected from the group consisting of IgG1, igG2, igG3, igG4, and IgA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
The term "antibody" is used herein in its broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, such as specifically binding to an HRP-II antigen or fragment thereof. An "antibody fragment" includes a portion of a full length antibody, preferably an antigen binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.
Typically, the variable regions VH/VL of the heavy and light chains of an antibody are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
The invention is realized in the following way:
the present invention provides a binding protein that specifically binds to MRP1 protein, the binding protein comprising an antigen binding domain comprising 6 complementarity determining regions:
CDR-VH1：SDFAWT；
CDR-VH2：FLSYSGSTTYNPSLKS；
CDR-VH3：WGYFGLY；
CDR-VL1：RSSQSIVHSNGNTYLE；
CDR-VL2：KVSNRFS；
CDR-VL3：FQGSYVPWT。
the binding protein provided by the invention endows the binding protein with higher activity and higher affinity with MRP1 protein through the 6 complementary determining regions formed by the specific amino acid sequences, can be used for developing corresponding detection reagents or kits for detecting the MRP1 protein, and can improve the sensitivity and specificity of detection. The binding protein can be used for diagnosing diseases taking MRP1 as a marker, provides more protein choices for detecting MRP1 and diagnosing diseases taking MRP1 as the marker, and enriches the types of monoclonal antibodies aiming at MRP1 proteins.
In a preferred embodiment of the present invention, the binding protein and MRP1 protein are used in a ratio of K.ltoreq.7.68X10 9 L/mol affinity binding.
For example K.ltoreq.6X10 9 Affinity binding of L/mol, or K.ltoreq.2X10) 9 Affinity binding of L/mol, e.g.K.ltoreq.8X10 8 Affinity binding of L/mol, e.g. K.ltoreq.9X10 8 L/mol，K≤7×10 8 L/mol，K≤6×10 8 L/mol，K≤5×10 8 L/mol，K≤4×10 8 L/mol，K≤3×10 8 L/mol，K≤2×10 8 L/mol or K is less than or equal to 1X 10 8 L/mol affinity binding.
The affinity constant is calculated by the formula k= (N-1)/(n×ab' -AB). AB', AB is the concentration of antibody (mol/L) that gives half-absorbance at the corresponding antigen concentration AG. n=ag/AG '(AG > AG').
In an alternative embodiment, the binding protein is an antibody or a functional fragment;
in an alternative embodiment, the functional fragment includes, but is not limited to, any of F (ab ') 2, fab', fab, fv, scFv, and bispecific antibodies.
The functional fragment of the above antibody generally has the same binding specificity as the antibody from which it is derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the antibodies described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In a preferred embodiment of the invention, the binding protein comprises the light chain framework regions FR1-L, FR2-L, FR-L and FR4-L, which are shown in sequence SEQ ID NO. 1-4, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, which are shown in sequence SEQ ID NO. 5-8.
The sequences of SEQ ID NOS 1-8 are shown in the following table:
the invention also provides a hybridoma cell strain, and the name of the cell strain is 3C4.
The invention also provides application of the binding protein in preparing a reagent or a kit for diagnosing tumor or MRP1 immunodetection.
In preferred embodiments of the invention, the tumor includes, but is not limited to, an epithelial tumor; MRP1 immunoassays are where tissue cells are labeled with binding proteins.
In preferred embodiments of the invention, epithelial tumors include, but are not limited to, papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, or squamous carcinoma.
The gastrointestinal cancer is selected from esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, small intestine cancer, colorectal cancer and anal cancer, optionally wherein the colorectal cancer is selected from colon cancer and rectal cancer.
In an alternative embodiment, the adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma, or pancreatic carcinoma;
in an alternative embodiment, adenomas include, but are not limited to, cystic, fibroadenomas, polymorphic adenomas, or polypoidal adenomas.
In an alternative embodiment, the breast cancer includes, but is not limited to, ductal breast cancer, epithelial breast cancer, or lobular breast cancer. Breast cancer, preferably stage II to IV and/or poorly differentiated invasive ductal cancer, acne cancer, and medullary cancer (preferably grade 2).
Ovarian cancer, serous and mucinous cancers (preferably stage Ic to stage IIIb), granulosa cell tumors, superficial epithelial-mesenchymal tumors (adenocarcinomas), cystic adenocarcinomas and endometrioid tumors.
Uterine cancer, preferably comprising endometrioid adenocarcinoma (preferably stage I to stage IIIc).
Bladder cancer, preferably comprising transitional cell carcinoma (preferably stage II to stage IV).
Lung cancer, preferably including small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma), and large cell lung cancer.
Squamous carcinoma is one or more of oral squamous carcinoma, pharyngeal squamous carcinoma, laryngeal carcinoma, esophageal squamous carcinoma (e.g., esophageal squamous carcinoma), squamous cell carcinoma of the lips, uterine squamous carcinoma, vaginal squamous carcinoma, and skin squamous carcinoma.
Squamous cell carcinoma of the oral cavity (oral squamous cell carcinoma, OSCC) is also known as oral squamous carcinoma. Oral squamous carcinomas include, for example, but are not limited to, tongue squamous carcinomas.
In another embodiment, the squamous cell carcinoma may be a squamous cell carcinoma formed by squamous metaplasia of a part such as bronchi, bladder, or renal pelvis.
In a preferred embodiment of the invention, the tissue cells are at least one of colon, liver, pancreas, ovary, endometrium, cervix, lung, thyroid and mammary epithelium, bronchi, bladder, renal pelvis.
The invention also provides a reagent or a kit for diagnosing tumor or MRP1 immunodetection, which contains the binding protein.
In an alternative embodiment, epithelial tumors include, but are not limited to papilloma, gastrointestinal cancer, ovarian cancer, endometrial tumor, cervical cancer, lung cancer, adenocarcinoma, adenoma, or squamous carcinoma; in an alternative embodiment, the adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma, or pancreatic carcinoma;
in an alternative embodiment, adenomas include, but are not limited to, cystic, fibroadenomas, polymorphic adenomas, or polypoidal adenomas;
in an alternative embodiment, breast cancer includes, but is not limited to, ductal breast cancer, epithelial breast cancer, or lobular breast cancer;
in an alternative embodiment, the MRP1 immunoassay is labeling tissue cells with a binding protein;
in an alternative embodiment, the tissue cell is at least one of colon, liver, pancreas, ovary, endometrium, cervix, lung, thyroid and mammary epithelium, bronchi, bladder, renal pelvis.
In an alternative embodiment, the above-described reagent or kit further comprises a label that is labeled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the detectable label includes, but is not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), etc., or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc., or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy3, etc., or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc., or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polymethine (cp), etc.).
In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids. Nanoparticles include, but are not limited to: organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
The invention also provides a vector comprising a nucleic acid encoding the binding protein described above.
The invention also provides a host cell which contains the vector.
The present invention also provides a method for producing the binding protein of the previous embodiment, comprising:
culturing the host cell of the previous embodiment, and isolating and purifying the binding protein from the culture medium or from the cultured host cell.
The method of production may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions to express the binding protein. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The binding proteins may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.
Construction of a suitable vector containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailing and religated as desired. Mutations may be introduced into the coding sequence by any method to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions, etc.
Based on the present disclosure of the amino acid sequence of an antibody or functional fragment thereof, it is readily apparent to a person skilled in the art that the preparation of the antibody or functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), e.g., isolation and purification from a culture product of recombinant cells capable of recombinantly expressing an antibody or functional fragment thereof as described in any of the above, is within the scope of the present disclosure, irrespective of the technique used to prepare the antibody or functional fragment thereof.
A method of detecting MRP1, comprising: mixing the binding protein of any of the preceding embodiments with a test sample.
In alternative embodiments, the above-described methods are for the purpose of diagnosis of non-disease.
It should be noted that, a person skilled in the art can perform qualitative or quantitative detection of MRP1 protein in a sample to be detected based on the characteristics of antibody/antigen binding to form an immune complex.
The invention has the following beneficial effects:
the binding protein provided by the invention has higher activity and higher affinity with MRP1 protein, can be used for developing corresponding detection reagents or kits, is used for detecting the expression level of MRP1 protein in relevant tumor tissues, and can improve the sensitivity and specificity of detection. The binding protein can be used for diagnosing diseases taking MRP1 as a marker, provides more protein choices for the detection of MRP1 and the diagnosis of diseases taking multi-drug resistance related protein MRP1 as a marker, and enriches the types of monoclonal antibodies aiming at MRP1 proteins.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of detection of MRP1 protein in A549 cells (human non-small cell lung cancer cells) identified when cell line 3C4 cell supernatants were subjected to Immunocytochemistry (ICC) detection;
fig. 2 is: immunohistochemical staining of gastric adenocarcinoma, breast cancer section tissue micrograph;
fig. 3 is: immunohistochemical staining lung adenocarcinoma section experiment result diagram.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example demonstrates the preparation of monoclonal antibodies specific for the MRP1 protein.
1. Mice were immunized.
Mice were immunized with the immunogen using a universal method. The immunogen is recombinant human MRP1 protein with His tag, and is used as detection antigen to measure and screen serum titer and hybridoma, and the high-purity antigen can increase the opportunity of obtaining the required monoclonal antibody and reduce the screening amount. 5 mice were immunized, each immunized 50ug MRP1 antigen. The antigen protein solution was prepared with PBS. And (3) placing a proper amount of antigen protein, PBS and Freund's adjuvant into a syringe, plugging a water outlet of the syringe by a plug, and placing the syringe on an emulsifying instrument for stirring and fully emulsifying to ensure that the antigen adjuvant forms a stable water-in-oil solution. The first tail blood serum titer detection is carried out 7-10 days after primary and secondary immunity, and good titer is obtained after 2-4 times of booster immunization. And selecting serum immune mice with high serum titer to perform cell fusion after abdominal cavity termination.
The sequence of the recombinant human MRP1 protein is as follows:
SSEMETNIVA VERLKEYSET EKEAPWQIQE TAPPSSWPQV GRVEFRNYCL RYREDLDFVL RHINVTINGG EKVGIVGRTG AGKSSLTLGL FRINESAEGE IIIDGINIAK IGLHDLRFKI TIIPQDPVLF SGSLRMNLDP FSQYSDEEVW TSLELAHLKD FVSALPDKLD HECAEGGENL SVGQRQLVCL ARALLRKTKI LVLDEATAAV DLETDDLIQS TIRTQFEDCT VLTIAHRLNT IMDYTRVIVL DKGEIQEYGA PSDLLQQRGL FYSMAKDAGL。
2. hybridoma cell fusion and screening were performed.
The following preparation is needed before cell fusion:
(1) Culturing mouse myeloma cells SP2/0 to log phase of growth;
(2) One negative mouse was sacrificed the day before fusion, and mouse peritoneal trophoblast cells were taken from the mouse peritoneal cavity with HAT medium injected into the mouse peritoneal cavity in a sterile environment and plated in 96-well plates, 100ul per well, with these cells promoting hybridoma cell growth.
Immunized mice were sacrificed, spleens were removed under sterile conditions, splenocytes and SP2/0 myeloma cells were chemically fused with PEG, appropriate HAT medium was added according to the number of plates to be plated, and finally the fused cells were plated on trophoblast cell culture plates at 100ul per well.
The growth of surviving hybridoma cells was observed under a microscope after 7-10 days, and after two weeks of plating, the supernatants from each well were collected and hybridoma cells were screened using the human MRP1-his protein antigen by ELISA. The method comprises the following steps:
the ELISA plate was coated with 100ul 2ug/ml of human MRP1-his protein antigen in PBS for two hours at 37 ℃. After 3 PBST washes, 150 ul/well of 3% nonfat dry milk in PBS was added and blocked overnight at 4 ℃. Plates were washed three more times, 80 ul/well hybridoma supernatant was added, incubated for 1 hour at 37℃and plates were washed three more times. Add 1 at 100 ul/well: and 7000 dilution of horseradish peroxidase-labeled goat anti-mouse secondary antibody, incubation at 37 ℃ for 45 minutes, plate washing for 3 times and drying. 100 ul/well TMB color development was added, developed at room temperature for 5-10 minutes, stopped with 2M sulfuric acid solution, and absorbance at 450nm was measured for each well. Positive hybridoma cells were selected.
ELISA positive fusion wells were selected and then subjected to immunocytochemistry staining (ICC), and positive wells were selected for subsequent experiments.
ICC experiment steps are as follows:
(1) The adherent cells A549 cells (human non-small cell lung cancer cells) are digested by pancreatin digestive juice and spread in 96-well plates a few days before the experiment, and the adherent cells are grown to be full of the plates;
(2) Taking out the 96-well plate, pouring out the culture medium, and washing with PBS for 3 times;
(3) Adding 4% paraformaldehyde, fixing at room temperature for 15-30 min, and washing the plate with PBS for three times;
(4) 0.2% triton X-100 room temperature for 10-15min, PBS rinse three times;
(5) 3% hydrogen peroxide for 15min, and rinsing with PBS for 3 times; 3% goat serum was blocked at 37 ℃ for 1 hour;
(6) 80ul of the fusion well ELISA positive cell supernatant was added to the icc assay plate overnight at 4 ℃;
(7) After PBST rinsing, adding secondary antibody to react for 40min at room temperature;
(8) Developing DAB;
(9) Hematoxylin staining;
(10) PBST anti-blue observation. The experimental results are shown with reference to fig. 1. FIG. 1 shows that when cell supernatants of the above fusion cell lines are used for Immunocytochemistry (ICC) detection, MRP1 protein in A549 cells (human non-small cell lung cancer cells) can be identified.
Fusion cells which are positive in combination are selected through ELISA and ICC experiments, cloned through a limiting dilution method, and each positive strain is paved into a 48/96-well plate for continuous culture. And performing secondary screening by ELISA method, screening out hybridomas which specifically recognize MRP1 protein and can block MRP1 binding, subcloning by limiting dilution method, and obtaining a monoclonal cell strain 3C4. Expanding the monoclonal cell line to collect about 1×10 6 Individual cells are injected into selected mice (the mice need to be injected with paraffin oil in the abdominal cavity in advance for one week), and after waiting for 7-10 days, the mice generate ascites, and the ascites is collected for antibody purification. After purification, a mouse monoclonal antibody against the MRP1 protein is obtained.
Example 2
This example performs DNA cloning and sequencing of the mouse monoclonal antibody selected in example 1 above, and gene sequencing of the variable region of the anti-human MRP1 monoclonal antibody.
Total RNA was extracted from the mouse monoclonal cell line using Trizol reagent. Cells cultured in 9cm dishes were collected in 1.5ml centrifuge tubes and the supernatant was aspirated to dryness. 1ml of Trizol reagent was added and the cells were blown up uniformly to lyse the cells, and the lysed sample or homogenate was left at room temperature for 5-10min to allow complete separation of nucleoprotein from nucleic acid. 0.2ml of chloroform was added thereto, and the mixture was vigorously shaken for 15sec and left at room temperature for 3min. Centrifuge at 12000rpm at 4℃for 10min. The upper aqueous phase is sucked and transferred into a clean centrifuge tube, added with isopropyl alcohol with equal volume, evenly mixed and placed for 20min at room temperature. Centrifuge at 12000rpm at 4℃for 10min, discard supernatant. The precipitate was washed with 1ml of 75% ethanol. Centrifuge at 12000rpm at 4℃for 3min, discard supernatant. Drying at room temperature for 5-10min. Adding 30-50ul RNase-free ddH 2 O. The resulting RNA solution was stored at-70℃or used in subsequent experiments.
The total RNA is reverse transcribed into cDNA by using AMV first-strand cDNA synthesis kit. The experimental system was configured as follows, with 6ul total RNA+1ul Oligo dT+4ul RNase-free water (11 ul total). After gentle mixing, the mixture was centrifuged for 3-5s, and after 5mi of pre-denaturation in a warm bath at 65℃the reaction mixture was ice-cooled for 30s, and then centrifuged for 3-5s followed by 2min in an ice-cooled bath. Under ice bath, 4ul of 5 Xbuffer+1 ul of dNTP mixture+1 ul of RNase inhibitor+1 ul of reverse transcriptase (total 20ul system) were added, and after gentle mixing, the mixture was centrifuged for 3-5s, and on a PCR instrument, the mixture was subjected to 42℃for 50 minutes and 85℃for 5 minutes to complete cDNA synthesis. Random primers are suitable for the synthesis of short-chain cDNA below 500bp, and the transcribed RNA template can transcribe 5' -terminal regions without poly (A) tail.
PCR amplification of light and heavy chains. For amplifying antibody light chain variable region sequences, a PCR reaction system was configured: 2 XTaq enzyme buffer 25ul+FP-VL 1ul+RP-VL 1ul+cDNA 2ul+ddH 2 And O21 ul. For amplifying antibody heavy chain variable region sequences, a PCR reaction system was configured: 2 XTaq enzyme buffer 25ul+FP-VH1ul+RP-VH1ul+cDNA 2ul+ddH 2 And O21 ul. The temperature cycle for PCR amplification of the heavy and light chain variable regions is as follows (wherein steps 2 to 4, 35 cycles are repeated):
step 1, pre-denaturation at 94 ℃ for 4min;
step 2-denaturation 94℃for 30sec;
step 3-annealing at 55 ℃ for 45sec;
step 4-extending at 72 ℃ for 60sec;
step 5-72 ℃ for 10min;
step 6-storing at 4 ℃.
The PCR products were analyzed by 1% agarose gel electrophoresis, bands of DNA segments of the corresponding sizes (about 355bp for VH and about 335bp for VL) were excised and DNA extracted using the SanPrep column DNA gel recovery kit. The following is a brief description: cutting off a gel block containing the target fragment from agarose gel, and weighing; adding buffer B2 with the weight 3-6 times of that of the rubber block, and carrying out water bath at 50 ℃ for 5-10 minutes to obtain sol; transferring the sol into an adsorption column, and centrifuging 8000g for 30 seconds; pouring out the liquid in the collecting pipe; adding 500ul wash Solution,9000g into the column, centrifuging for 30 seconds, and pouring out the liquid in the collecting pipe; repeatedly adding the wash solution once, and pouring out the liquid; centrifuging the hole adsorption column at 9000g for 1 min; the column was placed in a clean 1.5ml centrifuge tube, 15-40 g ul Elution Buffer g was added to the center of the adsorption membrane, and after 1 min of standing at room temperature, it was centrifuged for 1 min. The prepared DNA solution is obtained, and the variable region sequence of the antibody is obtained by purifying the PCR product and sequencing.
Experimental example 1
This experimental example detects the affinity and sensitivity of the monoclonal antibody prepared in example 1.
1. The recombinant human MRP1 protein of example 1 was plated at 3mg/L, 1.5mg/L, 0.75mg/L, 0.375 mg/L.
2. The concentration of the monoclonal antibody produced in example 1 was adjusted to 10 -7 mol/L level (1X 10 -7 To 5x 10 -7 mol/L). Then diluted 1:2-1:256 in a multiple ratio and added to wells with different antigen coating amounts.
3. Adding secondary antibody and developing TMB. Absorbance at 450nm was measured and absorbance data is shown in table 1.
4. And (3) according to the antigen-antibody combination S curve, determining the antibody concentration of half-absorbance values under different antigen concentrations. Thus there are four antibody concentrations (mol/L). 9.501*10 -10 mol/L、5.268*10 -10 mol/L、3.3334*10 -10 mol/L、2.377*10 -10 mol/L。
5. The affinity constant is calculated by substituting the formula k= (N-1)/(n×ab' -AB). AB', AB is the concentration of antibody that gave half-absorbance at the corresponding antigen concentrations AG (3 mg/L, 1.5ml/L, 0.75mg/L, 0.375 mg/L). n=ag/AG '(AG > AG').
6. When n=2, three K values 9.652, 7.143, 7.042 can be obtained. When n=4, two K values 7.821, 7.075 can be obtained. When n=8, a K value 7.356 is obtained, an average of 6K values is obtained, 7.682×10 9 。
Table 1 shows statistics of absorbance at 450 nm.
Experimental example 2
The binding capacity of the purified antibodies MRP1-3C4 to MRP1 protein was determined based on Immunohistochemistry (IHC).
Baking gastric adenocarcinoma, lung adenocarcinoma or breast cancer slices in a 60 ℃ incubator for 60 minutes, soaking the slices in xylene I for 15 minutes, replacing xylene II, and then soaking for 15 minutes, wherein the slices are respectively soaked in absolute ethyl alcohol (1) for 5 minutes, absolute ethyl alcohol (2) for 5 minutes, 95% ethyl alcohol for 5 minutes, 85% ethyl alcohol for 5 minutes and 75% ethyl alcohol for 5 minutes; ddH 2 Soaking in O for 5 minutes, and cleaning for 3 times; adding 10mmol/L citrate buffer solution (pH 6.0) which is enough to submerge slices into a pressure cooker for antigen retrieval (boiling method), heating to boiling, placing the slices on a heat-resistant material slice frame, placing into the cooker, covering a cooker cover, fastening a pressure valve, continuing heating, setting pressure maintaining for 4 minutes, opening a deflation valve for deflation after the time is up, opening the cooker cover after the pressure is zeroed, taking out the inner cooker, and cooling at room temperature.
Taking out the slice (about 40 min) after the solution is cooled to room temperature; ddH 2 O is soaked for 5 minutes, washed for 2 times, PBST is soaked for 5 minutes, and washed for 2 times; the sections were placed in 20ml of 3% H 2 O 2 -in methanol solution, protected from light, treated at room temperature for 10 minutes; PBST is soaked for 5 minutes and is washed for 3 times; one drop of goat serum blocking solution was added to each tissue group and incubated in a wet box for 45 minutes at room temperature; PBST was soaked for 5 minutes and washed 3 times.
The treated tissue sections were compared with CK7 antibody from Roche, and the remaining sections were subjected to MRP1-3C4 purification. Incubation overnight in a wet box at 4 ℃; taking out from the refrigerator at the temperature of 4 ℃ and incubating for 60 minutes at room temperature; PBST is gently washed and then soaked for 5 minutes, and the PBST is washed for 3 times; each tissue group was incubated with HRP-labeled Changdao secondary antibody (CAT#:) 25ul at room temperature for 45 min; washing; preparing DAB color development liquid, reacting for 10-15min in dark, then dripping the DAB color development liquid onto a slice, and developing for 1-5 min; terminating the color reaction with distilled water; dropping 50ul hematoxylin dye solution into each tissue group, dyeing for 5-10 minutes, and washing with distilled water; the slices are put into 1 percent hydrochloric acid-ethanol for decoloration for 2 to 3 seconds, then are quickly taken out and put into distilled water for termination, and then are put into PBST (pH 8.0) for blue reflection for 5 to 10 minutes; soaking in 75% ethanol for 5 min: soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 min: soaking in absolute ethanol for 5 minutes. Soaking in xylene for 10min, and soaking for 10min after changing xylene; dripping a neutral resin sealing piece, and covering a glass slide sealing piece; microscope imaging. As shown in fig. 2 and 3.
Immunohistochemical neutralization assay: a certain amount of MRP1 antigen protein is taken and mixed with MRP1-3C4 purified antibody, and neutralization reaction is carried out for 1 hour at 37 ℃. The method comprises the steps of performing immunohistochemical experiments, dripping the antibody serving as a primary antibody to lung adenocarcinoma slices, overnight at 4 ℃, performing immunohistochemical staining according to the same steps, and taking a picture.
FIGS. 2 and 3 show the immunohistochemical test of MRP1, and the test specimens are stomach adenocarcinoma, breast carcinoma and lung adenocarcinoma tissues of different patients. In FIG. 2, A is stomach adenocarcinoma tissue, B is breast carcinoma tissue, and the antibody is MRP1-3C4 antibody we produce. FIG. 3 shows lung adenocarcinoma tissue, A is MRP1-3C4 antibody of the present invention, and B is MRP1 antibody of a biological company in China in FIG. 3. As can be seen from the staining results of FIG. 3, the MRP1-3C4 antibodies provided by the present invention stained more strongly than antibodies from a biological company. The MRP1 antibody provided by the invention has application prospect in preparing a kit.
The neutralization test results are shown as C in FIG. 3. The antigen protein reaction in the neutralization assay binds to the MRP1 antibody, which cannot bind to the antigen in the cancerous tissue, leaving the tissue almost free of staining.
It can be seen that the anti-MRP 1 protein monoclonal antibodies MRP1-3C4 provided in the embodiment 1 of the invention can recognize and combine with MRP1 protein in human cells, and make cytoplasms in lung adenocarcinoma tissues brown in subsequent IHC staining experiments, and have stronger color than MRP1 antibodies of a certain biological company in China.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A binding protein that specifically binds to MRP1 protein, said binding protein comprising an antigen binding domain comprising 6 complementarity determining regions:
CDR-VH1：SDFAWT；
CDR-VH2：FLSYSGSTTYNPSLKS；
CDR-VH3：WGYFGLY；
CDR-VL1：RSSQSIVHSNGNTYLE；
CDR-VL2：KVSNRFS；
CDR-VL3：FQGSYVPWT。
2. the binding protein for specifically binding to MRP1 protein according to claim 1, wherein K.ltoreq.7.68X10 of the binding protein to MRP1 protein 9 L/mol affinity binding.
3. The binding protein that specifically binds to MRP1 protein of claim 2, wherein said binding protein is an antibody or a functional fragment thereof.
4. The binding protein that specifically binds to MRP1 protein of claim 3, wherein said functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, scFv, and bispecific antibodies.
5. The binding protein of any one of claims 1 to 4, wherein the binding protein comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, respectively, having the sequences shown in SEQ ID NO. 1-4, respectively.
6. Use of a binding protein according to any one of claims 1 to 4 for the preparation of a reagent or kit for the diagnosis of a tumor or MRP1 immunoassay.
7. The use according to claim 6, wherein the tumour is selected from epithelial tumours; the MRP1 immunoassay is to label tissue cells with the binding protein.
8. The use according to claim 7, wherein the epithelial tumor is selected from papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma or squamous carcinoma.
9. The use according to claim 8, wherein the adenocarcinoma is selected from lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma or pancreatic carcinoma;
the adenoma is selected from the group consisting of cystic, fibroadenoma, polymorphic or polypoidal adenoma;
the breast cancer is selected from ductal breast cancer, epithelial breast cancer or lobular breast cancer;
the squamous carcinoma is one or more of oral squamous carcinoma, pharyngeal squamous carcinoma, laryngeal carcinoma, esophageal squamous carcinoma, squamous cell carcinoma of lip, uterine squamous carcinoma, vaginal squamous carcinoma and skin squamous carcinoma.
10. The use according to claim 7, wherein the tissue cells are at least one of colon, liver, pancreas, ovary, endometrium, cervix, lung, thyroid and mammary epithelium, bronchi, bladder, renal pelvis.
11. A reagent or kit for the diagnostic of a tumor or an MRP1 immunoassay, characterized in that it comprises a binding protein according to any one of claims 1-4.
12. The reagent or kit for diagnosing a tumor or MRP1 immunoassay according to claim 11, wherein the epithelial tumor is selected from papilloma, gastrointestinal cancer, ovarian cancer, endometrial tumor, cervical cancer, lung cancer, adenocarcinoma, adenoma or squamous carcinoma.
13. The reagent or kit for diagnosing a tumor or MRP1 immunoassay according to claim 12, wherein the adenocarcinoma is selected from lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma or pancreatic carcinoma;
the adenoma is selected from the group consisting of cystic, fibroadenoma, polymorphic or polypoidal adenoma;
the breast cancer is selected from ductal breast cancer, epithelial breast cancer or lobular breast cancer;
the MRP1 immunoassay is to label tissue cells with the binding protein.
14. The reagent or kit for diagnosing a tumor or MRP1 immunoassay according to claim 13, wherein the tissue cells are at least one of colon, liver, pancreas, ovary, endometrium, cervical, lung, thyroid and mammary epithelium, bronchi, bladder, renal pelvis.
15. A vector comprising a nucleic acid encoding the binding protein of any one of claims 1-4.
16. A host cell comprising the vector of claim 15.
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