A human chitinase-3-like protein 1 monoclonal antibody 1A9, recombinant vector, recombinant cell and method and application

Abstract

The present application belongs to the technical field of antibody preparation, and belongs to a human chitinase-3-like protein 1 monoclonal antibody 1A9, a recombinant carrier, a recombinant cell and a method and application. The present application provides a monoclonal antibody 1A9 capable of specifically recognizing CHI3L1, wherein the monoclonal antibody 1A9 comprises a heavy chain and a light chain; a variable region of the heavy chain comprises three heavy chain complementarity determining regions, and amino acid sequences of the three heavy chain complementarity determining regions are shown as SEQ ID NO. 1 to SEQ ID NO. 3; a light chain variable region of the monoclonal antibody 1A9 comprises three light chain complementarity determining regions, and amino acid sequences are shown as SEQ ID NO. 4 to SEQ ID NO. 6. The monoclonal antibody has good titer, and the expression carrier and the recombinant cell for preparing the monoclonal antibody are easy to store and modify.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibody preparation, and particularly relates to a human chitinase-3-like protein 1 monoclonal antibody 1A9, a recombinant vector, a recombinant cell, a method and application. BACKGROUND

[0002] Chitinase-3-like protein 1 (CHI3L1) is one of the members of chitinase glycoside hydrolase 18 family, with a size of about 40 kDa, and is expressed and secreted by various cells such as macrophages, chondrocytes, fibroblast-like synoviocytes, vascular smooth muscle cells and hepatic stellate cells. CHI3L1 is one of the glycoprotein members of human glycosyl hydrolase 18 family, has chitin binding activity, but lacks chitinase activity. Current studies have shown that the expression of the protein is closely related to the occurrence and development process of inflammatory diseases, extracellular tissue remodeling, fibrosis, solid cancer and asthma-related diseases.

[0003] Chitinase-3-like protein 1 can promote the migration of primary astrocytes. The absence of the protein can change the inflammatory response of glial cells, promote the phagocytosis of amyloid beta by astrocytes and microglia, reduce the formation of amyloid plaques, and affect the disease progression of Alzheimer's disease (AD); in the occurrence and development process of traumatic brain injury (TBI)-related neuroinflammatory response, its absence will cause more severe neuropathological phenomena and more significant glial pathogenesis; in addition, high expression of CHI3L1 can also promote the accelerated development of aortic atherosclerotic plaque. CHI3L1 shows great development potential in medical applications, and with further research, CHI3L1 may become a new target for diagnosis and treatment of central nervous system-related diseases. CHI3L1 detection antibodies can be used for CHI3L1 antigen detection and other scientific research, but most of the antibodies for CHI3L1 detection at present come from hybridoma cells, and traditional hybridoma cells are not easy to save, and will appear cell state deterioration, difficult to recover and other situations after long-term use. SUMMARY

[0004] The purpose of the present application is to provide a human chitinase-3-like protein 1 monoclonal antibody 1A9, a recombinant vector, a recombinant cell, a method and application. The monoclonal antibody has good titer, and the expression vector and recombinant cell used for preparing the monoclonal antibody are easy to save and modify.

[0005] The application provides a monoclonal antibody 1A9 capable of specifically recognizing chitinase-3-like protein 1, the monoclonal antibody 1A9 comprising a heavy chain and a light chain; a variable region of the heavy chain comprises three heavy chain complementarity determining regions, and amino acid sequences of the three heavy chain complementarity determining regions are shown as SEQ ID NO. 1 to SEQ ID NO. 3; a light chain variable region of the monoclonal antibody 1A9 comprises three light chain complementarity determining regions, and amino acid sequences are shown as SEQ ID NO. 4 to SEQ ID NO. 6.

[0006] Preferably, an amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 7; and an amino acid sequence of the light chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 9.

[0007] Preferably, a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 8; and a nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 10.

[0008] The application further provides a recombinant vector for expressing the monoclonal antibody 1A9, the recombinant vector comprising a vector carrying a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 and a vector carrying a nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9; a base vector of the vector carrying the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 comprises pFUSE-CHIg-m2a, and a base vector of the vector carrying the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 comprises pFUSE2ss-CLIg-mk; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 8; and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 10.

[0009] Preferably, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 1A9 is connected between EcoRI and NheI of pFUSE-CHIg-m2a; and the nucleotide sequence of the light chain variable region of the monoclonal antibody 1A9 is connected between EcoRI and NheI of pFUSE2ss-CLIg-mk.

[0010] The application further provides a recombinant cell for expressing the monoclonal antibody 1A9, the recombinant cell containing the recombinant vector in the technical solution, and a base cell of the recombinant cell comprises a mammalian cell.

[0011] The application further provides a construction method of the recombinant cell, comprising the following steps: mixing the recombinant vectors, and then transfecting the base cell to obtain the recombinant cell.

[0012] The application further provides a method for preparing the monoclonal antibody 1A9, comprising the following steps: culturing the recombinant cell for 48 hours, centrifuging, and collecting the supernatant.

[0013] The application further provides an application of the monoclonal antibody 1A9, the recombinant vector, the recombinant cell or the recombinant cell constructed by the construction method, or the monoclonal antibody 1A9 prepared by the method in the preparation of a reagent for detecting CHI3L1.

[0014] The application further provides an application of the monoclonal antibody 1A9, the recombinant vector, the recombinant cell or the recombinant cell constructed by the construction method, or the monoclonal antibody 1A9 prepared by the method in the preparation of a reagent for detecting CHI3L1.

[0015] The application provides a human chitinase-3-like protein 1 monoclonal antibody 1A9. The monoclonal antibody 1A9 has good titer, and based on the complementarity determining region sequences of the variable regions of the heavy chain and the light chain, a framework expression vector is constructed, cells are transfected, and the expression of the monoclonal antibody 1A9 can be realized. The monoclonal antibody 1A9 vector is easy to store and easy to control the quality of the antibody production process, and it is also convenient to make a series of modifications to the antibody, to make more in-depth research and more extensive application. It is verified by the examples that the monoclonal antibody 1A9 or the monoclonal antibody 1A9 produced by the recombinant cell can recognize CHI3L1 protein and has biological activity. The monoclonal antibody 1A9 can be used for CHI3L1 antigen detection and other scientific research, and has very good application value and very important scientific research guiding significance. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 A CBA method provided by the present application for detecting the antibody titer of serum of immunized mice;

[0018] Figure 2 A CBA method provided by the present application for screening positive hybridoma cells;

[0019] Figure 3 A monoclonal antibody subtype identification result provided by the present application; wherein each group of data from left to right represents IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, kappa and lambda;

[0020] Figure 4 A monoclonal antibody antibody titer determination result provided by the present application;

[0021] Figure 5 An immunofluorescence verification monoclonal antibody antigen epitope distribution situation diagram provided by the present application; wherein (a) the antibody recognition site is located in the 293-383 interval of the amino acid sequence schematic diagram; (b) the antibody recognition site is located on the 311-382 amino acid fragment schematic diagram;

[0022] Figure 6 A WB verification recombinant monoclonal antibody 1A9 action characteristic result provided by the present application. DETAILED DESCRIPTION

[0023] The present application provides a monoclonal antibody 1A9 capable of specifically recognizing human chitinase-3-like protein 1, the monoclonal antibody 1A9 comprising a heavy chain and a light chain; the variable region of the heavy chain comprises three heavy chain complementarity determining regions, the amino acid sequences of the three heavy chain complementarity determining regions are shown as SEQ ID NO. 1-SEQ ID NO. 3; the light chain variable region of the monoclonal antibody 1A9 comprises three light chain complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO. 4-SEQ ID NO. 6.

[0024] In the present application, the amino acid sequences of the three heavy chain complementarity determining regions are as follows:

[0025] CDR1 (SEQ ID NO. 1): NYGMN;

[0026] CDR2 (SEQ ID NO. 2):

[0027] WINTYTGEPTYADDFKG;

[0028] CDR3 (SEQ ID NO. 3): TGLKYYFDY;

[0029] In the present application, the amino acid sequences of the 3 light chain complementarity determining regions are as follows:

[0030] CDR1 (SEQ ID NO. 4):

[0031] KASQNVGTHVA;

[0032] CDR2 (SEQ ID NO. 5): SASYRYS;

[0033] CDR3 (SEQ ID NO. 6): QQYYSYPLT;

[0034] In the present application, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A9 is as shown in SEQ ID NO. 7 (MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARTGLKYYFDYWGQGTTLTVSS); and the amino acid sequence of the light chain variable region of the monoclonal antibody 1A9 is as shown in SEQ ID NO. 9 (MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTHVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSESGTDFTLTISNVQSEDLAEYFCQQYYSYPLTFGGGTKLEIK).

[0035] In the present application, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 8 (atggcttgggtgtggaccttgctattcctgatggcagctgcccaaagtgcccaagcacagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctggatataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgctgatgacttcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaaggacgggcttgaagtactactttgactactggggccaaggcaccactctcacagtctcctca); the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 is shown as SEQ ID NO. 10 (atggagtcacagactcaggtctttgtatacatgttgctgtggttgtctggtgttgatggagacattgtgatgacccagtctcaaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtactcatgtagcctggtatcaacagaaaccagggcaatctcctaaagcactgatttactcggcatcctaccggtacagtggagtccctgatcgcttcacaggcagtgaatctgggacagatttcactctcaccatcagcaatgtgcagtctgaagacttggcagagtatttctgtcagcaatattacagctatcctctgacgttcggtggaggcaccaagctggaaatcaaa).

[0036] In the present application, the monoclonal antibody 1A9 of chitinase-3-like protein 1, the heavy chain belongs to IgG2b, and the light chain is kappa.

[0037] The monoclonal antibody 1A9 is screened from hybridoma cells. In the present application, the method for constructing the hybridoma cells preferably comprises constructing a prokaryotic expression vector comprising the sequence of the human chitinase-3-like protein 1 gene (NM_001276.4, SEQ ID NO. 11), transforming the prokaryotic expression vector into TOP10 competent cells, picking single colonies, extracting plasmids, and then transforming the plasmids into E. coli BL21(DE3) expression competent cells, picking single colonies, culturing and centrifuging to collect the supernatant, and purifying to obtain the purified antigen; immunizing mice with the purified antigen, and then taking the spleen of a mouse with good immunization effect to prepare spleen cells; fusing the spleen cells with hybridoma cells, and screening ELISA-positive hybridoma cells. In the present application, the monoclonal antibody is prepared by ascites, and the monoclonal antibody 1A9 is obtained after purification.

[0038] In the present application, the antibody epitope amino acid fragment position information that can be recognized by the monoclonal antibody 1A9 is located at the 311-328 amino acid fragment of CHI3L1, and the specific nucleotide sequence is shown in SEQ ID NO. 19: atcctcggccagcaggtcccctatgccaccaagggcaaccagtgggtaggatac.

[0039] The present application also provides a recombinant vector for expressing the monoclonal antibody 1A9, which comprises a vector carrying a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 and a vector carrying a nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9; the base vector of the vector carrying the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 comprises pFUSE-CHIg-m2a, and the base vector of the vector carrying the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 comprises pFUSE2ss-CLIg-mk; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO. 8; and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO. 10. In the present application, the pFUSE-CHIg-m2a is preferably purchased from the invivogen company. In the present application, the pFUSE2ss-CLIg-mk is preferably purchased from the invivogen company.

[0040] In the present application, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 1A9 is preferably connected between EcoRI and NheI of pFUSE-CHIg-m2a to form a recombinant vector 1A9m2a; and the light chain variable region of the monoclonal antibody 1A9 is preferably connected between EcoRI and NheI of pFUSE2ss-CLIg-mk to form a recombinant vector 1A9mk. The method for inserting the nucleotide sequence into the vector is not particularly limited in the present application, and preferably includes double enzyme digestion.

[0041] The present application also provides a recombinant cell for expressing the monoclonal antibody 1A9, wherein the recombinant cell contains the recombinant vector described in the above technical solution, and the base cell of the recombinant cell includes a mammalian cell.

[0042] The present application also provides a construction method of the recombinant cell described in the above technical solution, which includes the following steps: mixing the recombinant vectors described in the above technical solution and then transfecting the base cell to obtain the recombinant cell. The type of the mammalian cell is not particularly limited in the present application, and preferably includes 293T cells, CHO cells or other mammalian cells, etc. In the embodiments of the present application, the 293T cells purchased from the ATCC cell bank are taken as an example for illustration. The method for extracting the plasmid (recombinant vector) is not particularly limited in the present application, and preferably uses the kit method. In the embodiments, the TIANGEN BIOTECH plasmid extraction kit is selected. The present application uses mixed plasmids to transfect the base cell, and the method for transfection is not particularly limited in the present application.

[0043] The present application also provides a method for preparing the monoclonal antibody 1A9 described in the above technical solution, which includes the following steps: culturing the recombinant cell described in the above technical solution for 48 hours, centrifuging and collecting the supernatant. The monoclonal antibody 1A9 is contained in the supernatant. The present application uses the vector to transfect the cell to express the monoclonal antibody, which is beneficial to cell preservation, antibody quality control and later antibody modification research. In the present application, the culture condition is preferably 37℃ and 5% CO2 environment.

[0044] The present application also provides the application of the monoclonal antibody 1A9 described in the above technical solution or the recombinant vector described in the above technical solution or the recombinant cell constructed by the construction method described in the above technical solution or the monoclonal antibody 1A9 prepared by the method described in the above technical solution in the preparation of a reagent for detecting chitinase-3-like protein 1. In the present application, the detection method preferably includes ELISA method and / or CBA method. The monoclonal antibody described in the present application or the monoclonal antibody produced by the recombinant cell can recognize CHI3L1 protein and has biological activity.

[0045] The application further provides application of the monoclonal antibody 1A9 prepared by the method in the preparation of a reagent for diagnosing and / or judging the prognosis of a nervous system disease. In the application, the reagent is preferably used for diagnosis and / or prognosis judgment by ELISA and / or CBA. In the application, the chitinase-3-like protein 1 is related to the occurrence, development and prognosis judgment of a nervous system disease, especially Alzheimer's disease and a disease related to a gliocyte pathological change, and therefore the monoclonal antibody 1A9 can be used for preparing a related reagent.

[0046] In order to further illustrate the application, the human CHI3L1 monoclonal antibody 1A9, the recombinant vector, the recombinant cell and the method and application provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0047] Example 1

[0048] Preparation of CHI3L1 protein

[0049] Step 1, construction of a CHI3L1 prokaryotic expression vector

[0050] 1. The human CHI3L1 gene sequence (SEQ ID NO. 11) was searched from a GenBank sequence database, and the sequence number was NM_001276.4. The gene sequence was synthesized between NdeI and NotI of a pET-32a vector, and the target gene CHI3L1 was connected between EcoRI and NotI of a pGEX 4t-1 vector.

[0051] 2. The primer was designed, and the target band was amplified by PCR.

[0052] The upstream primer CHI3L1-F (SEQ ID NO. 12) is:

[0053] ggatctggttccgcgtggatccccggaattcatgggtgtgaaggcgtctc;

[0054] The downstream primer CHI3L1-R (SEQ ID NO. 13) is:

[0055] atgcactcgctgcaacgtaggcggccgcatcgtgactgactgacgatctg。

[0056] PCR amplification system (50 μl): template 50 ng, CHI3L1-F (10 μM) 1 μl, CHI3L1-R (10 μM) 1 μl, FastPfu DNA Polymerase (2.5 units) 1 μl, 5x FastPfu buffer 10 μl, 2.5 mM dNTP 4 μl, and the rest of Nuclease-free Water;

[0057] PCR amplification program: 95°C pre-denaturation 5 min; 95°C denaturation 20 s, 58°C annealing 20 s, 72°C extension 40 s, denaturation to extension 35 cycles; 72°C re-extension 5 min; 4°C storage.

[0058] 3. The PCR product was subjected to agarose gel electrophoresis, the gel was cut and recovered, and the target fragment and the vector were respectively digested with corresponding restriction enzymes and recovered;

[0059] 4. The target fragment and the vector were ligated with a homologous recombination enzyme at 50°C for 15 min;

[0060] 5. The ligation product was transformed into TOP10 competent cells and cultured in a 37°C incubator overnight;

[0061] 6. Single colonies were picked into corresponding Amp-resistant LB medium and cultured in a 37°C shaker overnight;

[0062] 7. The plasmid was extracted and sequenced, and the results were compared.

[0063] Step two, CHI3L1 protein expression and purification

[0064] 1. The constructed and correctly sequenced plasmid was transformed into E. coli BL21 (DE3) expression competent cells;

[0065] 2. Single colonies were picked into LB medium and cultured in a 37°C shaker to an OD value of 0.5-1, and IPTG was added for induction, and expressed overnight at 16°C;

[0066] 3. The bacterial cells were collected, ultrasonically broken, and the supernatant was collected after centrifugation. The CHI3L1 protein expressed by pET-32a was purified by Ni column affinity chromatography, and the purified protein was denoted as CHI3L1-6his. The CHI3L1 protein expressed by pGEX4t-1 was purified, and the purified protein was denoted as CHI3L1-strep;

[0067] The purified protein was dialyzed and concentrated to obtain high-concentration CHI3L1-6his protein as an antigen for standby, and CHI3L1-strep protein was used to coat ELISA plates to detect CHI3L1 antibody.

[0068] Example 2

[0069] Obtaining of hybridoma cells

[0070] Step one, immunizing mice with CHI3L1 antigen

[0071] The purified CHI3L1-6his antigen was mixed with complete Freund's adjuvant at a volume ratio of 1:1 to obtain a final concentration of 40 μg antigen / 100 μL; five 6-8 week old female Balb / c mice were injected with 100 μL (containing 40 μg antigen) of the mixed antigen in the left hind leg muscle; three weeks later, the second immunization was performed, 40 μg antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1, and 100 μL (containing 40 μg antigen) of the mixed antigen was injected into the right hind leg muscle of the mice;

[0072] Step two, detecting antibody production of the immunized mice

[0073] The mice were immunized twice, and three weeks later, the tail blood of the mice was collected, the serum was collected by centrifugation, and the antibody production of the mice was detected, which was implemented as follows:

[0074] 1. ELISA method for detecting the antibody titer of the mice: the ELISA plate was coated with the purified CHI3L1-strep protein, 100 ng / well, 4°C overnight (the coating solution was 25 mL carbonate buffer, pH 9.6, containing Na2CO30.03975 g, NaHCO30.07325 g, and KH2PO40.00625 g); the next day, the plate was washed with PBST solution for 3 times, 3 min each time, and then patted dry; 2% BSA was added to the plate to block, and the plate was incubated at 37°C for 1 h; after the incubation, the plate was washed with PBST for 3 times, 3 min each time, and then patted dry to remove the excess water; the serum was diluted with PBST, and 100 μL of the diluted serum was added to each well, and the plate was incubated at 37°C for 1 h; the plate was washed with PBST for 3 times, 3 min each time, and then patted dry; the goat anti-mouse IgG-HRP secondary antibody was diluted at a ratio of 1:5000, and then added to the plate, and the plate was incubated at 37°C for 30 min; after the incubation, the plate was washed with PBST for 3 times, patted dry, and then TMB was added for color development for 10 min, and 2M H2SO4 was added to stop the reaction, and the absorbance value was measured at 450 nm. The No. 3 mouse with the highest antibody titer was selected for the subsequent experiment, and the ELISA titer of the antibody was 656.1 million.

[0075] 2. CBA method for detecting mouse antibody titer: CHI3L1 (CHI3L1-6his) is constructed on eukaryotic expression vector pcDNA3.1, and the plasmid is prepared for standby; the glass slides are placed in 10 cm cell culture dishes, and after polylysine treatment, 293T cells are evenly seeded in the dishes and cultured overnight in a 37℃, 5% CO2 cell incubator; the above successfully constructed CHI3L1-6his containing plasmid is transfected into the above prepared 293T cells, and after 48h, the culture solution is discarded and fixed with acetone for 30min, and the culture box is dried for standby; the serum is diluted with diluent in proportion, diluted by 1:100, 1:1000, 1:10000, 1:100000, and the serum diluted according to the above proportion is used to incubate the above slides for 1h at room temperature; PBST is washed for 3 times, and the second antibody is incubated for 30min; PBST is washed for 3 times, and the results are observed under a microscope, and the antibody CBA titer of the No. 3 mouse is about 1000, as shown in Figure 1

[0076] Step three, cell fusion

[0077] 1. Preparation of myeloma cells: resuscitate SP2 / 0 cells, culture with DMEM medium and 15% FBS for one week, and select logarithmic growth period myeloma cells for cell fusion.

[0078] 2. Preparation of spleen cells: select mice with good immune effect, collect blood by enucleation method, centrifuge to separate serum; sacrifice the mouse by cervical dislocation method, soak in 75% (v / v) alcohol for 5min for disinfection; transfer to a clean bench for subsequent processing, fix the mouse, remove the spleen, remove the fat tissue and connective tissue attached to the cells; wash the obtained spleen with serum-free culture medium, place it on a cell filter, gently grind it with a syringe core, and gently wash the filter with serum-free culture medium, collect the washed liquid; centrifuge the collected spleen cell suspension at 500g for 5min, collect the cell precipitate, wash the cells 3 times, discard the supernatant, and suspend the cell precipitate in serum-free DMEM medium, count and reserve.

[0079] ​3. Cell fusion: ultraclean table in advance ultraviolet sterilization water bath, adjust the water bath to 37℃ for standby, the prepared SP2 / 0 cells and spleen cells are added into 50 mL centrifuge tube at a ratio of 1:10, mixed well. 500g centrifugation for 10 min, gently aspirate and discard the supernatant, gently tap the bottom of the centrifuge tube to slightly loosen the cell sediment; slowly drop 1 mL of 45% PEG1450 solution preheated to 37℃ within 90s; and constantly gently shake the centrifuge tube; the whole process is carried out in 37℃ water bath; then gradually add DMEM medium to the above PEG1450 cell mixed solution at the following rate, that is: 1 mL is added dropwise at 1 min, 2 mL is added at 2 min, 3 mL is added at 3 min, 4 mL is added at 4 min, 5 mL is added at 5 min, and shake in 37℃ water bath. Then incubate at 37℃ for 15 min, centrifuge at 500g for 5 min, discard the supernatant; add 5 mL of DMEM medium containing HAT to the collected cell sediment, suspend the sedimented cells gently, and finally supplement DMED medium containing HAT to a total volume of about 100 mL. Distribute in 96-well cell culture plate with macrophages at 100 μL / well, then place the culture plate in 37℃, 5% CO2 incubator for culture.

[0080] Step four, positive hybridoma cell screening

[0081] 1. ELISA method for screening positive hybridoma cells

[0082] ELISA plate coated with CHI3L1 protein expressed by pGEX4t-1 vector, 100 ng / well, 4℃ overnight coating.

[0083] Observe the growth of hybridoma cells, after seven days, when the cell culture supernatant turns yellow, take an appropriate amount of culture supernatant for ELISA detection of the corresponding antibody, and carry out the first subclone screening, the results are shown in Table 1; after seven to ten days, the second subclone screening is carried out again, ELISA detection of the antibody, select the clones with high OD value in the first screening, and plate in 96-well plates with about 1 cell per well, culture for seven to ten days, then ELISA detection of the antibody, the experimental results are shown in Table 2; select the clones with high OD value in the second screening for the third subclone screening, plate in 96-well plates with about 1 cell per well. Select the clones with high OD value, and finally screen out one positive clone cell 1A9-D8-F6, named 1A9. The experimental results are shown in Table 3.

[0084] Table 1 Experimental results of first ELISA method for screening positive monoclonal

[0085]

[0086]

[0087] Table 2 ELISA screening results of positive monoclonal antibodies

[0088] 1A9 1 2 3 4 5 6 7 8 9 10 11 12 A 0.053 0.053 0.066 0.089 0.047 0.052 0.05 0.085 0.053 0.053 0.054 0.047 B 0.054 0.056 0.054 0.056 0.048 0.064 0.065 0.046 0.053 0.047 0.075 0.054 C 0.052 0.06 0.051 0.053 0.042 0.062 0.119 0.598 0.05 0.08 0.053 0.074 D 0.061 0.06 0.048 0.055 0.084 0.057 0.048 1.342 0.06 0.061 0.052 0.062 E 0.084 0.065 0.051 0.048 0.045 0.051 0.049 0.05 0.047 0.049 0.062 0.07 F 0.086 0.059 0.054 0.053 0.067 0.057 0.056 0.056 0.056 0.053 0.06 0.057 G 0.058 0.058 0.056 0.048 0.05 0.064 0.052 0.059 0.047 0.061 0.053 0.051 H 0.071 0.055 0.049 0.051 0.047 0.057 0.051 0.054 0.048 0.064 0.047 0.055

[0089] Table 3 ELISA screening results of positive monoclonal antibodies

[0090] 1A9D8 1 2 3 4 5 6 7 8 9 10 11 12 A 0.05 0.088 0.09 0.593 0.667 0.165 0.053 0.085 0.053 0.053 0.054 0.085 B 0.05 0.31 0.049 0.665 0.049 0.334 0.047 0.046 0.053 0.047 0.075 0.046 C 0.051 0.169 0.049 0.076 0.168 0.463 0.08 0.598 0.05 0.08 0.053 0.598 D 0.064 0.081 0.048 0.894 0.5 0.521 0.061 0.032 0.06 0.061 0.052 0.032 E 0.05 0.732 0.193 0.265 0.207 0.06 0.049 0.05 0.047 0.049 0.062 0.05 F 0.048 0.05 0.52 0.125 0.48 0.904 0.053 0.056 0.056 0.053 0.06 0.056 G 0.121 0.0449 0.077 0.634 0.131 0.305 0.061 0.059 0.047 0.061 0.053 0.059 H 0.053 0.057 0.049 0.408 0.618 1.053 0.064 0.054 0.048 0.064 0.047 0.054

[0091] 2. CBA method for verifying the screening results of positive hybridoma cells

[0092] CHI3L1 (CHI3L1-6his) was constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was prepared for use. The slides were placed in 10 cm cell culture dishes, and the 293T cells were evenly seeded in the dishes after being treated with polylysine. The dishes were incubated in a 37°C, 5% CO2 cell incubator overnight. The plasmid containing CHI3L1-6his was transfected into the prepared 293T cells, and after 48 hours, the culture medium was discarded and the slides were fixed with acetone for 30 minutes. The slides were dried in the incubator and stored for use. The slides were incubated with the culture supernatant of the screened hybridoma cells, and the culture supernatant of SP2 / 0 cells was used as a control group. The slides were incubated at room temperature for 1 hour, washed with PBST for 3 times, and incubated with a secondary antibody for 30 minutes. The slides were washed with PBST for 3 times, and the results were observed under a microscope. The culture supernatant of the control group could not bind to the cells overexpressing CHI3L1 protein to produce a positive signal, but the culture supernatant of the 1A9 hybridoma cells could produce a positive signal. The results are shown in Table 3, indicating that the screened positive hybridoma cell 1A9 can secrete antibodies that bind to CHI3L1. Figure 2

[0093] Example 3

[0094] Monoclonal antibody typing identification

[0095] CHI3L1 expressed by pGEX 4t-1 vector was coated on ELISA plates at 100 ng / well overnight. The coated ELISA plates were washed with PBST for 3 times, and blocked with 2% BSA for 1 hour. The culture supernatant of the positive monoclonal cells screened in Example 2 was added at 100 μL, and the culture supernatant of SP2 / 0 cells was used as a negative control group (NC group), and the eyeball blood of immunized mice was used as a positive control group. The plates were incubated at 37°C for 1 hour. The plates were washed with PBST for 3 times, and incubated with HRP-labeled secondary antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM, IgG kappa chain, lambda) at 37°C for 30 minutes. The plates were washed with PBST for 3 times, and developed with TMB. The absorbance value was measured at 450 nm. The results are shown in Table 4, indicating that the heavy chain of 1A9 monoclonal antibody is IgG2b, and the light chain is kappa. Figure 3 ​​

[0096] Example 4

[0097] Monoclonal antibody sequence determination

[0098] RNA was extracted from the positive monoclonal cells screened by Example 2; the antibody heavy chain and light chain variable region fragments were amplified by 5' RACE method; the amplified fragments were connected to pEASY-Blunt vector, plasmid was extracted, and sequencing was performed; the light chain and heavy chain nucleotide sequences are SEQ ID NO. 8 and SEQ ID NO. 10, and the corresponding amino acid sequences are SEQ ID NO. 7 and SEQ ID NO. 9; the CDR regions of the antibody amino acid sequences were marked using the Kabat method (SEQ ID NO. 1-SEQ ID NO. 6).

[0099] Example 5

[0100] Recombinant monoclonal antibody and its effect

[0101] Step one, preparation of recombinant monoclonal antibody

[0102] The monoclonal antibody 1A9 heavy chain variable region (SEQ ID NO. 8) determined by sequencing was connected to the antibody expression vector pFUSE-CHIg-m2a, and was recorded as 1A9m2a; the light chain variable region (SEQ ID NO. 10) was connected to the antibody expression vector pFUSE2ss-CLIg-mk, and was recorded as 1A9mk. After sequencing, a large amount of plasmid was extracted for cell transfection to prepare the antibody.

[0103] The glass was placed in a 10 cm cell culture dish, and after polylysine treatment, the 293T cells were evenly seeded in the dish and cultured overnight at 37°C, 5% CO2 cell incubator; the constructed recombinant antibody expression plasmid 1A9m2a, 1A9mk was transfected into the above prepared 293T cells, and cultured at 37°C, 5% CO2 for 48h. The supernatant of the cultured cells was collected, centrifuged, and the supernatant was reserved.

[0104] Step two, detection of recombinant monoclonal antibody titer

[0105] ELISA plate was coated with purified CHI3L1-strep protein, 100 ng / well, 4°C overnight coating (coating solution is 25 mL carbonate buffer, pH 9.6, containing Na2CO30.03975 g, NaHCO30.07325 g, KH2PO40.00625 g); the next day, wash 3 times with PBST solution, 3 min each time, and dry each time; add 2% BSA to fill the hole and block at 37°C for 1 h; after incubation, wash 3 times with PBST, 3 min each time, and remove excess water by tapping the plate each time; dilute the supernatant collected above with diluent by fold dilution, and the dilution ratio is 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, respectively, add 100 μL to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, 3 min each time, and dry each time; dilute goat anti-mouse IgG-HRP secondary antibody at a ratio of 1:5000, add to the plate, and incubate at 37°C for 30 min; after incubation, wash 3 times with PBST, dry, add TMB for color development for 10 min, stop with 2M H2SO4, and measure the absorbance at 450 nm as shown in Table 4 and Figure 4

[0106] Table 4. Recombinant monoclonal antibody titer detection experiment results (OD 450 )

[0107] Dilution fold NC 1A9 1:1 0.051 1.262 1:3 0.048 0.769 1:9 0.050 0.748 1:27 0.045 0.642 1:81 0.048 0.303 1:243 0.05 0.131 1:729 0.051 0.064 1:2187 0.044 0.060

[0108] The results are shown in Figure 4 and Table 4. The recombinant monoclonal antibodies obtained by experimental screening have good titer and are of significance for further research.

[0109] Step three, recombinant monoclonal antibody property test

[0110] 1. Immunofluorescence verification of recombinant monoclonal antibody epitope

[0111] ​CHI3L1 (CHI3L1-6his), CHI3L1 front section (CHI3L1-22-111-6his, SEQ ID NO. 14), CHI3L1 middle front section (CHI3L1-112-201-6his, SEQ ID NO. 15), CHI3L1 middle back section (CHI3L1-202-292-6his, SEQ ID NO. 16), CHI3L1 back section (CHI3L1-293-383-6his, SEQ ID NO. 17) were respectively constructed on the eukaryotic expression vector pcDNA3.1, and the plasmids were prepared for standby use; the glass slides were placed in 10 cm cell culture dishes, and the 293T cells were evenly seeded in the dishes after being treated with polylysine, and then the dishes were placed in a 37 °C, 5% CO2 cell incubator for overnight culture; the plasmids containing CHI3L1-6his, CHI3L1-22-111-6his, CHI3L1-112-201-6his, CHI3L1-202-292-6his, and CHI3L1-293-383-6his fragments were transfected into the above prepared 293T cells, and pcDNA3.1 was transfected as a blank control group; 48 h after transfection, the culture solution was discarded, and the cells were fixed with acetone for 30 min, and then the dishes were dried in the incubator for standby use; the supernatant of the collected recombinant monoclonal cells in step one was used as the primary antibody to incubate the above slides, and the incubation was performed at room temperature for 1 h; the slides were washed with PBST for 3 times, and the secondary antibody was incubated for 30 min; the slides were washed with PBST for 3 times, and the results were observed under a microscope. The experimental results are shown in Figure 5 As shown in (a) of FIG. 1, the recombinant 1A9 monoclonal antibody was successfully expressed in 293T cells, and was successfully secreted in the cell supernatant. The recombinant monoclonal antibody can recognize CHI3L1 and has biological activity. In addition, positive signals were detected only in the cells overexpressing the CHI3L1 back section CHI3L1-293-383-6his and the complete CHI3L1 sequence, indicating that the antibody recognition site is located in the 293-383 interval of the amino acid sequence. To further determine the recognition site, the fragment was further divided for study.

[0112] The CHI3L1 segment (CHI3L1-293-383-6his, SEQ ID NO. 17) was divided into five segments, CHI3L1 (CHI3L1-293-310, SEQ ID NO. 18), CHI3L1 (CHI3L1-311-328, SEQ ID NO. 19), CHI3L1 (CHI3L1-329-346, SEQ ID NO. 20), CHI3L1 (CHI3L1-347-364, SEQ ID NO. 21), and CHI3L1 (CHI3L1-365-383, SEQ ID NO. 22), each with 18 amino acids, and each segment was constructed on the eukaryotic expression vector pcDNA3.1 by deleting each segment, and the plasmid was prepared for use; the glass was placed in a 10 cm cell culture dish, and after being treated with polylysine, the 293T cells were evenly seeded in the dish and incubated overnight in a 37°C, 5% CO2 cell incubator; the empty plasmid pcDNA3.1, Q CHI3L1-293-310-6his plasmid, Q CHI3L1-311-328-6his plasmid, Q CHI3L1-329-346-6his plasmid, Q CHI3L1-347-364-6his plasmid, Q CHI3L1-365-383-6his plasmid, plasmid containing CHI3L1-293-383-6his fragment, and plasmid containing the complete CHI3L1-6his sequence were transfected into the prepared 293T cells; the above steps were repeated, and the results were observed under a microscope.

[0113] The experimental results are shown in (a) of FIG. 6, wherein the recombinant 1A9 monoclonal antibody was successfully expressed in 293T cells and successfully secreted in the cell supernatant. The recombinant monoclonal antibody can recognize CHI3L1 and has biological activity, and the recognition site of the antibody is located in the 293-383 interval of the amino acid sequence. Figure 5 The further experimental results are shown in (b) of FIG. 6, wherein the control group transfected with the empty pcDNA3.1 plasmid had no fluorescence signal, and the group incubated with the anti-his antibody had a signal, indicating that the plasmid transfection and overexpression operation was successful; in the segment deletion groups, except for the group with the deletion of the 311-328 amino acid interval, the other segment groups could be recognized by the CHI3L1 antibody and produced a fluorescence signal, indicating that the deletion of the 311-328 fragment directly affected the recognition process of the antibody, further proving that the recognition site of the antibody is located in the 311-328 amino acid fragment. Figure 5

[0114] 2. WB verifies the characteristics of the recombinant monoclonal antibody

[0115] ​The monkey brain tissue protein and serum samples were used as Western Blot loading samples for SDS-PAGE electrophoresis to verify the action characteristics of the recombinant monoclonal antibody; after electrophoresis, wet transfer was used, the transfer conditions were 200 mA for 60 min; 5% skimmed milk powder was blocked at room temperature for 1 h; the recombinant monoclonal cell 1A9 culture supernatant was used as the primary antibody, and the commercially available CHI3L1 antibody (Affinity Biosciences Cat# DF7223, RRID: AB_2839164) was used as the control group primary antibody, and incubated at room temperature for 2 h; TBST was washed for 3 times, each time for 5 min; the HRP-labeled secondary antibody was added and incubated at room temperature for 1 h; TBST was washed for 3 times, each time for 5 min; the chemiluminescence solution was added for color development and photography, and the results were observed, and the experimental results are shown in Figure 6 The signal in the serum can be recognized by the CHI3L1 antibody, and there is no signal in the monkey brain tissue protein; 1A9 has no signal in the two groups of samples. According to the above experimental results, the recombinant monoclonal antibody 1A9 does not recognize the linear structure, and the antibody is a space conformation antibody.

[0116] Although the above embodiment describes the present application in detail, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which belong to the protection scope of the present application.

Claims

1. A monoclonal antibody 1A9 that can specifically recognize chitinase-3-like protein 1, characterized in that: The monoclonal antibody 1A9 comprises a heavy chain and a light chain; the variable region of the heavy chain comprises three heavy chain complementary determining regions, wherein the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO.3; the light chain variable region of the monoclonal antibody 1A9 comprises three light chain complementary determining regions, wherein the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO.

6.

2. The monoclonal antibody 1A9 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO.

9.

3. The monoclonal antibody 1A9 according to claim 1, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO.8; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1A9 is shown in SEQ ID NO.

10.

4. A recombinant vector expressing monoclonal antibody 1A9, characterized in that: The recombinant vector includes a vector carrying a nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1A9 and a vector carrying a nucleotide sequence encoding the light chain variable region of monoclonal antibody 1A9; the basic vector of the vector carrying the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1A9 includes pFUSE-CHIg-m2a, and the basic vector of the vector carrying the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1A9 includes pFUSE2ss-CLIg-mk; the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1A9 is shown in SEQ ID NO.8; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1A9 is shown in SEQ ID NO.

10.

5. The recombinant vector according to claim 4, characterized in that The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 1A9 was linked between EcoRI and NheI of pFUSE-CHIg-m2a; the light chain variable region of the monoclonal antibody 1A9 was linked between EcoRI and NheI of pFUSE2ss-CLIg-mk.

6. A recombinant cell expressing the monoclonal antibody 1A9, characterized in that: The recombinant cell contains the recombinant vector according to claim 4 or 5, and the basic cells of the recombinant cell include mammalian cells.

7. The method for constructing the recombinant cell according to claim 6, comprising the following steps: The recombinant vectors according to claim 4 or 5 are mixed and then transfected into the basic cells to obtain recombinant cells.

8. A method for preparing the monoclonal antibody 1A9 according to any one of claims 1 to 3, comprising the following steps: The recombinant cell according to claim 6 is cultured for 48 hours, centrifuged, and the supernatant is collected.

9. Use of the monoclonal antibody 1A9 according to any one of claims 1 to 3, the recombinant vector according to claim 4 or 5, the recombinant cell according to claim 6, the recombinant cell constructed by the construction method according to claim 7, or the monoclonal antibody 1A9 prepared by the method according to claim 8 in the preparation of a reagent for detecting CHI3L1.

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