A human CHI3L1 monoclonal antibody 1D7, a recombinant vector, a recombinant plasmid, a method and an application

Abstract

The present application belongs to the technical field of antibody preparation, and belongs to a human CHI3L1 monoclonal antibody 1D7, a recombinant carrier, a recombinant plasmid and a method and application. The present application provides a monoclonal antibody 1D7 capable of specifically recognizing CHI3L1, wherein the monoclonal antibody 1D7 comprises a heavy chain and a light chain; a variable region of the heavy chain comprises three heavy chain complementarity determining regions, and the amino acid sequences of the three heavy chain complementarity determining regions are shown as SEQ ID NO. 1-SEQ ID NO. 3 or SEQ ID NO. 11-SEQ ID NO. 13; and a light chain variable region of the monoclonal antibody 1D7 comprises three light chain complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO. 4-SEQ ID NO. 6. The monoclonal antibody provided in the present application 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 CHI3L1 monoclonal antibody 1D7, a recombinant vector, a recombinant plasmid, a method and application. BACKGROUND

[0002] Chitinase-3-like protein 1 (CHI3L1) is one of the members of the chitinase glycoside hydrolase 18 family. CHI3L1 is a secreted glycoprotein with a size of about 40 kDa, which is expressed and secreted in various cells such as macrophages, chondrocytes, fibroblast-like synoviocytes, vascular smooth muscle cells and hepatic stellate cells. CHI3L1 has two isoforms: one is a full-length isoform containing all 10 exons, and the other is a short variant with exon 8 selectively spliced and deleted. There are eight members of the chitinase glycoside hydrolase 18 family in the human body, and CHI3L1 is one of the glycoprotein members of the human glycosyl hydrolase 18 family, which has chitin binding activity but lacks chitinase activity. The protein is composed of one (β / α) 8 barrel domain and one second domain composed of six anti-parallel β chains, with one α helix (α+β) domain inserted after chain β7, and the C-terminal of the β chain in the (β / α) 8 barrel structure has a carbohydrate binding site containing 43 residues, and the side edge stacking can control the protein-carbohydrate interaction. This structure determines that CHI3L1 can activate the innate defense as a sensor and at the same time regulate the inflammatory response caused by infection, and may also contribute to the occurrence of tumors. Current studies have shown that the expression of the protein is closely related to the occurrence and development of inflammatory diseases, extracellular tissue remodeling, fibrosis, solid cancer and asthma-related diseases.

[0003] CHI3L1 plays an important role in the development of nervous system diseases. The expression of CHI3L1 is controlled by NFI-X3, STAT3 and AP-1, and can promote the migration of primary astrocytes. Its absence can change the inflammatory response of glial cells, promote the phagocytosis of amyloid plaques by astrocytes and microglia, reduce the formation of amyloid plaques, and affect the disease progression of Alzheimer's disease (AD); CHI3L1 can regulate the neuroinflammatory response related to traumatic brain injury (TBI), and its absence leads to more severe neuropathological phenomena and more significant glial pathogenesis; in addition, the high expression of CHI3L1 can significantly increase the content of aortic atherosclerotic plaques, increase the neovascularization of carotid artery plaques, and significantly reduce the collagen content in carotid artery plaques. In early human carotid artery plaques and mouse model plaques, CHI3L1 expression promotes a significant increase in lesion area, while significantly inhibiting macrophage apoptosis. CHI3L1 shows great potential for medical applications, and with further research, CHI3L1 may become a diagnostic marker and new target for the 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 current CHI3L1 detection antibodies come from hybridoma cells, and traditional hybridoma cells are not easy to store, and will be in poor condition after a long period of use, and are difficult to recover. SUMMARY

[0004] The present application aims to provide a human CHI3L1 monoclonal antibody 1D7, a recombinant vector, a recombinant plasmid, and a method and application. The monoclonal antibody has good titer, and the expression vector and recombinant cells used to prepare the monoclonal antibody are easy to store and modify.

[0005] The present application provides a monoclonal antibody 1D7 that can specifically recognize CHI3L1, which comprises a heavy chain and a light chain; the variable region of the heavy chain comprises three heavy chain complementarity determining regions, and the amino acid sequences of the three heavy chain complementarity determining regions are shown in SEQ ID NO. 1-3 or SEQ ID NO. 11-13; the variable region of the light chain of the monoclonal antibody 1D7 comprises three light chain complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 4-6.

[0006] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO. 7 or SEQ ID NO. 14; and the amino acid sequence of the light chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO. 9.

[0007] Preferably, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 is as shown in SEQ ID NO. 8 or as shown in SEQ ID NO. 15; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7 is as shown in SEQ ID NO. 10.

[0008] The present application further provides a recombinant vector for expressing the monoclonal antibody 1D7, wherein the recombinant vector comprises a vector carrying a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 and a vector carrying a nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7; the base vector of the vector carrying the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 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 1D7 comprises pFUSE2ss-CLIg-mk; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 is as shown in SEQ ID NO. 8; and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7 is as shown in SEQ ID NO. 10.

[0009] Preferably, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 1D7 is connected between EcoRI and NheI of pFUSE-CHIg-m2a; and the nucleotide sequence of the light chain variable region of the monoclonal antibody 1D7 is connected between EcoRI and NheI of pFUSE2ss-CLIg-mk.

[0010] The present application further provides a recombinant cell for expressing the monoclonal antibody 1D7, wherein the recombinant cell comprises the recombinant vector described in the above technical solution, and the base cell of the recombinant cell comprises a mammalian cell.

[0011] The present application further provides a construction method of the recombinant cell described in the above technical solution, comprising the following steps: mixing the recombinant vector described in the above technical solution, and then transfecting the base cell to obtain the recombinant cell.

[0012] The present application further provides a method for preparing the monoclonal antibody 1D7 described in the above technical solution, comprising the following steps: culturing the recombinant cell described in the above technical solution for 48 hours, centrifuging, and collecting the supernatant.

[0013] The present application further provides an application of the monoclonal antibody 1D7 described in the above technical solution, the recombinant vector described in the above technical solution, the recombinant cell described in the above technical solution, the recombinant cell constructed by the construction method described in the above technical solution, or the monoclonal antibody 1D7 prepared by the method described in the above technical solution in the preparation of a reagent for detecting CHI3L1.

[0014] The application further provides application of the monoclonal antibody 1D7 prepared by the method in the preparation of a reagent for diagnosing and / or judging the prognosis of a nervous system disease.

[0015] The application provides a human CHI3L1 monoclonal antibody 1D7. The monoclonal antibody 1D7 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 and transfected cells can realize expression of the monoclonal antibody 1D7. The monoclonal antibody 1D7 vector is easy to preserve, easy to control the quality of the antibody production process, and also facilitates a series of modifications of the antibody, more in-depth research and more extensive application. It is verified by examples that the monoclonal antibody 1D7 or the monoclonal antibody 1D7 produced by the recombinant cells can recognize CHI3L1 protein and has biological activity. The monoclonal antibody 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 following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0017] Figure 1 A CBA method provided by the application for detecting serum antibody titer of immunized mice is shown in the result graph;

[0018] Figure 2 A CBA method provided by the application for screening positive hybridoma cells is shown in the result graph;

[0019] Figure 3 A monoclonal antibody subtype identification result graph provided by the application is shown, 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 graph provided by the application is shown;

[0021] Figure 5The immunofluorescence verification monoclonal antibody antigen epitope distribution provided by the present application is shown in the figure; 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 329-346 amino acid fragment schematic diagram;

[0022] Figure 6 The WB verification recombinant monoclonal antibody effect characteristic result graph provided by the present application is shown in the figure. DETAILED DESCRIPTION

[0023] The present application provides a monoclonal antibody 1D7 capable of specifically recognizing CHI3L1, the monoclonal antibody 1D7 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 being shown in SEQ ID NO.1-SEQ ID NO.3, or shown in SEQ ID NO.11-SEQ ID NO.13; the light chain variable region of the monoclonal antibody 1D7 comprises three light chain complementarity determining regions, the amino acid sequences being shown in 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): GFWMS;

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

[0027] DINSDGSTINYAPSIKD;

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

[0029] Or,

[0030] CDR1 (SEQ ID NO.11): VFWMS;

[0031] CDR2 (SEQ ID NO.12):

[0032] DIISDGSVINYAPSIKD;

[0033] CDR3 (SEQ ID NO.13): YKGAY;

[0034] In the present application, CDR1 (SEQ ID NO.11), CDR2 (SEQ ID NO.12) and CDR3 (SEQ ID NO.13) are alternative heavy chain complementarity determining regions, when the monoclonal antibody 1D7 heavy chain variable region sequence is replaced, the same experimental results can still be achieved.

[0035] In the present application, the amino acid sequences of the 3 light chain complementarity determining regions are as follows:

[0036] CDR1 (SEQ ID NO. 4):

[0037] KSSQSLLDSDGKTYLN;

[0038] CDR2 (SEQ ID NO. 5): LVSKLDS;

[0039] CDR3 (SEQ ID NO. 6): WQGTHFPYT.

[0040] In the present application, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D7 is as shown in SEQ ID NO. 7 (MEWELSLIFIFALVKDVQCEVQLLETGGGLVQPGGSRGLSCEGSGFTFSGF WMSWVRQTPGKTLEWIGDINSDGSTINYAPSIKDRFTIFRDNDKSTLYLQMSN VRSEDTATYFCMRYKGAHWGQGTLVTVSA) or as shown in SEQ ID NO. 14 (MEWELSLIFIFALLKDVQCEVKLLETGGGLVQPGGSRGLSCEGSGFTFSVF WMSWVRQTPGKTLEWIGDIISDGSVINYAPSIKDRFTIFRDNDKSTLYLQMSN VRSEDTATYFCLRYKGAYWGQGTTLTVSS); the amino acid sequence of the light chain variable region of the monoclonal antibody 1D7 is as shown in SEQ ID NO. 9 (MMSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK).

[0041] In the present application, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 is shown as SEQ ID NO. 8 (atggagtgggaactgagcttaattttcatttttgctcttgtaaaagatgtccagtgtgaagtgcagctgttggagactggaggaggcttggtgcaacctggggggtcacggggactctcttgtgaaggctcagggtttacttttagtggcttctggatgagttgggttcgacagacacctgggaagaccctggagtggattggagacattaattctgatggcagtacaataaactacgcaccatccataaaggatcgattcactatcttcagagacaatgacaagagcaccctgtacctgcagatgagcaatgtgcgatctgaggacacagccacgtatttctgtatgagatataaaggggcccactggggccaagggactctggtcactgtctctgca) or SEQ ID NO. 15 (atggagtgggaactgagcttaattttcatttttgctcttttaaaagatgtccagtgtgaagtgaagctgttggagactggaggaggcttggtgcaacctggggggtcacggggactctcttgtgaaggtcaggttttacttttagtgtcttctggatgagctgggttcgacagacacctgggaagaccctggagtggattggagacattatttctgatggcagtgtaataaactacgcaccatccataaaggatcgattcactatcttcagagacaatgacaagagcaccctgtacctgcagatgagcaatgtgcgatctgaggacacagccacgtatttctgtttgagatataagggggcctactggggccaaggcaccactctcacagtc tcctca); the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7 is shown as SEQ ID NO.10 (atgatgagtcctgcccagttcctgtttctgttagtgctctggattcgggaaaccaacggtgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttccgtacacgttcggaggggggaccaagctggaaataaaa).

[0042] In the present application, the CHI3L1 monoclonal antibody 1D7 has a heavy chain belonging to IgG2b and a light chain of kappa.

[0043] The monoclonal antibody 1D7 in the present application is screened from hybridoma cells. In the present application, the construction method of the hybridoma cells preferably comprises constructing a prokaryotic expression vector containing the sequence of human CHI3L1 gene (NM_001276.4, SEQ ID NO. 16), 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 for culture and centrifuging to collect supernatant, and purifying to obtain a purified antigen; immunizing mice with the purified antigen, taking the spleen of a mouse with better immunization effect to prepare spleen cells; fusing the spleen cells with hybridoma cells, and screening ELISA positive hybridoma cells. In the present application, ascites is used to prepare the monoclonal antibody, and the monoclonal antibody 1D7 is obtained after purification.

[0044] In the present application, the antibody epitope amino acid fragment position information that can be recognized by the monoclonal antibody 1D7 is located at the 329-346 amino acid fragment of CHI3L1, and the specific nucleotide sequence is shown in SEQ ID NO. 25: gacgaccaggaaagcgtcaaaagcaaggtgcagtacctgaaggacaggcagctg.

[0045] The present application also provides a recombinant vector for expressing monoclonal antibody 1D7, which comprises a vector carrying a nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D7 and a vector carrying a nucleotide sequence encoding the light chain variable region of monoclonal antibody 1D7; the base vector of the vector carrying the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D7 comprises pFUSE-CHIg-m2a, and the base vector of the vector carrying the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1D7 comprises pFUSE2ss-CLIg-mk; the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D7 is shown in SEQ ID NO. 8; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1D7 is shown in SEQ ID NO. 10. In the present application, the pFUSE-CHIg-m2a is preferably purchased from invivogen company. In the present application, the pFUSE2ss-CLIg-mk is preferably purchased from invivogen company.

[0046] In the present application, the nucleotide sequence of the heavy chain variable region of monoclonal antibody 1D7 is preferably connected between EcoRI and NheI of pFUSE-CHIg-m2a to form a recombinant vector 1D7m2a; and the light chain variable region of monoclonal antibody 1D7 is preferably connected between EcoRI and NheI of pFUSE2ss-CLIg-mk to form a recombinant vector 1D7mk. The method for inserting the nucleotide sequence into the vector is not particularly limited in the present application, and preferably comprises double enzyme digestion.

[0047] The present application also provides a recombinant cell for expressing monoclonal antibody 1D7, which comprises the recombinant vector described in the above technical solution, and the base cell of the recombinant cell comprises a mammalian cell.

[0048] The present application also provides a construction method of the recombinant cell described in the above technical solution, which comprises the following steps: mixing the recombinant vector described in the above technical solution and transfecting the base cell to obtain the recombinant cell. In the present application, the mass ratio of the vector carrying the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D7 and the vector carrying the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1D7 is preferably 1:1. The type of the mammalian cell is not particularly limited in the present application, and preferably comprises 293T cell, CHO cell or other mammalian cell, etc., and in the embodiment of the present application, the 293T cell purchased from ATCC cell library is taken as an example for illustration. The method for extracting the plasmid (recombinant vector) is not particularly limited in the present application, and preferably utilizes kit method, and in the embodiment, TIANGEN BIOTECH plasmid extraction kit is selected. The base cell is transfected by mixing the plasmids in the present application, and the method for transfection is not particularly limited in the present application.

[0049] The application further provides a method for preparing the monoclonal antibody 1D7, which comprises the following steps: culturing the recombinant cell for 48 hours, centrifuging, and collecting the supernatant. The monoclonal antibody 1D7 is contained in the supernatant. The application uses a vector to transfect a cell to express the monoclonal antibody, which is beneficial to cell preservation, antibody quality control and later antibody modification research. In the application, the culture condition is preferably culturing at 37 DEG C and in a 5% CO2 environment.

[0050] The application further provides application of the monoclonal antibody 1D7, the recombinant vector, the recombinant cell or the recombinant cell prepared by the construction method or the monoclonal antibody 1D7 prepared by the method in preparation of a reagent for detecting CHI3L1. In the application, the detection method preferably comprises an ELISA method and / or a CBA method. The monoclonal antibody or the monoclonal antibody produced by the recombinant cell can recognize CHI3L1 protein and has biological activity.

[0051] The application further provides application of the monoclonal antibody 1D7, the recombinant vector, the recombinant cell or the recombinant cell prepared by the construction method or the monoclonal antibody 1D7 prepared by the method in preparation of a reagent for diagnosing and / or judging prognosis of a nervous system disease. In the application, the reagent preferably adopts an ELISA method and / or a CBA method for diagnosis and / or prognosis judgment. In the application, CHI3L1 is related to occurrence, development and prognosis judgment of a nervous system disease, especially Alzheimer's disease and a related disease of a glial cell lesion, so that the monoclonal antibody 1D7 can be used for preparing a related reagent.

[0052] In order to further illustrate the application, the application of a human CHI3L1 monoclonal antibody 1D7, a recombinant vector, a recombinant plasmid and a method and application are described in detail in combination with the drawings and examples, but they should not be understood as limitation to the protection scope of the application.

[0053] Example 1

[0054] Preparation of CHI3L1 protein

[0055] Step one, construction of a CHI3L1 prokaryotic expression vector

[0056] 1. The human CHI3L1 gene sequence (SEQ ID NO. 16) was obtained from the GenBank sequence database, the sequence number is NM_001276.4, and the gene sequence was synthesized into the pET-32a vector between NdeI and NotI, and the target gene CHI3L1 was connected into the pGEX 4t-1 vector between EcoRI and NotI;

[0057] 2. The primer was designed, and the target band was amplified by PCR.

[0058] The upstream primer CHI3L1-F (SEQ ID NO. 17) is:

[0059] ggatctggttccgcgtggatccccggaattcatgggtgtgaaggcgtctc;

[0060] The downstream primer CHI3L1-R (SEQ ID NO. 18) is:

[0061] atgcactcgctgcaacgtaggcggccgcatcgtgactgactgacgatctg.

[0062] The PCR amplification system (50 μl) is as follows: 50 ng of template, 1 μl of CHI3L1-F (10 μM), 1 μl of CHI3L1-R (10 μM), 1 μl of FastPfu DNA Polymerase (2.5 units), 10 μl of 5×FastPfu buffer, 4 μl of 2.5 mM dNTP, and the rest of Nuclease-free Water;

[0063] The PCR amplification program is as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 20 s, 58°C annealing for 20 s, 72°C extension for 40 s, denaturation to extension for 35 cycles; 72°C extension for 5 min again; and 4°C storage.

[0064] 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 the corresponding restriction endonuclease and recovered;

[0065] 4. The target fragment and the vector were connected with the homologous recombination enzyme, and the connection was performed at 50°C for 15 min;

[0066] 5. The ligation product was transformed into TOP10 competent cells, and the cells were cultured in a 37°C incubator overnight;

[0067] 6. The single colony was picked into the corresponding Amp-resistant LB medium, and the cells were cultured in a 37°C shaker overnight;

[0068] 7. The plasmid was extracted, sequenced, and the results were compared.

[0069] Step two, CHI3L1 protein expression and purification

[0070] 1. The constructed and sequenced plasmid was transferred into E. coli BL21 (DE3) expression competent cells;

[0071] 2. The single colony was picked and inoculated in LB medium, and shaken at 37°C until the OD value was 0.5-1. IPTG was added for induction, and the expression was performed at 16°C overnight;

[0072] 3. The bacterial cells were collected, and broken by ultrasonic. After centrifugation, the supernatant was collected. The CHI3L1 protein expressed by pET-32a was purified by Ni column affinity chromatography, and the purified protein was recorded as CHI3L1-6his. The CHI3L1 protein expressed by pGEX4t-1 was purified, and the purified protein was recorded as CHI3L1-strep;

[0073] 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 for coating ELISA plate to detect CHI3L1 antibody.

[0074] Example 2

[0075] Obtaining of hybridoma cells

[0076] Step one, immunizing mice with CHI3L1 antigen

[0077] 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 into the left hind leg muscle. Three weeks later, the second immunization was performed, and 40 μg antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1. The mixed antigen was injected into the right hind leg muscle of the mice at a dose of 100 μL (containing 40 μg antigen);

[0078] Step two, detecting antibody production in immunized mice

[0079] The mice were immunized twice, and three weeks later, the tail blood was collected, and the serum was collected by centrifugation. The antibody production in the mice was detected, and the specific implementation was as follows:

[0080] 1. ELISA method for detecting mouse antibody titer: the purified CHI3L1-strep protein was used to coat the ELISA plate, 100 ng / well, 4°C overnight coating (coating solution was 25 mL carbonate buffer, pH 9.6, containing Na2CO30.03975 g, NaHCO30.07325 g, KH2PO40.00625 g); the next day, wash with PBST solution for 3 times, 3 min each time, and pat dry each time; add 2% BSA to fill the well for blocking, incubate at 37°C for 1 h; after incubation, wash with PBST for 3 times, 3 min each time, and pat the plate to remove excess water each time; dilute the serum with diluent, add 100 μL of the diluted serum to each well, and incubate at 37°C for 1 h, wash with PBST for 3 times, 3 min each time, and pat dry each time; dilute the 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 with PBST for 3 times, pat dry, add TMB for color development for 10 min, terminate with 2M H2SO4, and measure the absorbance value at 450 nm. The No. 3 mouse with good titer was selected for subsequent experiments, and the antibody ELISA titer was 6.561 million.

[0081] 2. CBA method for detecting mouse antibody titer: CHI3L1 (CHI3L1-6his) was constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was prepared for use; the glass slides were placed in a 10 cm cell culture dish, and after polylysine treatment, 293T cells were uniformly seeded in the dish and incubated overnight at 37°C in a 5% CO2 cell incubator; the successfully constructed plasmid was transfected into the prepared 293T cells, and after 48 h, the culture medium was discarded and fixed with acetone for 30 min, and the culture dish was dried in the incubator for standby; the serum was diluted with diluent at a ratio of 1:100, 1:1000, 1:10000, and 1:100000, and the above slides were incubated with the serum diluted according to the above ratios at room temperature for 1 h; wash with PBST for 3 times, incubate with secondary antibody for 30 min; wash with PBST for 3 times, and observe the results under a microscope. The No. 3 mouse immunofluorescence signal result is shown in Figure 1 , and the antibody CBA titer was about 1000.

[0082] Step three, cell fusion

[0083] 1. Preparation of myeloma cells: resuscitate SP2 / 0 cells, culture with DMEM medium and 15% FBS for one week, and select the logarithmic growth period of myeloma cells for cell fusion.

[0084] 2. Preparation of spleen cells: select mice with good immune effect, take blood by enucleation, centrifugal separation of serum; decapitation method to kill mice, 75% (v / v) alcohol immersion 5 min disinfection; transfer to clean bench for subsequent processing, fixed mice, remove the spleen, remove the fat tissue and connective tissue of the adherent cells; the obtained spleen was washed with serum-free culture medium, placed on a cell filter, gently ground with a syringe core, and gently washed the filter with serum-free culture medium, and collected the washed liquid; centrifuge the collected spleen cell suspension at 500g for 5 min, 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.

[0085] 3. Cell fusion: ultraclean bench, ultraviolet sterilization water bath in advance, adjust the water bath to 37℃ for standby, add the prepared SP2 / 0 cells and spleen cells in a ratio of 1:10 into a 50mL centrifuge tube, mix well. 500g centrifugation for 10 min, gently suck out and discard the supernatant, gently tap the bottom of the centrifuge tube to slightly loosen the cell precipitate; slowly drop 1mL of 45% PEG1450 solution preheated to 37℃ within 90s; and constantly gently shake the centrifuge tube; the whole process is carried out in a 37℃ water bath; then gradually add DMEM medium to the above PEG1450 cell mixed solution at the following rate: 1mL at a time, 2mL at 2min, 3mL at 3min, 4mL at 4min, 5mL at 5min, shake in 37℃ water bath. Then incubate at 37℃ for 15 min, centrifuge at 500g for 5 min, discard the supernatant; add 5mL of DMEM medium containing HAT to the collected cell precipitate, gently suspend the precipitated cells, and finally supplement DMED medium containing HAT to a total volume of about 100mL. Place in a 96-well cell culture plate coated with macrophages at 100μL / well, then place the culture plate in a 37℃, 5% CO2 incubator for culture.

[0086] Step four, screening of positive hybridoma cells

[0087] 1. ELISA method for screening positive hybridoma cells

[0088] ELISA plate coated with CHI3L1 (CHI3L1-strep) protein expressed by pGEX4t-1 vector, 100ng / well, 4℃ overnight coating.

[0089] The growth of the hybridoma cells was observed, and after seven days when the cell culture supernatant turned yellow, an appropriate amount of the culture supernatant was taken for ELISA detection of the corresponding antibody, and first subcloning screening was performed, and the results are shown in Table 1; after seven to ten days, second subcloning screening was performed, and the antibody was detected by ELISA, and the clones with high OD values in the first screening were selected, and were plated in 96-well plates at about 1 cell per well, and after seven to ten days of culture, the antibody was detected by ELISA, and the experimental results are shown in Table 2; the clones with high OD values in the second screening were selected for third subcloning screening, and were plated in 96-well plates at about 1 cell per well. The clones with high OD values were selected, and finally one positive clone cell 1D7-C5-D5 was screened, and was named 1D7. The experimental results are shown in Table 3.

[0090] Table 1 Experimental results of first ELISA screening of positive monoclonal antibodies

[0091]

[0092]

[0093] Table 2 Experimental results of second ELISA screening of positive monoclonal antibodies

[0094] 1D7 1 2 3 4 5 6 7 8 9 10 11 12 A 0.055 0.059 0.062 0.064 0.059 0.054 0.05 0.085 0.054 0.057 0.035 0.051 B 0.042 0.054 0.053 0.045 0.059 0.062 0.065 0.046 0.053 0.047 0.062 0.069 C 0.034 0.06 0.051 0.053 0.954 0.052 0.072 0.598 0.045 0.051 0.078 0.062 D 0.035 0.062 0.078 0.054 0.049 0.053 0.056 0.042 0.059 0.052 0.054 0.059 E 0.029 0.062 0.075 0.041 0.045 0.057 0.059 0.05 0.062 0.065 0.049 0.06 F 0.057 0.047 0.051 0.052 0.502 0.058 0.056 0.056 0.065 0.058 0.053 0.052 G 0.058 0.047 0.046 0.039 0.062 0.064 0.049 0.059 0.046 0.056 0.056 0.046 H 0.07 0.055 0.049 0.049 0.045 0.057 0.052 0.054 0.048 0.056 0.042 0.052

[0095] Table 3 Experimental results of third ELISA screening of positive monoclonal antibodies

[0096] 1 D7 C5 1 2 3 4 5 6 7 8 9 10 11 12 A 0.075 0.154 0.229 0.075 0.062 0.067 0.05 0.085 0.054 0.057 0.035 0.051 B 0.138 0.279 1.103 0.905 0.573 1.502 0.065 0.046 0.053 0.047 0.062 0.069 C 0.089 0.067 0.139 0.088 1.225 1.055 0.072 0.598 0.045 0.552 0.078 0.211 D 0.601 1.405 1.286 0.786 1.624 0.088 0.056 0.042 0.123 0.052 0.112 0.075 E 0.06 1.243 0.26 0.943 1.296 1.504 0.059 0.05 0.120 0.065 0.049 0.061 F 1.349 0.378 1.141 1.272 0.088 0.528 0.056 0.056 0.079 1.037 0.053 0.052 G 0.408 0.18 0.742 1.159 0.132 1.23 0.049 0.059 0.046 0.056 0.056 0.048 H 0.058 0.069 0.376 0.272 0.873 0.072 0.052 0.023 0.120 0.056 0.042 0.059

[0097] 2. CBA method for verifying the screening results of positive hybridoma cells

[0098] CHI3L1 (CHI3L1-6his) was constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was prepared for later use; the slides were plated in 10 cm cell culture dishes, and after polylysine treatment, 293T cells were uniformly seeded in the dishes and incubated overnight in a 37°C, 5% CO2 cell incubator; the successfully constructed plasmid was transfected into the above prepared 293T cells, and after 48 h, the culture medium was discarded and fixed with acetone for 30 min, and then dried in the incubator for later use; the above 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, and incubated at room temperature for 1 h; washed with PBST for 3 times, and incubated with the secondary antibody for 30 min; washed with PBST for 3 times, and the results were observed under a microscope, wherein the control group supernatant could not bind to the CHI3L1 protein overexpressing cells to produce a positive signal, but the 1D7 hybridoma cell supernatant could detect a positive signal, and the experimental results are shown in Table 4, indicating that the screened positive hybridoma cells can secrete antibodies that bind to CHI3L1. Figure 2

[0099] Example 3​

[0100] Monoclonal antibody typing identification

[0101] Coat ELISA plate with CHI3L1-strep, 100 ng / well, overnight; wash coated ELISA plate with PBST for 3 times, block with 2% BSA for 1 hour; add 100 μL of positive monoclonal cell culture supernatant screened in Example 2, SP2 / 0 cell culture supernatant as negative control group (NC group), eyeball blood of immunized mouse as positive control group, incubate at 37°C for 1 hour; wash for 3 times with PBST; incubate HRP-labeled secondary antibody (IgG1, IgG2a, IgG2b, IgG3, IgM, IgG kappa chain, lambda) at 37°C for 30 minutes. Wash for 3 times with PBST, develop with TMB, and measure absorbance at 450 nm. The experimental results are shown in Figure 3 Figure 1, the heavy chain of the 1D7 monoclonal antibody is IgG2b, and the light chain is kappa.

[0102] Example 4

[0103] Monoclonal antibody sequence determination

[0104] Extract RNA from the positive monoclonal cell 1D7 screened in Example 2; amplify the variable region fragments of the heavy chain and light chain of the antibody by 5' RACE method; connect the amplified fragments to the pEASY-Blunt vector, extract the plasmid, and sequence; the nucleotide sequences of the light chain and heavy chain 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; use the Kabat method to mark the CDR regions of the antibody amino acid sequence (SEQ ID NO. 1-SEQ ID NO. 6).

[0105] Example 5

[0106] Recombinant monoclonal antibody and its effect

[0107] Step one, preparation of recombinant monoclonal antibody

[0108] Connect the sequenced monoclonal antibody 1D7 heavy chain variable region (SEQ ID NO. 8) to the antibody expression vector pFUSE-CHIg-m2a, and mark it as 1D7m2a; connect the 1D7 light chain variable region (SEQ ID NO. 10) to pFUSE2ss-CLIg-mk, and mark it as 1D7mk. After sequencing, extract a large amount of plasmid for cell transfection to prepare the antibody.

[0109] The glass slides were placed in 10 cm cell culture dishes, and 293T cells were evenly seeded in the dishes after polylysine treatment, and then the dishes were incubated overnight at 37°C in a 5% CO2 cell incubator. The constructed recombinant antibody expression plasmids 1D7m2a and 1D7mk were introduced into the prepared 293T cells, and the cells were incubated at 37°C in 5% CO2 for 48 h. The supernatant of the cultured cells was collected by centrifugation, and the supernatant of the screened positive monoclonal cells was collected as a positive control.

[0110] Step two, detection of the titer of the recombinant monoclonal antibody

[0111] The ELISA plate was coated with the purified CHI3L1-strep protein at 100 ng / well at 4°C overnight (the coating solution was 25 mL carbonate buffer, pH 9.6, containing Na2CO3 0.03975 g, NaHCO3 0.07325 g, and KH2PO4 0.00625 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 fill the wells for blocking, and the plate was incubated at 37°C for 1 h; after incubation, the plate was washed with PBST for 3 times, 3 min each time, and then patted dry to remove excess water; the collected supernatant was diluted with diluent at a ratio of 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, and 100 μL of each dilution was added to each well, and the plate was incubated at 37°C for 1 h; 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 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 at 450 nm was measured as shown in Table 4 and Figure 4

[0112] Table 4 Experimental results of detection of the titer of the recombinant monoclonal antibody (OD 450 )

[0113] Dilution factor NC 1 D7 hybridoma 1D7 1:3 0.051 0.737 1.935 1:9 0.048 0.639 2.142 1:27 0.05 0.505 2.229 1:81 0.045 0.169 1.658 1:243 0.05 0.106 0.924 1:729 0.051 0.062 0.327 1:2187 0.044 0.055 0.156

[0114] The results are shown in Figure 4 and Table 4, and the titer of the obtained recombinant monoclonal antibody is good, which has significance for further research.

[0115] Step three, property test of the recombinant monoclonal antibody

[0116] 1. Immunofluorescence verification of the epitope of the recombinant monoclonal antibody

[0117] ​CHI3L1 (CHI3L1-6his), CHI3L1 front section (CHI3L1-22-111-6his, SEQ ID NO. 19), CHI3L1 middle front section (CHI3L1-112-201-6his, SEQ ID NO. 20), CHI3L1 middle back section (CHI3L1-202-292-6his, SEQ ID NO. 21), CHI3L1 back section (CHI3L1-293-383-6his, SEQ ID NO. 22) were respectively constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was prepared for standby; the glass was placed in a 10 cm cell culture dish, and after polylysine treatment, 293T cells were evenly seeded in the dish and cultured overnight in a 37°C, 5% CO2 cell incubator; the plasmid containing CHI3L1-6his, CHI3L1-22-111-6his, CHI3L1-112-201-6his, CHI3L1-202-292-6his, CHI3L1-293-383-6his fragment was transfected into the above prepared 293T cells, and pcDNA3.1 was transfected as a negative control group; 48 h after transfection, the culture solution was discarded and fixed with acetone for 30 min, and the incubator was dried for standby; the incubation of anti-his antibody group was set to verify whether the overexpression of plasmid transfection was successful; then the supernatant of the collected recombinant monoclonal cells in step one was used as a primary antibody to incubate the above slides, and the supernatant of the positive hybridoma cells was used as a control primary antibody, and incubated at room temperature for 1 h; PBST was washed for 3 times, and the secondary antibody was incubated for 30 min; PBST was washed for 3 times, and the results were observed under a microscope. The results showed that, except for the pcDNA3.1 group, the cells incubated with his antibody showed positive results, indicating that the corresponding protein was successfully overexpressed by plasmid transfection; in addition, positive signals were detected only in cells overexpressing CHI3L1 gene back section CHI3L1-293-383-6his and 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.

[0118] CHI3L1-293-310 (SEQ ID NO. 23), CHI3L1-311-328 (SEQ ID NO. 24), CHI3L1-329-346 (SEQ ID NO. 25), CHI3L1-347-364 (SEQ ID NO. 26), CHI3L1-365-383 (SEQ ID NO. 27), and the plasmids containing each fragment and the plasmid containing the complete CHI3L1-6his sequence were constructed on the eukaryotic expression vector pcDNA3.1, and the plasmids were prepared for use; the 293T cells were uniformly seeded in the dishes after being treated with polylysine, and the dishes were 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, the plasmid containing the CHI3L1-293-383-6his fragment, and the 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.

[0119] The experimental results are shown in (a) of FIG. 6. The recombinant 1D7 monoclonal antibody was successfully expressed in the 293T cells and was successfully secreted in the cell supernatant. The recombinant monoclonal antibody can recognize CHI3L1 and has biological activity. The antibody recognition site is located in the 293-383 interval of the CHI3L1 amino acid sequence. Figure 5 The experimental results are shown in (b) of FIG. 6. 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 groups with the deletion of the fragments, except for the group with the deletion of the 329-346 amino acid interval, the other groups could be recognized by the CHI3L1 antibody and produced a fluorescence signal, indicating that the deletion of the 329-346 fragment directly affected the recognition process of the antibody, and further proving that the antibody recognition site is located in the 329-346 amino acid fragment. Figure 5 2. WB verification of the characteristics of the recombinant monoclonal antibody

[0120]

[0121] ​The purified CHI3L1 protein, CHI3L1 cell protein overexpressed and cell control protein transfected with empty pcDNA3.1 plasmid were used as Western Blot loading samples for SDS-PAGE electrophoresis to verify the properties of the recombinant monoclonal antibody; after electrophoresis, wet transfer was used, the transfer conditions were 200 mA, 60 min; 5% skim milk was blocked at room temperature for 1 h; the recombinant monoclonal cell supernatant was used as the primary antibody, and 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; washed with TBST for 3 times, 5 min each time; HRP-labeled secondary antibody was added and incubated at room temperature for 1 h; washed with TBS T for 3 times, 5 min each time; chemiluminescence solution was added for color development and photography, and the results were observed.

[0122] The experimental results are shown in Table 1. Figure 6 As shown in Table 1, the CHI3L1 antibody can detect the purified CHI3L1 protein; the recombinant monoclonal antibody 1D7 can achieve the same experimental results; the cell protein overexpressing CHI3L1 may not be detected due to low concentration. In summary, the recombinant monoclonal antibody 1D7 can recognize the linear structure of CHI3L1 protein and has good performance and can be used as Western Blot experiment.

[0123] 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 are within the protection scope of the present application.

Claims

1. A monoclonal antibody 1D7 that specifically recognizes CHI3L1, characterized in that: The monoclonal antibody 1D7 includes a heavy chain and a light chain; the variable region of the heavy chain includes 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 1D7 includes 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 1D7 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO.

9.

3. The monoclonal antibody 1D7 according to claim 1, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO.8; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO.

10.

4. A recombinant vector expressing monoclonal antibody 1D7, characterized in that: The recombinant vector includes a vector carrying a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 and a vector carrying a nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7; the basic vector of the vector carrying the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 includes pFUSE-CHIg-m2a, and the basic vector of the vector carrying the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7 includes pFUSE2ss-CLIg-mk; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID NO.8; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D7 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 1D7 was linked between EcoRI and NheI of pFUSE-CHIg-m2a; the light chain variable region of the monoclonal antibody 1D7 was linked between EcoRI and NheI of pFUSE2ss-CLIg-mk.

6. A recombinant cell expressing the monoclonal antibody 1D7, 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 1D7 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 1D7 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 1D7 prepared by the method according to claim 8 in the preparation of a reagent for detecting CHI3L1.

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