Monoclonal antibody specifically recognizing hcg p-39 and its preparation method and application

Abstract

The application provides a monoclonal antibody specifically recognizing HCGP-39 and a preparation method and application thereof, and relates to the technical field of biological medicines.The application provides two monoclonal antibodies 6F6 and 6D9 of HCGP-39, and the titers are good.Based on the complementarity determining region sequences of the variable regions of the heavy chains and the light chains of the monoclonal antibodies, the variable region sequences are respectively constructed on corresponding antibody expression vectors, and the recombinant cells expressing the recombinant monoclonal antibodies can be obtained by transfecting cells.The monoclonal antibody vector obtained by the application is easy to preserve and easy to control the quality in the antibody production process; the monoclonal antibody can recognize the HCGP-39 protein, has biological activity, can be used for HCGP-39 antigen detection and the like, and has very good application value and very important scientific research guiding significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a monoclonal antibody specifically recognizing HCGP-39, a preparation method and application thereof. BACKGROUND

[0002] Human cartilage glycoProtein-39 (HCGP-39) was first obtained from the supernatant of cultured joint cartilage cells in 1993 by denaturing polyacrylamide gel electrophoresis. HCGP-39 is classified into the chitinase (also known as chitinase) family, also known as chitinase-3-like protein-1 (CHI3LI) and cartilage glycoProtein-39 (GP-39). HCGP-39 is a secreted glycoprotein with a size of about 40 kDa. In vivo studies have found that the expression level of HCGP-39 is increased in the serum, synovial fluid and cartilage of rheumatoid arthritis patients, the serum of alcoholic cirrhosis patients, recurrent breast cancer and colorectal cancer patients, and the macrophages of atherosclerotic plaques. It is speculated that HCGP-39 plays a role in inflammation, tissue reconstruction and tumor metastasis. Recent studies have shown that HCGP-39 can stimulate human connective tissue cell proliferation and activate the extracellular signal-regulated kinase (ERK) and protein kinase B (pKB) mediated signaling pathways, suggesting the growth factor-like role of HCGP-39. Other studies have shown that the expression of the protein is closely related to the occurrence and development of inflammatory, extracellular tissue remodeling, fibrosis, solid cancer and asthma-related diseases.

[0003] A study comparing the expression differences of 10,000 genes between 19 glioma samples and normal brain tissue samples in a gene profile screening of glioma samples and normal brain tissue showed that HCGP-39 is the gene with the most significant expression difference, and the expression of HCGP-39 at the mRNA level is significantly increased in glioma samples compared with normal brain tissue. In the disease progression of Alzheimer's disease (AD), HCGP-39 can promote the migration of primary astrocytes, change the inflammatory response of glial cells, promote the phagocytosis of Aβ by astrocytes and microglial cells, and reduce the formation of amyloid plaques. In addition, the high expression of HCGP-39 can significantly increase the content of aortic atherosclerotic plaques, increase the neovascularization of carotid artery plaques, and significantly reduce the collagen content. HCGP-39 has good development potential and research prospects. With further research, HCGP-39 may become a new diagnostic marker and therapeutic target for central nervous system-related diseases.

[0004] The preparation of the conventional monoclonal antibody at present mainly depends on hybridoma cells, but the hybridoma cells are not easy to preserve, and the cell state will become poor after long time use, and it is difficult to recover. SUMMARY

[0005] The present application aims to provide a monoclonal antibody specifically recognizing HCGP-39, and a preparation method and application thereof, the monoclonal antibody has good titer, and the expression vector and recombinant cell based on the monoclonal antibody are easy to preserve and modify.

[0006] The present application provides a monoclonal antibody specifically recognizing HCGP-39, the monoclonal antibody comprises 6F6 and 6D9;

[0007] The heavy chain variable region of 6F6 comprises three complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 1-3; the light chain variable region comprises three complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 4-6;

[0008] The heavy chain variable region of 6D9 comprises three complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 11-13; the light chain variable region comprises three complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 14-16.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of 6F6 is shown in SEQ ID NO. 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of 6D9 is shown in SEQ ID NO. 17; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 18.

[0011] The present application further provides a recombinant vector for expressing the above monoclonal antibody.

[0012] Preferably, the basic vector of the recombinant vector comprises pFUSE-CHIg-m2a carrying the nucleotide sequence for coding the heavy chain variable region, and pFUSE2ss-CLIg-mk carrying the nucleotide sequence for coding the light chain variable region.

[0013] The present application further provides a hybridoma cell for expressing the above monoclonal antibody.

[0014] The present application further provides a recombinant cell for expressing the above monoclonal antibody.

[0015] The application also provides a method for constructing the recombinant cell, comprising the following steps: transfecting a base cell with a recombinant vector expressing a heavy chain variable region of a monoclonal antibody and a recombinant vector expressing a light chain variable region of the same monoclonal antibody after mixing the two vectors, so as to obtain a recombinant cell expressing the monoclonal antibody.

[0016] The application also provides a method for preparing the monoclonal antibody, comprising the following steps: culturing the recombinant cell for 48 hours, and collecting a supernatant, wherein the supernatant contains the corresponding monoclonal antibody.

[0017] The application also provides an application of the monoclonal antibody in preparing a reagent for detecting HCGP-39.

[0018] The application also provides an application of the monoclonal antibody in preparing a reagent for diagnosing and / or judging the prognosis of a nervous system disease.

[0019] Beneficial effects: the application provides two monoclonal antibodies 6F6 and 6D9 of HCGP-39, and the titers are good. Based on the complementarity determining region sequences of the heavy chain and light chain variable regions of the two monoclonal antibodies, the variable region sequences are respectively constructed into corresponding antibody expression vectors, and the recombinant cells expressing the recombinant monoclonal antibodies are obtained by transfecting cells. The monoclonal antibody vectors obtained by the application are easy to store and easy to control the quality of the antibody production process; it is also convenient to make a series of modifications to the antibodies, and the antibodies can be more deeply studied and more widely applied. It is verified by the examples that the monoclonal antibody 6F6, the monoclonal antibody 6D9 or the monoclonal antibody 6F6 and the monoclonal antibody 6D9 produced by the recombinant cells can recognize HCGP-39 protein, have biological activity, can be used for HCGP-39 antigen detection and the like, have very good application value and very important scientific research guiding significance, and have good deep application prospect and very important scientific research guiding significance. BRIEF DESCRIPTION OF DRAWINGS

[0020] 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.

[0021] Figure 1 CBA method for detecting immune mouse serum antibody titer result graph;

[0022] Figure 2 CBA method for screening positive hybridoma cell result graph;

[0023] Figure 3 Purified monoclonal antibody coomassie brilliant blue staining result graph;

[0024] Figure 4 Figure 6 is a graph of the results of monoclonal antibody subtype identification, with each set of data from left to right representing IgG, IgGl, IgG2a, IgG2b, IgG3, IgM, kappa and lambda;

[0025] Figure 5 Figure 7 is a graph of the results of monoclonal antibody antibody titer determination;

[0026] Figure 6 Figure 8 is a graph of the results of immunofluorescence verification of the monoclonal antibody antigen epitope distribution; (a) the antibody recognition site is located in the interval of 293 to 383 of the amino acid sequence; (b) more specifically, the recognition site of the antibody 6F6, antibody 6D9 is located on the amino acid fragment of 311-346;

[0027] Figure 7 Figure 9 is a graph of the results of WB verification of the action characteristics of the recombinant monoclonal antibodies 6F6 and 6D9. DETAILED DESCRIPTION

[0028] The present application provides a monoclonal antibody specifically recognizing HCGP-39, and the monoclonal antibody comprises 6F6 and 6D9;

[0029] The heavy chain variable region of 6F6 comprises three complementarity determining regions, and the amino acid sequences are respectively shown in SEQ ID NO. 1-SEQ ID NO. 3; the light chain variable region comprises three complementarity determining regions, and the amino acid sequences are respectively shown in SEQ ID NO. 4-SEQ ID NO. 6;

[0030] The heavy chain variable region of 6D9 comprises three complementarity determining regions, and the amino acid sequences are respectively shown in SEQ ID NO. 11-SEQ ID NO. 13; the light chain variable region comprises three complementarity determining regions, and the amino acid sequences are respectively shown in SEQ ID NO. 14-SEQ ID NO. 16.

[0031] The heavy chain variable region and the light chain variable region of the monoclonal antibody of the present application each comprise three complementarity determining regions, wherein the three complementarity determining region sequences of the heavy chain variable region of 6F6 are respectively:

[0032] CDR1 (SEQ ID NO. 1): SYWIQ;

[0033] CDR2 (SEQ ID NO. 2): AIYPGDGDTRYTQKFKG;

[0034] CDR3 (SEQ ID NO. 3): GNAY;

[0035] The three complementarity determining region sequences of the light chain variable region are respectively:

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

[0037] CDR2 (SEQ ID NO. 5): KVSNRFS;

[0038] CDR3 (SEQ ID NO. 6): FQGSHVPWT.

[0039] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6F6 according to the present application is preferably as shown in SEQ ID NO. 7: MECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWIQWVKKRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCASGNAYWGQGTTLTVSS, and the corresponding nucleotide sequence of the encoding gene is as shown in SEQ ID NO. 9.

[0040] The amino acid sequence of the light chain variable region of the monoclonal antibody 6F6 according to the present application is as shown in SEQ ID NO. 8: MKLPVRLLVLMFWIPVSSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSRSGTGTEFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK, and the corresponding nucleotide sequence of the encoding gene is as shown in SEQ ID NO. 10.

[0041] The 3 complementarity determining region sequences of the heavy chain variable region of the monoclonal antibody 6D9 according to the present application are respectively:

[0042] CDR1 (SEQ ID NO. 11): DYYMH;

[0043] CDR2 (SEQ ID NO. 12): WIDPENGDTEYAPKFQG;

[0044] CDR3 (SEQ ID NO. 13): WDLGGY;

[0045] The 3 complementarity determining region sequences of the light chain variable region are respectively:

[0046] CDR1 (SEQ ID NO. 14): RSSQNIVHSNGNTYLE;

[0047] CDR2 (SEQ ID NO. 15): KVSNRFS;

[0048] CDR3 (SEQ ID NO. 16): FQGSHIPPT.

[0049] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6D9 described in the application is shown in SEQ ID NO. 17: MKCSWVIFFLMAVVIGINSEVQLQQSGAELVRSGASVKLSCTASGFNIKD YYMHWVKQRPEQGLEWIGWIDPENGDTEYAPKFQGKAAMTADTSSNTAYL HLSSLTSEDTAVYYCNAWDLGGYWGQGTTLTVSS; the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO. 19.

[0050] The amino acid sequence of the light chain variable region of the monoclonal antibody 6D9 described in the application is shown in SEQ ID NO. 18: MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQNIV HSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDLGVYFCFQGSHIPPTFGAGTKLELK; the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO. 20.

[0051] The application also provides a hybridoma cell expressing the above-mentioned monoclonal antibody.

[0052] The construction method of the hybridoma cell described in the application is not particularly limited, and preferably comprises constructing a prokaryotic expression vector containing the human HCGP-39 gene sequence (NM_001276.4), 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 the supernatant, purifying to obtain a purified antigen; immunizing mice with the purified antigen, 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.

[0053] The application also provides a method for preparing a monoclonal antibody using the hybridoma cell, preferably comprising preparing a monoclonal antibody using ascites, and purifying to obtain the monoclonal antibody 6F6 and the monoclonal antibody 6D9.

[0054] The application also provides a recombinant vector expressing the above-mentioned monoclonal antibody.

[0055] The basic vector of the recombinant vector of the present application comprises pFUSE-CHIg-m2a carrying the nucleotide sequence encoding the heavy chain variable region and pFUSE2ss-CLIg-mk carrying the nucleotide sequence encoding the light chain variable region. The pFUSE-CHIg-m2a and pFUSE2ss-CLIg-mk of the present application are preferably both purchased from invivogen company, and the above-mentioned encoding genes are inserted into the corresponding basic vectors by double enzyme digestion method, such as the nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 19 are inserted into the EcoRI, NheI sites of pFUSE-CHIg-m2a to form the recombinant vectors 6F6m2a, 6D9m2a; the nucleotide sequences shown in SEQ ID NO. 10 and SEQ ID NO. 20 are inserted into the EcoRI, NheI sites of pFUSE2ss-CLIg-mk to form the recombinant vectors 6F6mk, 6D9mk.

[0056] The present application also provides a recombinant cell expressing the above-mentioned monoclonal antibody.

[0057] The basic cell of the recombinant cell of the present application preferably comprises a eukaryotic cell, and more preferably comprises a mammalian cell. The type of the mammalian cell of the present application is not particularly limited, and preferably comprises 293T cells, CHO cells or other mammalian cells, etc. In the embodiments of the present application, 293T cells purchased from ATCC cell bank are taken as an example for illustration, but it cannot be regarded as the whole protection scope of the present application.

[0058] The present application also provides a construction method of the above-mentioned recombinant cell, comprising the following steps: mixing the recombinant vector expressing the heavy chain variable region of the monoclonal antibody with the recombinant vector expressing the light chain variable region of the same monoclonal antibody, and then transfecting the basic cell to obtain the recombinant cell expressing the monoclonal antibody.

[0059] When the transfection of the present application is carried out, the plasmids 6F6m2a and 6F6mk are mixed and then transfected, and the plasmids 6D9m2a and 6D9mk are mixed and then transfected. The mixing ratio of 6F6m2a and 6F6mk and 6D9m2a and 6D9mk is preferably 1:1. The extraction method of the plasmid of the present application is not particularly limited, and the kit method is preferably used, and the TIANGEN BIOTECH plasmid extraction kit is selected in the embodiments. The basic cell is transfected by using the mixed plasmid, and the transfection method of the present application is not particularly limited, and the conventional transfection method in the art can be used.

[0060] The present application also provides a preparation method of the above-mentioned monoclonal antibody, comprising the following steps: culturing the above-mentioned recombinant cell for 48h, and collecting the supernatant, wherein the supernatant comprises the corresponding monoclonal antibody.

[0061] The culture preferably comprises culturing at 37℃ under 5% CO2, and collecting the cell supernatant after the end of the culture, and centrifuging to obtain the supernatant for use. The monoclonal antibodies obtained by the present application all have biological activity, and can specifically recognize HCGP-39, and the antibody epitope recognized by monoclonal antibody 6F6 and monoclonal antibody 6D9 corresponds to the amino acid nucleotide sequence shown in SEQ ID NO. 27 and SEQ ID NO. 28:

[0062] SEQ ID NO. 27: atcctcggccagcaggtcccctatgccaccaagggcaaccagtgggtaggatac

[0063] SEQ ID NO. 28: gacgaccaggaaagcgtcaaaagcaaggtgcagtacctgaaggacaggcagctg.

[0064] The monoclonal antibody 6F6 obtained by the present application has an IgG2b heavy chain and a kappa light chain; and the monoclonal antibody 6D9 has an IgG1 heavy chain and a kappa light chain.

[0065] The present application also provides the use of the above monoclonal antibodies in the preparation of reagents for detecting HCGP-39.

[0066] The monoclonal antibody 6F6 and monoclonal antibody 6D9 of the present application can both recognize HCGP-39 protein, have biological activity, and can be used for detecting HCGP-39. The method for the detection is not particularly limited in the present application, and conventional protein detection methods in the art can be used, such as ELISA method, CBA method and Western Blot method.

[0067] The present application also provides the use of the above monoclonal antibodies in the preparation of reagents for diagnosing and / or judging the prognosis of nervous system diseases.

[0068] In the present application, HCGP-39 is related to the occurrence, development and prognosis of nervous system diseases, especially Alzheimer's disease and related diseases of glial cell pathology, and thus the monoclonal antibody 6F6 and monoclonal antibody 6D9 of the present application can be used for preparing related diagnostic and / or prognostic reagents.

[0069] In order to further illustrate the present application, the specific monoclonal antibody for recognizing HCGP-39 provided by the present application, and the preparation method and application thereof will be described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0070] Example 1 Preparation of HCGP-39 protein

[0071] Step one, construction of HCGP-39 prokaryotic expression vector

[0072] 1. The human HCGP-39 gene sequence (SEQ ID NO. 21) was obtained from the GenBank sequence database, sequence number NM_001276.4, and the gene sequence was synthesized between Ndel and Notl of pET-32a vector, and the target gene HCGP-39 was connected between EcoRI and Notl of pGEX 4t-1 vector;

[0073] 2. The primers were designed, and the target band was amplified by PCR. The PCR amplification system (50 μl) was configured: template 50 ng, HCGP-39-F (10 μM) 1 μL, HCGP-39-R (10 μM) 1 μL, FastPfu DNA Polymerase (2.5 units) 1 μL, 5x FastPfu buffer 10 μL, 2.5 mM dNTP 4 μL, Nuclease-free Water to 50 μL;

[0074] The upstream primer HCGP-39-F (SEQ ID NO. 31) is: ggatctggttccgcgtggatccccggaattcatgggtgtgaaggcgtctc;

[0075] The downstream primer HCGP-39-R (SEQ ID NO. 32) is: atgcactcgctgcaacgtaggcggccgcatcgtgactgactgacgatctg.

[0076] The PCR amplification program is: 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, 34 cycles; 72°C re-extension for 5 min; 4°C storage.

[0077] 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;

[0078] 4. The target fragment and the vector were ligated with the homologous recombination enzyme, and the ligation was performed at 50°C for 15 min;

[0079] 5. The ligation product was transformed into TOP10 competent cells, and incubated in a 37°C incubator overnight;

[0080] 6. Single colonies were picked into the corresponding Amp-resistant LB medium and incubated in a 37°C shaker overnight;

[0081] 7. Extract plasmid, sequence, compare results.

[0082] Step two, HCGP-39 protein expression and purification

[0083] 1. The plasmid with correct sequence was transformed into E. coli BL21(DE3) expression competent cells;

[0084] 2. The single colony was inoculated in LB medium and incubated at 37°C until the OD value was 0.5-1. IPTG was added for induction and the expression was carried out at 16°C overnight.

[0085] 3. The bacterial cells were collected and disrupted by ultrasonic. The supernatant was collected after centrifugation. The HCGP-39 protein expressed by pET-32a was purified by Ni column affinity chromatography. The purified protein was denoted as HCGP-39-6his. The HCGP-39 protein expressed by pGEX4t-1 was purified. The purified protein was denoted as HCGP-39-strep.

[0086] The purified protein was dialyzed and concentrated to obtain high-concentration HCGP-39-6his protein as an antigen for storage. The HCGP-39-strep protein was used for coating ELISA plates to detect HCGP-39 antibodies.

[0087] Example 2: Obtaining hybridoma cells

[0088] Step one, immunizing mice with HCGP-39 antigen

[0089] 40 μg of purified HCGP-39-6his antigen was mixed with complete Freund's adjuvant at a ratio of 1:1. Five 6-8 week-old female Balb / c mice were injected with 100 μL (containing 40 μg of antigen) of the mixed antigen into the left hind leg muscle. Three weeks later, the mice were injected with 100 μL (containing 40 μg of antigen) of the mixed antigen into the right hind leg muscle for the second immunization.

[0090] Step two, detecting antibody production in immunized mice

[0091] The mice were immunized twice. Three weeks later, the mouse tail blood was collected and the serum was collected by centrifugation. The antibody production in the mice was detected. The specific implementation is as follows:

[0092] 1. ELISA method for detecting mouse antibody titer:

[0093] (1) The purified HCGP-39-strep protein was used to coat the ELISA plate at a dosage of 100 ng / well. The coating was carried out at 4°C overnight. The coating solution composition (25 mL carbonate buffer, pH = 9.6, composition: Na2CO30.03975 g, NaHCO30.07325 g, KH2PO40.00625 g).

[0094] (2) The next day, wash 3 times with PBST solution, 3 min each time, and pat dry each time;

[0095] (3) Add 2% BSA blocking, incubate at 37°C for 1 h; after incubation, wash 3 times with PBST, 3 min each time, and pat the plate to remove excess water each time;

[0096] (4) Dilute the serum with PBST solution, add 100 μL of the diluted serum to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, 3 min each time, and pat dry each time;

[0097] (5) Dilute the goat anti-mouse IgG-HRP secondary antibody at a ratio of 1:5000, add it to the plate, and incubate at 37°C for 30 min;

[0098] (6) After incubation, wash 3 times with PBST, pat dry, add TMB for color development for 10 min, terminate with 2M H2SO4, and measure the absorbance at 450 nm.

[0099] Experimental results: The No. 5 mouse with good screening titer was used for subsequent experiments, and the antibody ELISA titer was 6.561 million.

[0100] 2. CBA method for detecting mouse antibody titer:

[0101] (1) Construct HCGP-39 (HCGP-39-6his) on the eukaryotic expression vector pcDNA3.1, and prepare the plasmid for use;

[0102] (2) Place the slides in a 10 cm cell culture dish, treat with polylysine, and evenly seed 293T cells in the dish, and incubate overnight at 37°C in a 5% CO2 cell incubator;

[0103] (3) Transfect HCGP-39-6his into the above prepared 293T cells, 48 h later, discard the culture medium, and fix with acetone for 30 min, and dry in the incubator for standby;

[0104] (4) Dilute the serum with PBST solution according to the ratio, dilute at a ratio of 1:100, 1:1000, 1:10000, and 1:100000, and incubate the above slides with the serum diluted according to the above ratio at room temperature for 1 h;

[0105] (5) Wash 3 times with PBST, incubate the secondary antibody for 30 min; wash 3 times with PBST, and observe the results under a microscope.

[0106] Experimental results: The No. 5 mouse immunofluorescence signal results are as shown in Figure 1 , and the antibody CBA titer is about 1000.

[0107] Step three, cell fusion

[0108] 1. Preparation of myeloma cells:

[0109] Recovery of SP2 / 0 cells, subculture with DMEM medium, 15% FBS for one week. Select the logarithmic growth period of myeloma cells for cell fusion.

[0110] 2. Preparation of spleen cells:

[0111] (1) Select mice with good immune effect, take blood by enucleation, centrifuge to separate serum;

[0112] (2) The mice were sacrificed by cervical dislocation and transferred to 75% (v / v) alcohol for 5 min disinfection;

[0113] (3) Transfer the mouse to the clean bench for subsequent processing. After fixation, remove the spleen, remove the fat tissue and connective tissue adhered to the cells;

[0114] (4) The obtained spleen was washed with serum-free culture solution, placed on a cell filter, gently ground with a syringe core, and gently washed the filter with serum-free culture solution. Collect the washed liquid;

[0115] (5) 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.

[0116] 3. Cell fusion:

[0117] (1) Ultraviolet sterilization water bath in clean bench in advance, adjust the water bath to 37℃ for standby, add the above prepared SP2 / 0 cells and spleen cells to 50mL centrifuge tube at a ratio of 1:10, mix well, centrifuge at 500g for 10min;

[0118] (2) Gently aspirate and discard the supernatant, gently tap the bottom of the centrifuge tube to slightly loosen the cell precipitate;

[0119] (3) Slowly drop 1mL of 45% PEG1450 solution preheated to 37℃ in 90s in 37℃ water bath; and constantly gently shake the centrifuge tube;

[0120] (4) Then gradually add DMEM medium at an increasing rate (1mL in the first min; 2mL in the second min; 3mL in the third min; 4min in the fourth min; 5min in the fifth min) to the PEG1450 cell mixture solution, and shake under 37℃ water bath conditions;

[0121] (5) Incubate at 37°C for 15 min, centrifuge at 500g for 5 min, discard the supernatant;

[0122] (6) Add 5 mL of DMEM medium containing HAT to the collected cell precipitate, and suspend the precipitated cells gently;

[0123] (7) Supplement the DMED medium containing HAT to a total volume of about 100 mL. Distribute 100 μL / well into a 96-well cell culture plate in which macrophages have been plated, and then place the culture plate in a 37°C, 5% CO2 incubator.

[0124] Step four, screening of positive hybridoma cells

[0125] 1. ELISA method for screening positive hybridoma cells

[0126] (1) Coat the ELISA plate with HCGP-39 protein expressed by pGEX4t-1 vector, 100 ng / well, and incubate overnight at 4°C.

[0127] (2) Observe the growth of hybridoma cells, and 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 perform the first subcloning screening. The results are shown in Table 1. The OD values of F6 and D9 positions of the sixth well plate are high, and the cells of these two wells are selected for further screening, and are marked as 6F6 and 6D9.

[0128] Table 1. Results of the first ELISA screening of positive monoclonal cells

[0129] HCGP-39 1 2 3 4 5 6 7 8 9 10 11 12 A 0.061 0.059 0.056 0.075 0.053 0.058 0.054 0.057 0.057 0.056 0.059 0.058 B 0.052 0.05 0.049 0.163 0.169 0.052 0.086 0.05 0.053 0.169 0.049 0.077 C 0.054 0.162 0.09 0.196 0.052 0.053 0.074 0.057 0.053 0.066 0.059 0.052 D 0.054 0.053 0.069 0.06 0.052 0.226 0.051 0.05 1.145 0.203 0.066 0.055 E 0.052 0.228 0.183 0.053 0.06 0.063 0.051 0.141 0.097 0.227 0.05 0.217 F 0.052 0.056 0.064 0.077 0.844 1.248 0.051 0.085 0.055 0.076 0.079 0.068 G 0.052 0.057 0.051 0.058 0.051 0.081 0.061 0.055 0.133 0.052 0.074 0.117 H 0.082 0.062 0.062 0.064 0.05 0.054 0.056 0.095 0.089 0.105 0.052 0.058

[0130] (3) After 7-10 days, perform the second subcloning screening, and detect the antibody by ELISA. Select the clones 6F6 and 6D9 with high OD values in the first screening, plate in a 96-well plate with about 1 cell per well, and after 7-10 days of culture, detect the antibody by ELISA. The experimental results are shown in Table 2.

[0131] Table 2. Results of the second ELISA screening of positive monoclonal cells

[0132]

[0133]

[0134] (4) After 7-10 days, perform the third subcloning screening, and detect the antibody by ELISA. Select the clones 6F6-G3 and 6D9-B3 with high OD values in the second screening, plate in a 96-well plate with about 1 cell per well, and after 7-10 days of culture, detect the antibody by ELISA. The experimental results are shown in Table 3.

[0135] Table 3 Experimental results of the third ELISA method screening positive monoclonal

[0136]

[0137]

[0138] Finally, 6F6-G3-B9 and 6D9-B3-F4 with high OD values were screened out and named 6F6 and 6D9.

[0139] 2. CBA method for verifying the screening results of positive hybridoma cells

[0140] (1) Construct HCGP-39 (HCGP-39-6his) on eukaryotic expression vector pcDNA3.1, and prepare the plasmid for later use;

[0141] (2) Place the glass slides in a 10 cm cell culture dish, treat with polylysine, and then evenly seed 293T cells in the dish. Incubate overnight at 37°C in a 5% CO2 incubator;

[0142] (3) Transfect HCGP-39-6his into the prepared 293T cells, and after 48 hours, discard the culture medium and fix with acetone for 30 minutes. Dry in the incubator and reserve for later use;

[0143] (4) Incubate the above slides with the culture supernatant of the screened hybridoma cells for 1 hour at room temperature;

[0144] (5) Wash 3 times with PBST, incubate with secondary antibody for 30 minutes, wash 3 times with PBST, and observe the results under a microscope.

[0145] The experimental results are shown in Table 3, and the culture supernatant of 6F6 and 6D9 cells can produce a positive reaction with cells overexpressing HCGP-39, indicating that the culture supernatant contains HCGP-39 antibodies. Figure 2

[0146] Example 3 Preparation of monoclonal antibodies by ascites and purification of monoclonal antibodies

[0147] Step 1: Preparation of monoclonal antibodies by ascites

[0148] 1. Inject 300 μL of ascites adjuvant into the abdominal cavity of a 12-week-old Balb / c mouse.

[0149] 2. After two weeks, culture the hybridoma cells to the best cell activity state, adjust the cell number to about 1 x 10 6 μL of hybridoma cells to the abdominal cavity of the mouse that has been injected with ascites adjuvant.

[0150] 3. Collect the ascites after 7-10 days. ​

[0151] Step two, purification of monoclonal antibody

[0152] 1. Dilute ascites with PBS pH 7.4, centrifuge to get supernatant, purify with protein A affinity chromatography;

[0153] 2. Equilibrium: equilibrate the purification column with 0.4M PB buffer (pH 7.0);

[0154] 3. Column loading: slowly load the diluted ascites supernatant into the column to ensure better binding of the antibody to the protein A column;

[0155] 4. Washing: wash the column with the equilibration buffer;

[0156] 5. Elution: elute the antibody bound to the column with 0.1M glycine buffer (pH 2.7) and neutralize the glycine with 1M Tris-HCl (pH 8.0) to maintain a neutral pH suitable for antibody storage. Perform SDS-PAGE electrophoresis on the purified monoclonal antibody and control serum, respectively, and the experimental results are shown in Figure 3 , where (a) represents 6F6 and (b) represents 6D9. The heavy and light chains of the two antibodies can be clearly seen.

[0157] Example 4: Identification of monoclonal antibody typing

[0158] (1) Coat the ELISA plate with HCGP-39 expressed by pGEX 4t-1 vector, 100 ng / well, overnight;

[0159] (2) Wash the coated ELISA plate with PBST 3 times, block with 2% BSA for 1 hour;

[0160] (3) Add 100 μL of hybridoma cell supernatant, incubate at 37°C for 1 hour;

[0161] (4) Wash 3 times with PBST;

[0162] (5) Add HRP-labeled secondary antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM, IgG kappa chain, lambda) respectively, incubate at 37°C for 30 minutes;

[0163] (6) Wash 3 times with PBST, develop with TMB, measure absorbance at 450 nm.

[0164] The experimental results are shown in Figure 4 , where the heavy chain of 6F6 monoclonal antibody is IgG2b and the light chain is kappa Figure 4 (a); the heavy chain of 6D9 monoclonal antibody is IgG1 and the light chain is kappa Figure 4 (b).

[0165] Example 5 Sequence determination of monoclonal antibody

[0166] (1) Extract RNA from the hybridoma cells cultured in Example 2;

[0167] (2) Amplify the variable region fragments of the heavy chain and light chain of the antibody using 5' RACE method;

[0168] (3) Connect the amplified fragments to pEASY-Blunt vector, extract plasmid, and sequence;

[0169] The nucleotide sequences of the heavy chain and light chain of monoclonal antibody 6F6 are shown in SEQ ID NO. 9 and SEQ ID NO. 10, and the corresponding amino acid sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8;

[0170] The nucleotide sequences of the heavy chain and light chain of monoclonal antibody 6D9 are shown in SEQ ID NO. 19 and SEQ ID NO. 20, and the corresponding amino acid sequences are shown in SEQ ID NO. 17 and SEQ ID NO. 18;

[0171] The CDR regions of the amino acid sequence of antibody 6F6 (SEQ ID NO. 1-SEQ ID NO. 6) and the CDR regions of the amino acid sequence of antibody 6D9 (SEQ ID NO. 11-SEQ ID NO. 16) are marked using the Kabat method.

[0172] Example 6 Recombinant monoclonal antibody and its effect

[0173] Step 1, preparation of recombinant monoclonal antibody

[0174] (1) Connect the sequenced monoclonal antibody 6F6 heavy chain variable region (SEQ ID NO. 9) and light chain variable region (SEQ ID NO. 10); and monoclonal antibody 6D9 heavy chain variable region (SEQ ID NO. 19) and light chain variable region (SEQ ID NO. 20) to antibody expression vectors pFUSE-CHIg-m2a, pFUSE2ss-CLIg-mk, respectively, and mark them as 6F6m2a, 6F6mk, 6D9m2a, 6D9mk. After sequencing, extract a large amount of plasmid and group them for cell transfection to prepare antibodies.

[0175] (2) Place the glass in a 10 cm cell culture dish, treat with polylysine, and then evenly seed 293T cells in the dish, and incubate in a 37℃, 5% CO2 cell incubator overnight;

[0176] (3) The constructed recombinant antibody expression plasmids were divided into groups: 6F6m2a and 6F6mk, 6D9m2a and 6D9mk, and transferred into the prepared 293T cells. The cells were cultured at 37°C and 5% CO2 for 48 hours. The supernatant of the cultured cells was collected and centrifuged for later use.

[0177] Step 2: Recombinant monoclonal antibody titer detection

[0178] (1) ELISA plates were coated with purified HCGP-39-strep protein at 100 ng / well at 4°C overnight (the coating solution was 25 mL carbonate buffer, pH 9.6, containing 0.03975 g of Na2CO3, 0.07325 g of NaHCO3, and 0.00625 g of KH2PO4);

[0179] (2) After coating, wash with PBST solution three times, 3 min each time, and pat dry each time to remove excess water;

[0180] (3) Add 2% BSA to block the wells and incubate at 37°C for 1 h;

[0181] (4) After incubation, wash with PBST three times for 3 minutes each time, and tap the plate to remove excess water each time;

[0182] (5) The collected supernatant was serially diluted with diluent at ratios of 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, and 1:6561, respectively;

[0183] (6) Add 100 μL of diluted culture supernatant to each well and incubate at 37°C for 1 h;

[0184] (7) Wash with PBST three times, 3 min each time, and pat dry to remove excess water each time;

[0185] (8) Dilute goat anti-mouse IgG-HRP secondary antibody at a ratio of 1:5000, add it to the plate, and incubate at 37°C for 30 min;

[0186] (9) After incubation, the cells were washed three times with PBST, patted dry, and TMB was added for 10 min for color development. The cells were terminated with 2 M H2SO4. The absorbance values ​​at 450 nm were shown in Table 4 below.

[0187] Table 4 Recombinant monoclonal antibody titer detection experimental results (OD 450 )

[0188]

[0189]

[0190] Monoclonal antibody 6F6 and 6D9 titers were as shown in Figure 5

[0191] Step three, recombinant monoclonal antibody property test

[0192] 1. Immunofluorescence verification of recombinant monoclonal antibody epitope

[0193] (1) HCGP-39 (HCGP-39-6his) was divided into HCGP-39 front section (HCGP-39-22-111-6his, SEQ ID NO. 22), HCGP-39 middle front section (HCGP-39-112-201-6his, SEQ ID NO. 23), HCGP-39 middle back section (HCGP-39-202-292-6his, SEQ ID NO. 24), HCGP-39 back section (HCGP-39-293-383-6his, SEQ ID NO. 25);

[0194] (2) The above sequences were respectively constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was prepared for standby;

[0195] (3) The glass was 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, 5% CO2 cell incubator;

[0196] (4) HCGP-39-6his, HCGP-39-22-111-6his, HCGP-39-112-201-6his, HCGP-39-202-292-6his, HCGP-39-293-383-6his were respectively transfected into the above prepared 293T cells;

[0197] (5) After 48h, the culture solution was discarded and the above cells were fixed with acetone for 30min, and the incubator was dried for standby;

[0198] (6) The above pieces were incubated with the supernatant of each group of recombinant monoclonal cells collected in step one, respectively, at room temperature for 1h;

[0199] (7) PBST was washed for 3 times, and the second antibody was incubated for 30min;

[0200] (8) PBST was washed for 3 times, and the results were observed under a microscope.

[0201] The experimental results are as shown in Figure 6 ​As shown in (a), the recombinant 6F6 monoclonal antibody was successfully expressed in 293T cells and successfully secreted in the cell supernatant. The recombinant monoclonal antibody can recognize HCGP-39, and the recognition site is located in the amino acid sequence interval 293-383, and has biological activity.

[0202] (9) The above HCGP-39 late section (HCGP-39-293-383-6his, SEQ ID NO. 25) is divided into five fragments, HCGP-39-293-310 (SEQ ID NO. 26), HCGP-39-311-328 (SEQ ID NO. 27), HCGP-39-329-346 (SEQ ID NO. 28), HCGP-39-347-364 (SEQ ID NO. 29), and HCGP-39-365-383 (SEQ ID NO. 30), each 18 amino acids long;

[0203] (10) After deleting each fragment (denoted as: Q HCGP-39-deletion position-6his), construct on eukaryotic expression vector pcDNA3.1, and prepare the plasmid for use;

[0204] (11) The glass slides are placed in 10 cm cell culture dishes, and after polylysine treatment, 293T cells are evenly seeded in the dishes and incubated overnight in a 37°C, 5% CO2 cell incubator;

[0205] (12) Transfect Q HCGP-39-293-310-6his, Q HCGP-39-311-328-6his, Q HCGP-39-329-346-6his, Q HCGP-39-347-364-6his, and Q HCGP-39-365-383-6his into the above prepared 293T cells; repeat the above operation steps and observe the results under a microscope.

[0206] The experimental results are shown in Figure 6 As shown in (a), the recombinant 6F6 and 6D9 monoclonal antibodies were successfully expressed in 293T cells and successfully secreted in the cell supernatant. The recombinant monoclonal antibody can recognize HCGP-39, has biological activity, and the recognition site is located in the amino acid sequence interval 293-383; as shown in (b), except for the deletion of the amino acid interval 311-346 group, the remaining segmented groups can be recognized by the HCGP-39 antibody, indicating that the antibody recognition site is located on the 311-346 amino acid fragment. Figure 6

[0207] 2. WB verifies the characteristics of the recombinant monoclonal antibody

[0208] ​(1) Take the purified HCGP-39 protein, the overexpressed HCGP-39 cell protein and the cell control protein transfected with empty pcDNA3.1 plasmid as the Western Blot loading samples for SDS-PAGE electrophoresis to verify the action characteristics of the recombinant monoclonal antibody;

[0209] (2) After the electrophoresis, wet transfer is used, the transfer conditions are 200 mA, 60 min; 5% skimmed milk powder is blocked at room temperature for 1 h;

[0210] (3) The supernatant of the recombinant monoclonal cell is used as the primary antibody, and the commercially available HCGP-39 antibody is used as the control group primary antibody, which is incubated at room temperature for 2 h;

[0211] (4) TBST is washed for 3 times, each time for 5 min;

[0212] (5) The HRP-labeled secondary antibody is added, which is incubated at room temperature for 1 h;

[0213] (6) TBST is washed for 3 times, each time for 5 min;

[0214] (7) The chemiluminescence solution is added for color development and photography, and the results are observed.

[0215] The experimental results are shown in Table 1. Figure 7 As shown in Table 1, the HCGP-39 antibody can detect the purified HCGP-39 protein; the recombinant monoclonal antibody 6F6 and the recombinant monoclonal antibody 6D9 can detect the corresponding signals in the overexpressed HCGP-39 cell protein in addition to the purified HCGP-39 protein signals. According to the above experimental results, the recombinant monoclonal antibody 6F6 and the recombinant monoclonal antibody 6D9 obtained in this embodiment can recognize the linear structure of the HCGP-39 protein, and have good performance and can be used for Western Blot experiments.

[0216] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A monoclonal antibody that specifically recognizes HCGP-39, characterized in that: The monoclonal antibodies are 6F6 and 6D9; The heavy chain variable region of 6F6 includes three complementarity determining regions: H-CDR1, H-CDR2, and H-CDR3. The amino acid sequence of H-CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of H-CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO.

3. The light chain variable region of 6F6 includes three complementarity determining regions: L-CDR1, L-CDR2, and L-CDR3. The amino acid sequence of L-CDR1 is shown in SEQ ID NO. 4, the amino acid sequence of L-CDR2 is shown in SEQ ID NO. 5, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO.

6. The heavy chain variable region of 6D9 includes three complementarity determining regions: H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of H-CDR2 is shown in SEQ ID NO.12, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO.13; The light chain variable region of 6D9 includes three complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO.14, the amino acid sequence of L-CDR2 is shown in SEQ ID NO.15, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO.

16.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of 6F6 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of 6D9 is shown in SEQ ID NO.17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

18.

4. A recombinant vector expressing the monoclonal antibody according to any one of claims 1 to 3.

5. A recombinant cell expressing the monoclonal antibody according to any one of claims 1 to 3.

6. The method for constructing the recombinant cell according to claim 5, characterized in that: The method comprises the following steps: mixing a recombinant vector expressing a monoclonal antibody heavy chain variable region with a recombinant vector expressing a monoclonal antibody light chain variable region, and then transfecting the mixture into a basic cell to obtain a recombinant cell expressing the monoclonal antibody.

7. The method for preparing the monoclonal antibody according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: culturing the recombinant cell according to claim 5 for 48 hours, and collecting the supernatant, wherein the supernatant contains the corresponding monoclonal antibody.

8. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent for detecting HCGP-39.

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