A ykl-40 monoclonal antibody and a preparation method and application thereof

Abstract

The application provides a YKL-40 monoclonal antibody and a preparation method and application thereof, and relates to the technical field of biological medicines.The monoclonal antibody 10F1 provided by the application has good titer.The heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively inserted into different basic vectors, and the two kinds of recombinant vectors are mixed and then co-transfected into basic cells, so that a recombinant cell capable of expressing the monoclonal antibody 10F1 is obtained.The monoclonal antibody vector obtained by the application is easy to preserve and easy to control the quality of the antibody production process; the monoclonal antibody vector can recognize YKL-40 protein and has biological activity.The monoclonal antibody provided by the application can be used for YKL-40 antigen detection and other scientific researches, and has very good application value and very important scientific research guiding significance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a YKL-40 monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] YKL-40 belongs to Chitinase-Like Proteins (CLPs), and is also called CHI3L1 (CHI3L1). YKL-40 is composed of heparin with a molecular weight of about 40 kDa and chitin-binding glycoprotein with a highly conserved domain. Because the N-terminal also has the YKL sequence of tyrosine-lysine-leucine, it is named YKL-40. Mammals do not synthesize chitin, but can produce two chitinases and several CLPs, including YKL-40. The protein can be expressed and secreted by macrophages, chondrocytes, fibroblast-like synoviocytes, vascular smooth muscle cells and hepatic stellate cells, etc. YKL-40, as one of the glycoprotein members of human glycosyl hydrolase 18 family, has no enzyme activity, so it lacks the ability to degrade chitin, but still shows oligosaccharide binding ability. Modern research has found that YKL-40 is elevated in diseases characterized by inflammation and tissue reconstruction, such as metabolic diseases, cardiovascular diseases, acute and chronic inflammation, cancer, etc.

[0003] Nerve system related research shows that YKL-40 can promote the migration of primary astrocytes. The absence of the protein can change the inflammatory response of glial cells, promote the phagocytosis of astrocytes and microglia Aβ, reduce the formation of amyloid plaques, and affect the disease progression of Alzheimer's disease (AD); in the occurrence and development process of traumatic brain injury (TBI) related neuroinflammation, its absence will cause more severe neuropathological phenomena and more significant glial cell pathological process; in addition, high expression of YKL-40 can also promote the accelerated development of aortic atherosclerotic plaque. With further research, YKL-40 may become a new target for diagnosis and treatment of central nervous system related diseases. However, the current research on YKL-40 monoclonal antibody is relatively superficial, and cannot be directly applied to medical research. SUMMARY

[0004] The purpose of the present application is to provide a YKL-40 monoclonal antibody and a preparation method and application thereof. The monoclonal antibody has good titer, and the expression vector and recombinant cell constructed based on the monoclonal antibody are easy to save and modify.

[0005] The application provides a monoclonal antibody 10F1 which specifically recognizes YKL-40, wherein the variable region of the heavy chain of the monoclonal antibody 10F1 comprises three complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO. 1-3.

[0006] The variable region of the light chain of the monoclonal antibody 10F1 comprises three complementarity determining regions, and the amino acid sequences are shown as SEQ ID NO. 4-6.

[0007] Preferably, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 10F1 is shown as SEQ ID NO. 7, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 8.

[0008] The application also provides a recombinant vector for expressing the monoclonal antibody 10F1, wherein the base vector of the recombinant vector comprises pFUSE-CHIg-m2a carrying the nucleotide sequence for coding the variable region of the heavy chain and pFUSE2ss-CLIg-mk carrying the nucleotide sequence for coding the variable region of the light chain.

[0009] The application also provides a recombinant cell for expressing the monoclonal antibody 10F1, wherein the base cell of the recombinant cell comprises a eukaryotic cell.

[0010] The application also provides a construction method of the recombinant cell, which comprises the following steps: inserting the gene sequence for coding the variable region of the heavy chain of the monoclonal antibody 10F1 into pFUSE-CHIg-m2a to obtain 10F1m2a;

[0011] inserting the gene sequence for coding the variable region of the light chain of the monoclonal antibody 10F1 into pFUSE2ss-CLIg-mk to obtain 10F1mk;

[0012] mixing the plasmids of 10F1m2a and 10F1mk and then transfecting the eukaryotic cell to obtain the recombinant cell.

[0013] Preferably, the gene sequence for coding the variable region of the heavy chain of the monoclonal antibody 10F1 is inserted into pFUSE-CHIg-m2a between EcoRI and NheI;

[0014] The gene sequence for coding the variable region of the light chain of the monoclonal antibody 10F1 is inserted into pFUSE2ss-CLIg-mk between EcoRI and NheI.

[0015] The application also provides a preparation method of the monoclonal antibody 10F1, which comprises the following steps: culturing the recombinant cell for 48 hours, collecting the supernatant, and the monoclonal antibody 10F1 is contained in the supernatant.

[0016] The application also provides hybridoma cells expressing the monoclonal antibody 10F1.

[0017] The application also provides use of the monoclonal antibody 10F1 in preparation of a reagent for detecting YKL-40.

[0018] The application also provides use of the monoclonal antibody 10F1 in preparation of a reagent for diagnosing and / or judging prognosis of nervous system diseases.

[0019] Beneficial effects: The application provides a monoclonal antibody 10F1 specifically recognizing YKL-40, which has good titer. The application inserts the heavy chain variable region and the light chain variable region of the monoclonal antibody into different basic vectors respectively, and then transfects the two recombinant vectors into basic cells together, so as to obtain recombinant cells capable of expressing the monoclonal antibody 10F1. The monoclonal antibody vector obtained by the application is easy to store and quality control in the process of antibody production, and is also convenient for a series of modifications of the antibody, more in-depth research and more extensive application. It is verified by examples that the monoclonal antibody 10F1 described in the application or the monoclonal antibody 10F1 produced by using the recombinant cells can recognize YKL-40 protein and has biological activity. The monoclonal antibodies can be used for YKL-40 antigen detection and other scientific research, and have very good application value and very important scientific research guiding significance. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of 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 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 cells result graph;

[0023] Figure 3 Comassie brilliant blue staining result graph of monoclonal antibody after purification;

[0024] Figure 4 Monoclonal antibody subtype identification result graph, each group of data from left to right in the graph represents IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, kappa and lambda;

[0025] Figure 5 Monoclonal antibody antibody titer determination result graph;

[0026] Figure 6 For immunofluorescence to verify the distribution of the antigen epitope of the monoclonal antibody; (a) the antibody recognition site is located in the interval of 293-383 of the amino acid sequence; (b) more specifically, the antibody recognition site is located on the 293-310 amino acid fragment;

[0027] Figure 7 For WB to verify the results of the action characteristics of the recombinant monoclonal antibody 10F1. DETAILED DESCRIPTION

[0028] The present application provides a monoclonal antibody 10F1 which specifically recognizes YKL-40, the variable region of the heavy chain of the monoclonal antibody 10F1 includes three complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 1-SEQ ID NO. 3.

[0029] The light chain variable region of the monoclonal antibody 10F1 includes three complementarity determining regions, and the amino acid sequences are shown in SEQ ID NO. 4-SEQ ID NO. 6.

[0030] The monoclonal antibody 10F1 provided by the present application has a heavy chain belonging to IgG1, a light chain kappa, and the variable regions of the heavy chain and the light chain both include three complementarity determining regions, and the sequences are as follows.

[0031] Among them, the heavy chain variable region complementarity determining region is:

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

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

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

[0035] The light chain variable region complementarity determining region is:

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

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

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

[0039] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 10F1 according to the present application is preferably as shown in SEQ ID NO. 7 (MMVLSLLYLLTALPGILSEVQLQESGPSLVKPSQTLSLSCSVTGDSITS GYWNWIRKFPGNKLEYMGFISYSGNTYYNPSLKSRISITRDTSKNQYFLQLNS VTTEDTATYYCVRKGDYDGAWFAYWGQGTLVTVSA); the corresponding nucleotide sequence of the encoding gene is as shown in SEQ ID NO. 9 (ATGATGGTGTTAAGTCTTCTGTACCTGTT GACAGCCCTTCCGGGTATCCTGTCAGAGGTGCAGCTTCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCTCCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAGTACATGGGGTTCATAAGCTACAGTGGTAACACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGTAAGAAAAGGGGATTACGACGGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA); the amino acid sequence of the light chain variable region of the monoclonal antibody 10F1 is as shown in SEQ ID NO. 8 (MSVLTQVLALLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVP SRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPLTFGAGTKLELK); the corresponding nucleotide sequence of the encoding gene of the light chain variable region is as shown in SEQ ID NO.10 (ATGAGTGTGCT CACTCAGGTCCTGGCGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGAGTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA).

[0040] The application further provides a recombinant vector for expressing the monoclonal antibody 10F1, wherein a base vector of the recombinant vector comprises pFUSE-CHIg-m2a carrying a nucleotide sequence encoding a variable region of a heavy chain, and pFUSE2ss-CLIg-mk carrying a nucleotide sequence encoding a variable region of a light chain.

[0041] The pFUSE-CHIg-m2a and pFUSE2ss-CLIg-mk are preferably purchased from invivogen company, and in the construction of the recombinant vector, the nucleotide sequence shown in SEQ ID NO. 9 is preferably inserted into the EcoRI and NheI sites of the pFUSE-CHIg-m2a to form the recombinant vector 10F1m2a, and the nucleotide sequence shown in SEQ ID NO. 10 is inserted into the EcoRI and NheI sites of the pFUSE2ss-CLIg-mk to form the recombinant vector 10F1mk. The method for inserting the exogenous gene into the vector is not particularly limited in the application, and preferably comprises a double enzyme digestion method.

[0042] The application further provides a recombinant cell for expressing the monoclonal antibody 10F1, wherein a base cell of the recombinant cell comprises a eukaryotic cell.

[0043] The eukaryotic cell preferably comprises a mammalian cell, more preferably comprises a 293T cell, a CHO cell or other mammalian cell, etc., and in the embodiments of the application, the 293T cell purchased from the ATCC cell bank is taken as an example for illustration, but it cannot be merely regarded as the whole protection scope of the application.

[0044] The present application also provides a method for constructing the recombinant cell, comprising the following steps: inserting a gene sequence encoding the heavy chain variable region of the monoclonal antibody 10F1 into pFUSE-CHIg-m2a to obtain 10F1m2a;

[0045] inserting a gene sequence encoding the light chain variable region of the monoclonal antibody 10F1 into pFUSE2ss-CLIg-mk to obtain 10F1mk;

[0046] mixing the plasmids of 10F1m2a and 10F1mk and then transfecting eukaryotic cells to obtain the recombinant cell.

[0047] The method for constructing the recombinant vectors 10F1m2a and 10F1mk is preferably the same as described above, and will not be repeated here. The plasmids corresponding to the recombinant vectors are preferably mixed after being extracted, and the mixing ratio is preferably 1:1. The method for transfection is not particularly limited, and can be performed by using conventional methods in the art.

[0048] The present application also provides a method for preparing the monoclonal antibody 10F1, comprising the following steps: culturing the recombinant cell for 48 hours, and collecting the supernatant, wherein the monoclonal antibody 10F1 is contained in the supernatant.

[0049] The culture preferably comprises culturing at 37°C in a 5% CO2 environment for 48 hours, centrifuging, and collecting the supernatant of the cell culture, wherein the monoclonal antibody 10F1 is contained in the supernatant.

[0050] The monoclonal antibody 10F1 prepared by the method of the present application has biological activity, can specifically recognize and bind YKL-40, and the nucleotide sequence of the antibody epitope amino acid fragment that can be recognized by the monoclonal antibody 10F1 is preferably as shown in SEQ ID NO. 18.

[0051] The present application also provides a hybridoma cell expressing the monoclonal antibody 10F1.

[0052] The method for constructing the hybridoma cell is not particularly limited, and preferably comprises constructing a prokaryotic expression vector containing a human YKL-40 gene sequence (NM_001276.4, SEQ ID NO. 16), transforming the prokaryotic expression vector into a TOP10 competent cell, picking a single colony, extracting a plasmid, and then transferring the plasmid into an E. coli BL21 (DE3) expression competent cell, picking a single colony, culturing, and centrifuging to collect the supernatant, and purifying to obtain a purified antigen; immunizing a mouse 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-detecting positive hybridoma cells.

[0053] In the present application, the method for producing the monoclonal antibody 10F1 by using the hybridoma cell is not particularly limited, and preferably includes the preparation by using ascites, and the monoclonal antibody 10F1 is obtained after purification.

[0054] The present application also provides the use of the above-mentioned monoclonal antibody 10F1 in the preparation of a reagent for detecting YKL-40.

[0055] The method for detecting YKL-40 according to the present application preferably includes ELISA method, CBA method and / or Western Blot method.

[0056] The present application also provides the use of the above-mentioned monoclonal antibody 10F1 in the preparation of a reagent for diagnosing and / or judging the prognosis of nervous system diseases.

[0057] The nervous system diseases according to the present application preferably include the occurrence, development and prognosis judgment of Alzheimer's disease and gliosis-related diseases, and thus the monoclonal antibody 10F1 according to the present application can be used in the preparation of related reagents. The detection method preferably includes ELISA method, CBA method and / or Western Blot method.

[0058] In order to further illustrate the present application, a YKL-40 monoclonal antibody, a preparation method and application thereof according to the present application are described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0059] Example 1 Preparation of YKL-40 protein

[0060] Step one, construction of YKL-40 prokaryotic expression vector

[0061] 1. The human YKL-40 gene sequence (SEQ ID NO. 16) was searched from the GenBank sequence database, and the sequence number was NM_001276.4. The gene sequence was synthesized between NdeI and NotI of pET-32a vector, and the target gene YKL-40 was connected between EcoRI and NotI of pGEX 4t-1 vector;

[0062] 2. The primer was designed, and the target band was amplified by PCR.

[0063] Upstream primer YKL-40-F (SEQ ID NO. 11): ggatctggttccgcgtggatccccggaattcatgggtgtgaaggcgtctc;

[0064] Downstream primer YKL-40-R (SEQ ID NO. 12): atgcactcgctgcaacgtaggcggccgcatcgtgactgactgacgatctg.

[0065] PCR amplification system (50 μL) was configured: 50 ng of template, 1 μL of YKL-40-F (10 μM), 1 μL of YKL-40-R (10 μM), 1 μL of FastPfu DNA Polymerase (2.5 units), 10 μL of 5x FastPfu buffer, 4 μL of 2.5 mM dNTP, and nuclease-free water to 50 μL.

[0066] PCR amplification program: 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, 35 cycles; 72°C extension for 5 min; 4°C storage.

[0067] 3. The PCR product was subjected to agarose gel electrophoresis, the gel was cut and recovered, the target fragment and the vector were respectively digested with corresponding restriction enzymes, and were recovered;

[0068] 4. The target fragment and the vector were ligated with a homologous recombination enzyme at 50°C for 15 min;

[0069] 5. The ligation product was transformed into TOP10 competent cells, and was cultured in a 37°C incubator overnight;

[0070] 6. A single colony was picked into corresponding Amp-resistant LB medium, and was cultured in a 37°C shaker overnight;

[0071] 7. The plasmid was extracted, and the sequencing results were compared.

[0072] Step two, YKL-40 protein expression and purification

[0073] 1. The constructed and correctly sequenced plasmid was transformed into E. coli BL21 (DE3) expression competent cells;

[0074] 2. A single colony was picked into LB medium, and was shaken at 37°C until the OD value was 0.5-1, IPTG was added for induction, and expression was performed at 16°C overnight;

[0075] 3. The bacterial cells were collected, were broken by ultrasonic, and the supernatant was collected after centrifugation, the YKL-40 protein expressed by pET-32a was purified by Ni column affinity chromatography, and the purified protein was recorded as YKL-40-6his; the YKL-40 protein expressed by pGEX4t-1 was purified, and the purified protein was recorded as YKL-40-strep;

[0076] The purified protein was dialyzed and concentrated to obtain high-concentration YKL-40-6his protein as an antigen for storage, and YKL-40-strep protein was used to coat ELISA plates to detect YKL-40 antibodies.

[0077] Example 2: Obtaining hybridoma cells

[0078] Step 1: Immunizing mice with YKL-40 antigen

[0079] 40 μg of purified YKL-40-6his antigen was mixed with complete Freund's adjuvant at a ratio of 1:1; 15 female Balb / c mice aged 6-8 weeks were selected, and 100 μL (containing 40 μg of antigen) of the mixed antigen was injected into the left hind leg muscle of each mouse; three weeks later, the second immunization was performed, and 40 μg of antigen was mixed with incomplete Freund's adjuvant at a ratio of 1:1, and 100 μL (containing 40 μg of antigen) of the mixed antigen was injected into the right hind leg muscle of each mouse;

[0080] Step 2: Detecting antibody production in immunized mice

[0081] The mice were immunized twice, and three weeks later, tail blood was collected, centrifuged to collect serum, and the antibody production in the mice was detected, as follows:

[0082] 1. ELISA method for detecting mouse antibody titer: YKL-40-strep protein was used to coat ELISA plates, 100 ng / well, and the plates were coated overnight at 4°C (coating solution: 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 plates were washed with PBST solution for 3 times, each for 3 min, and then patted dry; 2% BSA was added to fill the wells and block at 37°C for 1 h; after incubation, the plates were washed with PBST for 3 times, each for 3 min, and then patted dry to remove excess water; the serum was diluted with PBST, and 100 μL of the diluted serum was added to each well and incubated at 37°C for 1 h; the plates were washed with PBST for 3 times, each for 3 min, and then patted dry; the goat anti-mouse IgG-HRP secondary antibody was diluted at a ratio of 1:5000, and added to the plates, which were incubated at 37°C for 30 min; after incubation, the plates were washed with PBST for 3 times, patted dry, and TMB was added for color development for 10 min, and then 2 M H2SO4 was added to stop the reaction, and the absorbance value was measured at 450 nm. The No. 6 mouse with the highest antibody titer was selected for further experiments, and the antibody ELISA titer was 2.187 million.

[0083] 2. CBA method for detecting mouse antibody titer: YKL-40 (YKL-40-6his) is constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid is prepared for standby; the glass slide is placed in a 10 cm cell culture dish, and after polylysine treatment, 293T cells are evenly seeded in the dish and incubated overnight in a 37°C, 5% CO2 cell incubator; YKL-40-6his is transfected into the above prepared 293T cells, and after 48h, the culture medium is discarded and fixed with acetone for 30min, and the culture box is dried for standby; the serum is diluted with PBST solution in proportion, and diluted by 1:100, 1:1000, 1:10000, 1:100000, respectively; incubate the above slides with the serum diluted according to the above proportions at room temperature for 1h; wash with PBST for 3 times, incubate with secondary antibody for 30min; wash with PBST for 3 times, observe the results under a microscope, and the results of the 6th mouse immunofluorescence signal are shown in Figure 1 , and the antibody CBA titer is about 1000.

[0084] Step three, cell fusion

[0085] 1. Preparation of myeloma cells: resuscitate SP2 / 0 cells, culture with DMEM medium and 15% FBS for one week, and select logarithmic growth period myeloma cells for cell fusion.

[0086] 2. Preparation of spleen cells: select the 6th mouse with good immune effect, take blood by enucleation method, centrifuge to separate serum; sacrifice the mouse by cervical dislocation method, soak in 75% (v / v) alcohol for 5min for disinfection; transfer to a clean bench for subsequent processing, fix the mouse, remove the spleen, remove the fat tissue and connective tissue attached to the spleen; wash the obtained spleen with serum-free culture medium, place it on a cell filter, gently grind it with a syringe core, and gently wash the filter with serum-free culture medium, collect the washed liquid; centrifuge the collected spleen cell suspension at 500g for 5min, collect the cell pellet, wash the cells 3 times, discard the supernatant, and suspend the cell pellet in serum-free DMEM medium, count and reserve.

[0087] 3. Cell fusion: ultraclean table in advance ultraviolet sterilization water bath, adjust the water bath to 37℃ for standby, the prepared SP2 / 0 cells and spleen cells are added to a 50mL centrifuge tube at a ratio of 1:10, mixed well. 500g centrifugation for 10min, gently aspirate and discard the supernatant, gently tap the bottom of the centrifuge tube to slightly loosen the cell pellet; 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, i.e.: drop 1mL at the first min, add 2mL at the second min, add 3mL at the third min, add 4mL at the fourth min, add 5mL at the fifth min, shake in a 37℃ water bath. Then incubate at 37℃ for 15min, 500g centrifuge for 5min, discard the supernatant; add 5mL of DMEM medium containing HAT to the collected cell pellet, suspend the cell pellet gently, and finally supplement the DMED medium containing HAT to a total volume of about 100mL. Place in a 96-well cell culture plate with macrophages at 100μL / well, then place the culture plate in a 37℃, 5% CO2 incubator.

[0088] Step four, positive hybridoma cell screening

[0089] 1. ELISA method for screening positive hybridoma cells

[0090] ELISA plates coated with YKL-40 protein expressed by pGEX4t-1 vector, 100ng / well, 4℃ overnight coating.

[0091] Observe the growth of hybridoma cells, after seven days when the cell culture supernatant turns yellow, take an appropriate amount of culture supernatant for ELISA detection of the corresponding antibody, and perform the first subcloning screening, the results are shown in Table 1, the well at position F1 of plate 10 has high absorbance value, selected for further screening.

[0092] Table 1 ELISA method for screening positive monoclonal experiment results

[0093] YKL-40 1 2 3 4 5 6 7 8 9 10 11 12 A 0.074 0.058 0.056 0.057 0.059 0.077 0.061 0.052 0.129 0.061 0.053 0.066 B 0.07 0.312 0.133 0.578 0.156 0.504 0.398 0.151 0.081 0.376 0.136 0.066 C 0.071 0.129 0.204 0.178 0.382 0.475 0.304 0.294 0.6 0.066 0.406 0.097 D 0.064 0.509 0.481 0.289 0.486 0.28 0.184 0.095 0.159 0.5 0.052 0.061 E 0.063 0.179 0.201 0.112 0.302 0.442 0.312 0.438 0.37 0.079 0.386 0.131 F 0.905 0.083 0.094 0.302 0.192 0.185 0.338 0.121 0.312 0.278 0.888 0.061 G 0.059 0.078 0.628 0.145 0.577 0.286 0.161 0.903 0.338 0.217 0.068 0.395 H 0.073 0.092 0.053 0.055 0.066 0.052 0.052 0.075 0.161 0.053 0.061 0.082

[0094] After seven to ten days, the second subcloning screening is performed, the antibody is detected by ELISA, and the F1 clone with high OD value in the first screening is selected and plated in a 96-well plate with about 1 cell per well. After seven to ten days of culture, the antibody is detected by ELISA, and the experimental results are shown in Table 2. The cell clone at position G8 has the highest OD value, which is recorded as 10F1-G8 and selected for further screening.

[0095] Table 2 ELISA method for screening positive monoclonal experiment results

[0096]

[0097]

[0098] The clones with high OD value in the second screening were selected for the third sub-cloning screening, and were plated in 96-well plates at about 1 cell per well. The clones with high OD value were selected, and finally one positive clone cell 10F1-G8-D9 was screened out, which was named 10F1. The experimental results are shown in Table 3.

[0099] Table 3 Experimental results of the third screening of positive monoclonal antibodies by ELISA method

[0100] 10F1 G8 1 2 3 4 5 6 7 8 9 10 11 12 A 0.901 0.053 0.889 0.085 0.889 0.169 0.936 0.092 0.054 0.714 0.053 0.081 B 0.086 0.053 0.594 0.046 0.594 0.799 0.999 0.219 0.075 0.8 0.047 1.026 C 0.817 0.05 0.078 0.598 0.078 0.592 0.992 0.093 0.053 0.342 0.08 0.181 D 0.823 0.06 0.189 0.032 0.189 0.992 0.944 0.239 1.136 0.764 0.061 1.058 E 0.799 0.047 0.822 0.05 0.822 0.903 0.148 0.995 0.062 0.738 0.049 1.078 F 0.606 0.056 0.256 0.056 0.256 0.358 0.96 1.043 0.06 0.683 0.053 0.644 G 1.048 0.047 0.099 0.059 0.099 0.92 1.015 0.621 0.053 0.747 0.061 1.032 H 0.996 0.048 0.135 0.054 0.135 0.28 0.854 1.018 0.047 0.181 0.064 0.274

[0101] 2. Verification of the screening results of positive hybridoma cells by CBA method

[0102] YKL-40 (YKL-40-6his) was constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was prepared for standby use; the glass slides were plated in 10 cm cell culture dishes, and after polylysine treatment, 293T cells were evenly seeded in the dishes and incubated overnight in a 37°C, 5% CO2 cell incubator; YKL-40-6his 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 standby use; the above slides were incubated with the culture supernatant of the screened hybridoma cells for 1 h at room temperature; washed with PBST for 3 times, and the secondary antibody was incubated for 30 min; washed with PBST for 3 times, and the results were observed under a microscope, wherein the positive signal results are shown in Figure 2 .

[0103] Example 3 Preparation of monoclonal antibody by ascites and purification of monoclonal antibody

[0104] Step one, preparation of monoclonal antibody by ascites

[0105] 1. Inject 300 μL of ascites adjuvant into the abdominal cavity of a 12-week-old Balb / c mouse.

[0106] 2. Two weeks later, the hybridoma cells were cultured to the best cell activity state, and the cell number was adjusted to about 1 × 10 6 cells / 100 μL, and 100 μL of hybridoma cells were inoculated into the abdominal cavity of the mouse which had been injected with ascites adjuvant.

[0107] 3. Collect the ascites after 7-10 days.

[0108] Step two, purification of monoclonal antibody

[0109] 1. Dilute the ascites with PBS pH 7.4, centrifuge to take the supernatant, and purify by protein G affinity chromatography.

[0110] 2. Equilibration: equilibrate the column with 0.4M PB buffer (pH 7.0);

[0111] 3. Column loading: load the diluted ascites supernatant slowly into the column to ensure better binding of the antibody to the protein G column;

[0112] 4. Washing: wash the column with the equilibration buffer again;

[0113] 5. Elution: elute the antibody bound to the column with 0.1M glycine buffer (pH 2.7), and add 1M Tris-HCl (pH 8.0) to neutralize the glycine to maintain a neutral pH suitable for antibody storage. Perform SDS-PAGE electrophoresis on the purified monoclonal antibody and control serum, respectively, and the results are shown in Figure 3 , where the heavy and light chains of the antibody can be clearly seen.

[0114] Example 4 Monoclonal antibody typing identification

[0115] Coat the ELISA plate with YKL-40 expressed by pGEX 4t-1 vector, 100 ng / well, overnight; wash the coated ELISA plate with PBST 3 times, block with 2% BSA for 1 hour; add 100 μL of hybridoma cell supernatant, incubate at 37°C for 1 hour; wash 3 times with PBST; incubate HRP-labeled secondary antibody (IgG1, IgG2a, IgG2b, IgG3, IgM, IgG kappa chain, lambda), incubate at 37°C for 30 minutes. Wash 3 times with PBST, develop with TMB, and measure the absorbance at 450 nm. The experimental results are shown in Figure 4 , where the heavy chain of the 10F1 monoclonal antibody is IgG1 and the light chain is kappa.

[0116] Example 5 Monoclonal antibody sequence determination

[0117] Extract RNA from the hybridoma cells cultured in Example 2; amplify the variable region fragments of the heavy and light chains of the antibody using the 5' RACE method; ligate the amplified fragments into the pEASY-Blunt vector, extract the plasmid, and sequence; the nucleotide sequences of the light and heavy chains are SEQ ID NO. 9 and SEQ ID NO. 10, and the corresponding amino acid sequences are SEQ ID NO. 7 and SEQ ID NO. 8; use the Kabat method to mark the CDR regions of the antibody amino acid sequence (SEQ ID NO. 1-SEQ ID NO. 6).

[0118] Example 6 Recombinant monoclonal antibody and its effect

[0119] Step one, preparation of recombinant monoclonal antibody

[0120] The sequencing-determined monoclonal antibody 10F1 heavy chain variable region (SEQ ID NO. 9) and light chain variable region (SEQ ID NO. 10) were connected to the antibody expression vectors pFUSE-CHIg-m2a, pFUSE2ss-CLIg-mk, respectively, and were recorded as 10F1m2a, 10F1mk. After sequencing, the plasmid was extracted in large quantities for cell transfection to prepare the antibody.

[0121] The 293T cells were evenly seeded in the dish after polylysine treatment, and were placed in a 37°C, 5% CO2 cell incubator overnight for culture; the constructed recombinant antibody expression plasmid 10F1m2a and 10F1mk were transferred into the above prepared 293T cells, and were cultured at 37°C, 5% CO2 for 48 h. The supernatant of the cultured cells was collected, and was centrifuged to obtain the supernatant for standby use.

[0122] Step two, recombinant monoclonal antibody titer detection

[0123] The ELISA plate was coated with the purified YKL-40-strep protein, 100 ng / well, 4°C overnight coating (coating solution was 25 mL carbonate buffer, pH 9.6); the next day, PBST solution was used to wash 3 times, 3 min each time, and was patted dry each time; 2% BSA was added to fill the hole for blocking, and was incubated at 37°C for 1 h; after incubation, PBST was used to wash 3 times, 3 min each time, and the plate was patted to remove excess water each time; the collected supernatant was diluted with diluent, and the dilution ratio was 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, 1:6561, respectively, 100 μL of the above diluent was added to each well, and was incubated at 37°C for 1 h, and was washed with PBST for 3 times, 3 min each time, and was patted dry each time; the goat anti-mouse IgG-HRP secondary antibody was diluted at a ratio of 1:5000, and was added to the plate, and was incubated at 37°C for 30 min; after incubation, PBST was used to wash 3 times, and was patted dry, TMB was added for color development for 10 min, 2M H2SO4 was used for termination, and the absorbance value at 450 nm was measured as shown in Table 4:

[0124] Table 4 Recombinant Monoclonal Antibody Titer Detection Experimental Results (OD 450 )

[0125]

[0126] The results are shown in Figure 5 .

[0127] Step three, recombinant monoclonal antibody property test

[0128] 1. Immunofluorescence verification of the epitope of the recombinant monoclonal antibody

[0129] YKL-40 (YKL-40-6his), YKL-40 front section ((YKL-40-22-111-6his, SEQ ID NO. 13), YKL-40 middle front section (YKL-40-112-201-6his, SEQ ID NO. 14), YKL-40 middle back section (YKL-40-202-292-6his, SEQ ID NO. 15), YKL-40 back section (YKL-40-293-383-6his, SEQ ID NO. 17) were respectively constructed on the eukaryotic expression vector pcDNA3.1, and the plasmids were prepared for standby; the glass slides were placed in 10 cm cell culture dishes, and after being treated with polylysine, 293T cells were evenly seeded in the dishes and cultured overnight in a 37°C, 5% CO2 cell incubator; YKL-40-6his, YKL-40-22-111-6his, YKL-40-112-201-6his, YKL-40-202-292-6his, YKL-40-293-383-6his were respectively transfected into the above prepared 293T cells; 48 h after transfection, the culture solution was discarded and the cells were fixed with acetone for 30 min, and then dried in the incubator for standby; the above slides were incubated with the supernatant of the recombinant monoclonal cells collected in step one, and incubated at room temperature for 1 h; washed with PBST for 3 times, and the secondary antibody was incubated for 30 min; washed with PBST for 3 times, and the results were observed under a microscope.

[0130] The YKL-40 rear segment (YKL-40-293-383-6his, SEQ ID NO. 17) was divided into five segments, each consisting of 18 amino acids: YKL-40-293-310 (SEQ ID NO. 18), YKL-40-311-328 (SEQ ID NO. 19), YKL-40-329-346 (SEQ ID NO. 20), YKL-40-347-364 (SEQ ID NO. 21), and YKL-40-365-383 (SEQ ID NO. 22). Each segment was deleted (indicated by: Q YKL-40-amino acid position interval-6his) was constructed on the eukaryotic expression vector pcDNA3.1, and the plasmid was extracted for use; a glass slide was spread on a 10 cm cell culture dish, and after polylysine treatment, 293T cells were evenly seeded in the dish and cultured in a 37°C, 5% CO2 cell culture incubator overnight; Q YKL-40-293-310-6his, Q YKL-40-311-328-6his, Q YKL-40-329-346-6his, Q YKL-40-347-364-6his and QYKL-40-365-383-6his were added to the prepared 293T cells. After 48 hours of transfection, the culture medium was discarded and the cells were fixed with acetone for 30 minutes. The cells were dried in an incubator and set aside. The slides were incubated with the supernatant of the recombinant monoclonal cells collected in step 1 at room temperature for 1 hour. The slides were washed three times with PBST and incubated with secondary antibodies for 30 minutes. The slides were washed three times with PBST and the results were observed under a microscope.

[0131] The experimental results are as follows Figure 6 As shown, the recombinant 10F1 monoclonal antibody was successfully expressed in 293T cells and successfully secreted into the cell supernatant. The recombinant monoclonal antibody can recognize YKL-40 and has biological activity. The antibody recognition site is located in the 293-383 interval of the amino acid sequence ( Figure 6 (a)); except for the group with missing amino acid interval 293-310, the rest of the segment groups can still be recognized by YKL-40 antibody, indicating that the antibody recognition site is located on the 293-310 amino acid fragment ( Figure 6 (b)).

[0132] 2. Western blot verification of recombinant monoclonal antibody characteristics

[0133] The purified YKL-40 protein was used as a sample for Western Blot to perform SDS-PAGE electrophoresis to verify the characteristics of the recombinant monoclonal antibody; after the electrophoresis was completed, wet transfer was used, the transfer conditions were 200 mA for 60 min; 5% skimmed milk was used for room temperature blocking for 1 h; the supernatant of the recombinant monoclonal cell was used as a primary antibody, and a commercially available YKL-40 antibody was used as a control group primary antibody, which was incubated at room temperature for 2 h; TBST was washed for 3 times, each time for 5 min; a HRP-labeled goat anti-mouse secondary antibody (1:5000 dilution) was added, which was incubated at room temperature for 1 h; TBST was washed for 3 times, each time for 5 min; a chemiluminescence solution was added for color development and photography, and the results were observed, and the experimental results are shown in Figure 7 As shown in the figure, the recombinant monoclonal antibody 10F1 and the commercially available YKL-40 antibody can both bind to the YKL-40 protein to produce a signal, and it can be known that the recombinant monoclonal antibody 10F1 can recognize a linear structure and can be used for Western Blot experiments.

[0134] 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 10F1 that specifically recognizes YKL-40, characterized in that: The heavy chain variable region of the monoclonal antibody 10F1 includes three complementarity 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 10F1 includes three complementarity 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 10F1 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 10F1 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.

3. A recombinant vector expressing the monoclonal antibody 10F1 according to claim 1 or 2, characterized in that: The basic vector of the recombinant vector includes pFUSE-CHIg-m2a carrying a nucleotide sequence encoding a heavy chain variable region, and pFUSE2ss-CLIg-mk carrying a nucleotide sequence encoding a light chain variable region.

4. A recombinant cell expressing the monoclonal antibody 10F1 according to claim 1 or 2, characterized in that: The base cells of the recombinant cells include eukaryotic cells.

5. The method for constructing the recombinant cell according to claim 4, characterized in that: The method comprises the following steps: inserting a gene sequence encoding a heavy chain variable region of the monoclonal antibody 10F1 into pFUSE-CHIg-m2a to obtain 10F1m2a; The gene sequence encoding the light chain variable region of the monoclonal antibody 10F1 was inserted into pFUSE2ss-CLIg-mk to obtain 10F1mk; The plasmids of 10F1m2a and 10F1mk are mixed and transfected into eukaryotic cells to obtain recombinant cells.

6. The construction method according to claim 5, characterized in that: The gene sequence encoding the heavy chain variable region of monoclonal antibody 10F1 was inserted between EcoRI and NheI of pFUSE-CHIg-m2a; The gene sequence encoding the light chain variable region of monoclonal antibody 10F1 was inserted between EcoRI and NheI of pFUSE2ss-CLIg-mk.

7. A method for preparing the monoclonal antibody 10F1 according to claim 1 or 2, characterized in that: The method comprises the following steps: culturing the recombinant cell according to claim 4 for 48 hours, and collecting the supernatant, wherein the supernatant contains the monoclonal antibody 10F1.

8. Use of the monoclonal antibody 10F1 according to claim 1 or 2 in the preparation of a reagent for detecting YKL-40.

Images