CN114149499B - Monoclonal antibodies against human EMC10 and their use in the treatment and/or prevention of obesity - Google Patents

Abstract

The invention discloses a monoclonal antibody against human EMC10 and application thereof in treating and/or preventing obesity. The invention firstly discloses a monoclonal antibody of anti-human EMC10, which can specifically recognize an epitope with an amino acid sequence shown as SEQ ID No. 5. The invention further provides application of the monoclonal antibody in preparing a product for treating or/and preventing obesity. The anti-EMC 10 monoclonal antibody provided by the invention can obviously reduce the weight of an obese mouse and improve obesity-related metabolic disorders, can be used for preparing products for treating or preventing obesity, and provides a brand-new treatment target for obesity.

Description

Monoclonal antibodies against human EMC10 and their use in the treatment and/or prevention of obesity

Technical Field

The invention belongs to antibody medicines in the biomedical industry, and particularly relates to an anti-human EMC10 monoclonal antibody and application thereof in treating and/or preventing obesity.

Background

The incidence of obesity increases year by year, due to factors such as dietary structure, lifestyle, and environment. Obesity can greatly increase the morbidity and mortality of type 2 diabetes, cardiovascular diseases and tumors, and bring a heavy burden to human health and economic development. Searching for new causes of obesity onset and elucidating the pathological mechanism thereof is particularly urgent for developing new drugs for intervention and treatment of obesity, and suppressing and reducing the harm of obesity to human beings, and has extremely important significance.

Fat is one of the major sources of energy in the body, and lipid metabolism disorders are the serious consequences of metabolic diseases and are also important factors in their pathogenesis. There are many hormones or secreted proteins in the body that regulate lipid metabolism, such as classical insulin, thyroid hormone, adrenoglucocorticoid, and growth hormone, etc., which play an important role in regulating glycolipid metabolism and energy homeostasis. In recent years, certain non-classical endocrine tissue organs have also been found to secrete certain hormones or cytokines, including fat-secreted fat factors such as: leptin, adiponectin, resistin (Resistin), TNFα, IL-6, PAI-1, MCP-1, and the like; liver secreted liver factors such as: fetuin A (Fetuin-A), FGF-21, sex hormone binding protein (SHBG), etc.; muscle factors secreted by muscles, such as: myostatin, tectorigenin, IL-6, IL-15, and the like; and cardiac factors secreted by the heart (e.g., atrial Natriuretic Peptide (ANP)); they also play an important role in regulating lipid metabolism, energy balance and the onset of obesity. Since these hormones or factors can be detected in serum or plasma, they are expected to be molecular markers for diagnosis of obesity and potential targets for therapy. It has been found in practice that some of the above-mentioned hormones or cytokines can be used as molecular markers useful for evaluating obesity, such as leptin and adiponectin, but few drugs are actually targets for treating obesity, so that there is a need to continue to find novel cytokines or secreted proteins that can be targets for treating obesity.

EMC10 was originally cloned from a cDNA library of human insulinoma tissue, and was named: the nucleotide sequence of INM02, INM02 (EMC 10) gene and the amino acid sequence encoded by it are as in GenBank database (accession number: AY 194293) (Wang XC, xu SY, wu XY, song HD, mao YF, fan HY, yu F, mou B, gu YY, xu LQ, zhou XO, chen Z, chen JL, hu RM.Gene expression profiling in human insulinoma tissue: genes involved in the insulin secretion pathway and cloning of novel full-length cDNAs.endocr Relat cancer.2004, 11:295-303.). Several studies have been conducted to date to reveal various biological functions of EMC 10. EL built by Wang X et alThe ISA method, we reported internationally for the first time that EMC10 is a secreted protein that can be detected in human serum, and found that the expression of Emc (Inm) gene in mouse islet β cells was regulated by glucose, suggesting that it may play an important role in glucose metabolism (Wang X, gong W, liu Y, yang Z, zhou W, wang M, yang Z, wen J, hu r.molecular cloning of a novel secreted peptide, INM02, and regulation of its expression by glucose.j endocrinol.2009,202:355-364. After that, another study group reported the cloning of EMC10 from human purified hematopoietic stem cells, which was named HSS1 and the other sheared isomer thereof was named HSM1; meanwhile, they found in vitro that EMC10 (HSS 1) can inhibit proliferation, migration and invasion of glioma cell lines and simultaneously inhibit neovascularization of endothelial cells, so that they considered EMC10 as a potential target for treatment of glioblastoma (Junes-Gill KS, galler TK, gluzman-Poltorak Z, miller JD, wheeler CJ, fan X, basile LA.hHSS1: a novel secreted factor and suppressor of glioma growth located at chromosome 19q13.33.J Neurooncol.2011,102 (2): 197-211.; junes-Gill KS, lawrence CE, wheeler CJ, cordner R, gill TG, mar V, shiri L, basile LA.Human Hematopoietic Signal peptide-containing Secreted 1 (hHSS 1) modulates genes and pathways in glioma: implications for the regulation of tumorigenicity and antigen ess.2014, 14:920.). It has also been found that elevated Mrita22 (a mouse homologous gene of human EMC 10) can inhibit the development of neuronal cell dendrites and spines in a schizophrenic mouse model, and that the defect in development of hippocampal neuronal dendrites and spines in the mouse model can be completely saved by decreasing the level of Mrita22, suggesting that EMC10 plays an important role in the formation of mouse neuronal dendrites and spines (Xu B, hsu PK, stark KL, karayiorgou M, gogos JA. Reduced micro-development of a neuronal inhibitor due to miRNA dysregulation in a schizophrenia-reduced micro-cell 2013,152 (1-2): 262-75.; diamantatopoulou A, sun Z, mukai J, xu B, fenelon K, karayiorgo JA M, goss-of-function mutation in Mirta/Emc 10 rescues specific schizophrenia-related phenotypes in a mouse mod) el of the 22q11.2 deletion.Proc Natl Acad Sci U S A.2017;114 (30) E6127-E6136.). Recently, researchers from Germany found that in a mouse model of myocardial infarction, emc deletion resulted in reduced angiogenesis in the infarct border region, impaired left ventricular contraction and relaxation, supplementation of EMC10 to myocardial infarction mice increased angiogenesis in the infarct border region, improved left ventricular function after myocardial infarction, suggesting that EMC10 is a growth factor with angiogenic function that promotes tissue repair after myocardial infarction (Reboll MR, korf-Klingebiel M, klene S, polten F, brinkmann E, reimann I,HJ, bobadilla M, faix J, kensah G, gruh I, klintschar M, gaestel M, niessen HW, pich A, bauersachs J, gogos JA, wang Y, wollert KC 10 (Endoplasmic Reticulum Membrane Protein Complex Subunit 10) Is a Bone Marrow-Derived Angiogenic Growth Factor Promoting Tissue Repair After Myocardial information. Circulation.2017;136 (19):1809-1823.). The Zhou Y et al study found that EMC10 deficiency resulted in male mouse sterility, and sperm lacking the Emc gene exhibited various defects including morphological abnormalities, impaired sperm motility, impaired sperm capacitation, and loss of acrosome response. Molecular mechanism studies have found that EMC10 deletion results in sodium/potassium-ATPase inactivation and HCO3 ^- The induced cAMP/PKA signaling pathway activates impaired and reduced levels of protein tyrosine phosphorylation associated with sperm capacitation (Zhou Y, wu F, zhang M, xiong Z, yin Q, ru Y, shi H, li J, mao S, li Y, cao X, hu R, view CW, ding Q, wang X, zhang Y.EMC10 governs male fertility via maintaining sperm ion balance.J Mol Cell biol.2018Dec1; 10 (6): 503-514.).

Despite these advances, it has not been reported whether there is a relationship between EMC10 and obesity.

Disclosure of Invention

The technical problem to be solved by the present invention is how to treat and/or prevent obesity.

In order to solve the technical problems, the invention firstly provides a monoclonal antibody of anti-human EMC 10.

The name of the monoclonal antibody against human EMC10 provided by the invention is 4C2, and the monoclonal antibody can specifically recognize an epitope with an amino acid sequence shown as SEQ ID No.5, namely VVGVSVVTHP.

The name of the monoclonal antibody against human EMC10 provided by the invention is 4C2, and the monoclonal antibody contains the name V _H Heavy chain variable region of (2) and designated V _L Light chain variable region of (V) _H And V _L Are composed of a determinant complementary region and a framework region; the V is _H And said V _L Is composed of CDR1, CDR2 and CDR 3;

The V is _H The amino acid sequence of CDR1 of SEQ ID No.1 is shown in positions 31-35;

the V is _H The amino acid sequence of CDR2 of SEQ ID No.1 is shown at positions 50-68;

the V is _H The amino acid sequence of CDR3 of SEQ ID No.1 is shown in 101-103;

the V is _L The amino acid sequence of CDR1 of SEQ ID No.2 is shown at positions 24-39;

the V is _L The amino acid sequence of CDR2 of SEQ ID No.2 is shown at positions 55-61;

the V is _L The amino acid sequence of CDR3 of SEQ ID No.2 is shown at positions 94-102.

In the above monoclonal antibody against human EMC10, the V _H And V _L Is derived from mice.

In the above monoclonal antibody against human EMC10, the V _H The amino acid sequence of (2) can be shown as SEQ ID No. 1; the V is _L The amino acid sequence of (2) can be shown as SEQ ID No. 2.

Wherein SEQ ID No.1 consists of 114 amino acid residues and SEQ ID No.2 consists of 113 amino acid residues.

The above monoclonal antibody against human EMC10 may be any of the following:

a) From said V _H And said V _L Connecting the obtained single-chain antibody;

b) A fusion antibody comprising a) said single chain antibody;

c) Containing said V _H And said V _L Fab of (d);

d) Containing said V _H And said V _L Is a whole antibody of (a);

e) Monoclonal antibody secreted by hybridoma cell strain 4C2 with collection number of CGMCC No. 19950.

The above monoclonal antibody against human EMC10 may be a murine monoclonal antibody.

Biological materials associated with the monoclonal antibodies described above, which may be any of B1) to B12), are also within the scope of the present invention:

b1 A nucleic acid molecule encoding said monoclonal antibody;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism comprising the recombinant vector of B4);

b9 A transgenic animal cell line comprising the nucleic acid molecule of B1);

b10 A transgenic animal cell line comprising the expression cassette of B2);

b11 A transgenic animal cell line comprising the recombinant vector of B3);

b12 A transgenic animal cell line comprising the recombinant vector of B4).

In the above biological material, the nucleic acid molecule may be DNA such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, the nucleic acid molecule of B1) may be a gene encoding the monoclonal antibody.

In the above biological material, the gene may be a DNA molecule as described in a) or B) below:

a) The V is _H The coding sequence of CDR1 of SEQ ID No.3 is shown at positions 91-105, V _H The coding sequence of CDR2 of SEQ ID No.3 is shown at positions 148-204, V _H The coding sequence of CDR3 of SEQ ID No.3 is shown at positions 301-309; the V is _L The coding sequence of CDR1 of SEQ ID No.4 is shown at positions 70-117, V _L The CDR2 encoding sequence of (2) is shown as 163-183 th bit of SEQ ID No.4, said V _L The coding sequence of CDR3 of SEQ ID No.4 is shown at positions 280-306;

b) A DNA molecule having more than 90% identity to the DNA molecule defined in a) and encoding said monoclonal antibody or antigen binding portion thereof.

Wherein SEQ ID No.3 consists of 342 nucleotides and SEQ ID No.4 consists of 339 nucleotides.

In the above biological material, the expression cassette of B2) means a DNA capable of expressing the monoclonal antibody or the antigen-binding portion thereof in a host cell, and the DNA may include not only a promoter for initiating gene transcription of the monoclonal antibody or the antigen-binding portion thereof but also a terminator for terminating gene transcription of the monoclonal antibody or the antigen-binding portion thereof. Further, the expression cassette may also include an enhancer sequence. Recombinant vectors containing the monoclonal antibody gene expression cassettes can be constructed using existing expression vectors.

In the above biological material, the recombinant vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the recombinant microorganism may be yeast, bacteria, algae or fungi.

In the above biological material, the transgenic animal cell line may be a non-propagating material.

In the above biological materials, "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the above biological material, the DNA molecules may be at least 91%, 92%, 95%, 96%, 98% or 99% identical to each other by 90% or more.

In order to solve the technical problems, the invention further provides application of the monoclonal antibody or the biological material in preparing products for treating and/or preventing obesity.

The use of the monoclonal antibodies of the invention or the above-described biomaterials in any of the following is also within the scope of the present invention:

m1) use in the preparation of a product for reducing the weight of an animal;

m2) use in the preparation of a product for reducing body fat content of an animal;

M3) use in the preparation of a product for reducing visceral fat volume in an animal;

m4) in the preparation of a product for promoting thermogenesis from brown fats of an active animal.

In order to solve the technical problems, the invention further provides a method for treating and/or preventing obesity.

The present invention provides methods for treating and/or preventing obesity comprising administering to a recipient animal the monoclonal antibodies described above.

In the above, the animal is a mammal, such as a human or a mouse.

In the above, the product may be a medicament, vaccine, reagent or kit.

In the present invention, the treatment and/or prevention of obesity is mainly manifested in the aspects of weight loss, reduction of body fat content and visceral fat volume, activation of brown fat function and increase of thermogenesis, reduction of blood sugar, serum insulin, free fatty acids, triglycerides and cholesterol, etc.

In such applications, the reduction in body fat content of the animal may be a reduction in volume and weight of white fat, including visceral and subcutaneous fat cells. The reduction in visceral fat volume of the animal may be reduction in volume of abdominal or epididymal fat cells. The activation of the animal's brown fat to promote thermogenesis may be a reduction of accumulation of brown fat lactone droplets in the animal, promoting energy intake and utilization by the brown fat.

The anti-EMC 10 monoclonal antibody provided by the invention can obviously reduce the weight of an obese mouse and improve obesity-related metabolic disorders, can be used for preparing products for treating or preventing obesity, and provides a brand-new treatment target for obesity.

Preservation description

Biological material of reference (strain): 4C2

Suggested class naming: mouse anti-human EMC10 monoclonal antibody hybridoma cell strain

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3

Preservation date: 2020, 6 and 18 days

Deposit number: CGMCC No.19950

Drawings

FIG. 1 is a graph showing the results of identifying purified proteins by the Dot blot method, wherein 1-9 represent the weights of the proteins of 30, 15, 7.5, 3.75, 1.875, 0.9375, 0.46875, 0.234375, 0.1171875ng, respectively.

FIG. 2 is a graph showing the results of identifying purified proteins by Western blot.

FIG. 3 is a graph showing the results of phosphorylation levels of CREB after simultaneous intervention of mouse EMC10 protein and different mouse anti-human EMC10 monoclonal antibodies in HeLa cells; in the figure, ctr represents that only EMC10 protein is contained, and no EMC10 antibody is present; 1. 2, 3, 4 respectively represent antibodies containing 1F12, 4B12-1, 4B12-2, 4C2, and EMC10 protein.

FIG. 4 shows the results of the detection of wild-type and different truncations of EMC10 protein by the anti-Flag-tagged antibody western blot, in which WT represents the wild-type, i.e., EMC10 protein; EMC10 truncations of amino acids 28-105, 66-145, 106-183, 146-225, 184-254, 146-175, 171-200, 196-225, 146-155, 156-165, 166-175 were deleted 28-105, 66-145, 106-183, 146-225, 184-254, 146-175, 171-200, 196-225, 146-155, 156-165, 166-175.

Figure 5 shows the weight gain of mice from the control IgG, 1F12 and 4C2 antibody groups, where P <0.05.

Fig. 6 shows the weight gain of mice from the control IgG, 1F12 and 4C2 antibody groups, where P <0.001 and P <0.0001.

Fig. 7 shows DEXA measurement of fat and non-fat weights of mice in the control IgG group, the 1F12 and 4C2 antibody groups, where P <0.05.

FIG. 8 is a graph showing the weights of mice sacrificed from the control IgG group, the 1F12 and 4C2 antibody groups, and the different tissues { Heart (Heart), liver (Liver), epididymal fat (eWAT), inguinal subcutaneous fat (iWAT), retroperitoneal fat (Retro), mesenteric fat (Mesen), brown fat (BAT), spleen (Spleen), kidney (Kidney), pancreas (Panc) }; in the figure, P <0.05.

Fig. 9 is a case of HE staining of brown fat, subcutaneous fat and epididymal adipose tissue sections of mice sacrificed in the control IgG group and the 4C2 antibody group after the end of the experiment.

FIG. 10 is blood glucose (6 hours on empty stomach) and non-empty stomach insulin, non-esterified fatty acids, triglycerides, cholesterol levels of control IgG, 1F12, and 4C2 antibody-interfered high fat diet mice; in the figure, P <0.05.

FIG. 11 is an energy metabolism profile of high fat diet mice with metabolism cage study control IgG and 4C2 antibody intervention, where IgG: mouse control IgG,4C2: mouse anti-human EMC10 monoclonal antibody 4C2.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 acquisition of monoclonal antibody 4C2 against human EMC10 and its use for weight loss and amelioration of obesity-related metabolic disorders

This example shows whether the mice can be reduced in weight and the obesity-related metabolic disorders can be ameliorated by intervention of obese mice with monoclonal antibodies against human EMC10.

1. Obtaining of monoclonal antibodies of murine anti-human EMC10

Preparation of (one) mouse anti-human EMC10 monoclonal antibody

8 strains of mouse anti-human EMC10 monoclonal antibodies were prepared. The method comprises the following specific steps:

1. construction of EMC10 eukaryotic expression recombinant plasmid

Construction of recombinant plasmid pRAG2a-EMC10: the DNA fragment shown in the 82 nd to 762 th positions of SEQ ID No.7 is used for replacing a small fragment between the cleavage sites NheI and XhoI of the eukaryotic expression vector pRAG2a, so as to obtain the recombinant plasmid pRAG2a-EMC10.

2. Transfection and expression

(1) Culturing>1×10 ⁸ HEK 293F cells were kept ready for use.

(2) 100. Mu.g of recombinant plasmid pRAG2a-EMC10 was diluted to 1ml with diluent (Opti-MEM) and gently mixed.

(3 dilution of 200. Mu.l Lipofectamine with diluent (Opti-MEM) ^TM 2000 final liposome volumes to 1ml. Mix gently and leave at room temperature for 5min.

(4) Adding diluted plasmid into diluted Lipofectamine ^TM 2000 liposomes were mixed gently to a final volume of 2 ml.

(5) Incubate at room temperature for 30min.

(6) Transfer 1X 10 ⁸ HEK 293F cells were added to 500ml shake flasks and the final volume was brought to 98ml by fresh, pre-warmed Expression Medium.

(7) 2ml of incubated DNA-Lipofectamine was added ^TM 2000 mixtures.

(8)8％CO ₂ The culture was carried out at 37℃and 125rpm in a concentrated incubator for 4-5 days.

(9) The supernatant was collected at 4 ℃. The supernatant contained EMC10 mature protein (shown as positions 28-254 of SEQ ID NO.6 of the sequence Listing) from which the signal peptide was cleaved.

3. Purified protein and SDS-PAGE identification

(1) The supernatant obtained in step 2 (9) was collected, and binding buffer (8M urea, 20mM sodium phosphate, 500mM NaCl,pH 7.8) was added thereto, followed by filtration through a 0.45 μm filter membrane, and the filtrate was collected.

(2) Balance: binding buffer equilibrates nickel columns for 5 column volumes.

(3) Loading: and (3) loading the filtrate obtained in the step (1).

(4) Washing: and 5 column volumes binding buffer are used for washing impurities until no substance flows out.

(5) Eluting: 5 column volumes of buffer (8M urea, 20mM NaH) ₂ PO ₄ 500mM NaCl,pH 4.0), and collecting the eluted product.

(6) SDS-PAGE detection

Only one band around 36KD is shown, indicating that the electrophoretically pure target protein is obtained.

4. Dot blot and Western blot identification

(1) And (3) identifying the purified protein obtained in the step (3) by adopting a Dot blot method. The anti-EMC 10 antibody is a rabbit anti-human EMC10 polyclonal antibody (a polyclonal antibody obtained by immunizing New Zealand white rabbits by using EMC10 protein shown as SEQ ID NO.6 of a sequence table as an immunogen); the secondary antibody was goat anti-rabbit HRP antibody (Thermo Fisher, catalog # 65-6120).

The results are shown in FIG. 1. As can be seen, a 2000-fold dilution, the sample protein diluted to about 0.47ng, resulted in a positive result (No. 7); about 0.23ng had no positive result (No. 8).

(2) And (3) identifying the purified protein obtained in the step (3) by adopting a Western blot method. The anti-EMC 10 antibody is a rabbit anti-human EMC10 polyclonal antibody (a polyclonal antibody obtained by immunizing New Zealand white rabbits by using EMC10 protein shown as SEQ ID NO.6 of a sequence table as an immunogen); the secondary antibody was goat anti-rabbit HRP antibody (Thermo Fisher, catalog # 65-6120).

The results are shown in FIG. 2. As can be seen, 10ng of the samples were loaded with positive bands around 36 KD.

The results of Dot blot and Western blot identification show that the eukaryotic expression of the EMC10 protein is successful.

5. Immunization of animals

6 female BALB/c mice with the age of 6-8 weeks are selected, the purified protein obtained in the step 3 and Freund's complete adjuvant are mixed according to the volume ratio of 1:1 for the first time, 100 mug is injected subcutaneously, the immunity is enhanced every 2-3 weeks, and 100 mug of mixture is injected subcutaneously. And (3) blood sampling detection after four-step, wherein the titer of the antiserum against EMC10 protein (the titer is expressed by the maximum dilution of serum with the OD value of a sample hole/the OD value of a negative hole being more than or equal to 2.1) is measured by an indirect ELISA method, and the titer is more than 1:10000, 1-2 mice were selected for cell fusion arrangement.

The procedure for determining the potency of antisera against EMC10 protein by the indirect ELISA method described above is specifically as follows:

(1) And (3) wrapping the plate: a self-prepared standard EMC10 (purified in step 3 of example 1) was pipetted into a 0.1M PBS buffer to prepare a coating solution at a concentration of 1. Mu.g/ml, 100. Mu.l/well, and coated overnight at 4 ℃.

(2) Washing the plate: the liquid in the wells was discarded, dried, the plate was washed 2 times, each time soaking for 1-2 minutes, about 200. Mu.L/well, dried and tapped on absorbent paper to dry the liquid in the wells.

(3) Closing: blocking solution 250. Mu.l/well, 37℃for 2h.

(4) Washing the plate: discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as the step (2).

(5) And (3) detection: the serum to be tested is sucked and dissolved in antibody diluent (0.1M PBS) to prepare working solutions with dilution times of 1000, 3000, 9000 and 27000 times, 100 mu L of working solutions with different concentrations are added into each hole, no bubbles are needed, the solution is added into the bottom of the ELISA plate during sample adding, the hole wall is not touched as much as possible, and the solution is gently shaken and mixed uniformly. The ELISA plate was covered and incubated at 37℃for 2h.

(6) Washing the plate: discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as the step (2).

(7) mu.L of rabbit anti-mouse-HRP was added to each well, and the mixture was incubated at 37℃for 1 hour.

(8) Discarding the liquid in the holes, spin-drying, washing the plate for 5 times, and the method is the same as the step (2).

(9) Color development: substrate development A, B liquid 1:1 (volume ratio), 100. Mu.L of each well was added, and the ELISA plate was covered with a membrane and incubated at 37℃for 15 minutes in the absence of light.

(10) The reaction was stopped by adding 50. Mu.L of stop solution to each well, and the blue color turned yellow immediately. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution.

(11) The optical density (OD value) of each well was measured immediately with a microplate reader at a dual wavelength of 450/630nm and read. The power supply of the enzyme label instrument should be opened in advance, the instrument is preheated, and a detection program is set.

(12) And (3) judging results: the OD value of the sample hole/the OD value of the negative hole (namely the blank control hole) is positive when the OD value is more than or equal to 2.1. The results showed positive sample wells with serum anti-EMC 10 antibody dilution of greater than 10,000, indicating an antibody titer of greater than 1:10000.

6. cell fusion

(1) Preparation of myeloma cells: one week prior to fusion, SP2/0 cells were grown expanded in DMEM medium containing 10% FBS. By the time of fusion, the cells grew to approximately 6T 25 cell flasks, SP2/0 cells were collected on the day of fusion into 50ml centrifuge tubes, 1000rpm, and centrifuged for 5 min. The supernatant was discarded, then 20ml DMEM basal medium was added, the cells were blown off and counted.

(2) Spleen cell preparation: serum ELISA titers after four immunizations were 1: mice with 10000 or more are finally exempted 3 days before fusion, and 100 mug of mixture of EMC10 protein and Freund's complete adjuvant with the volume ratio of 1:1 is injected into the abdominal cavity after purification in the step three. Mice to be fused were euthanized on the day of fusion by cervical dislocation. Soaking in 75% alcohol for 5min. Spleens were aseptically removed and placed in petri dishes with 10ml DMEM basal culture. The screen was placed in another plate, the spleen was transferred to the screen and the spleen was ground with the syringe core. DMEM was added to the screen and the screen was rinsed to allow more spleen cells to collect in the dish. The cells were transferred to a 10ml centrifuge tube, and splenocytes were washed twice with serum-free DMEM, centrifuged at 1000rpm for 5min, and collected for splenocyte counts.

(3) Cell fusion: myeloma cells and spleen cells were mixed such that the myeloma cell to spleen cell number ratio was 1:20 are preferable. The cells were placed in 50ml centrifuge tubes and diluted with DMEM basal mediumThen centrifuged at 1000rpm for 5min. The supernatant was discarded. The centrifuge tube was shaken to homogenize the cells. 0.8ml of 50% PEG was slowly added, reacted for 90 seconds, and then 20-30ml of DMEM medium was added to terminate the PEG. The fused cells were placed in a 37℃water bath for 10 minutes. Centrifuge at 1000rpm for 5min, discard supernatant and add HAT DMEM medium. The fused cells were plated into 96-well plates at 100 μl per well. The cell culture plate is then placed in CO ₂ Culturing in an incubator.

The hybridoma cell cloning efficiency is more than 50% after 4 days of fusion, a small amount of cell fragments exist, and the cell growth state is good. Screening assays were started 10 days after fusion.

7. Fusion screening and subcloning

(1) Fusion screening: on the day before the detection, 5. Mu.g/ml antigen (EMC 10 protein after purification step three) was coated with PBS on ELISA plates overnight. The ELISA detection is carried out by sucking 100 μl/well of cell supernatant in the next day, and positive wells (OD value of sample well/OD value of negative well (blank control well) is more than or equal to 2.1) are judged according to ELISA results, and positive wells are judged. And (3) picking the positive holes detected by the whole plate by using a single-channel pipette, and performing a second confirmation detection to further confirm the positive holes. The positive well cells after the determination were subcloned.

(2) Subcloning: blowing cells in the positive holes, counting, adding 4ml of DMEM culture medium into a centrifuge tube, taking 100 mu l of cell suspension into the centrifuge tube, keeping 1ml after blowing evenly, adding DMEM to 4ml, blowing evenly, and keeping 100 mu l (about 2 drops) at the bottom of the tube. Adding DMEM to 5ml into a centrifuge tube, uniformly mixing, dropwise adding into the first three rows of a 96-well plate, keeping about 1.8-2ml of DMEM to 5ml of DMEM at the bottom of each drip tube, uniformly blowing, dropwise adding into D, E, F three rows of the 96-well plate, keeping about 1.5-1.8ml of DMEM to about 2.8-3ml of DMEM at the bottom of each drip tube, uniformly blowing, dropwise adding into G, H rows of the 96-well plate, dropwise adding into each drip, observing under a microscope after 7-10 days, detecting a hole with clone growth, marking a monoclonal hole, picking positive monoclonal cells as far as possible for subcloning again, and picking out the monoclonal holes for enlarging culture strains after 100% of positivity is detected.

Finally 8 hybridoma cell lines capable of stably secreting monoclonal antibodies against EMC10 protein were obtained, numbered 8C11, 6B9, 1F12, 4B12-1, 1H11, 4C2, 4B12-2 and 8A3, respectively.

8. Ascites preparation and purification

(1) Preparing ascites: each mouse was intraperitoneally injected with 0.5ml of liquid paraffin, and the pretreated mice were intraperitoneally injected with hybridoma cells within 30 days after 7 days. Injection into each mouse was 1X 10 ⁶ Amount of individual cells, hybridoma cells were injected. From 7 to 10 days, as much liquid as possible was carefully withdrawn from the abdominal cavity with a syringe needle, and titer determination was performed by indirect ELISA (titer was expressed as the maximum dilution of serum with an OD value of the sample well/OD value of the negative well of 2.1 or more, and the negative well was a blank). Mice were sacrificed by cervical dislocation after the last harvest.

(2) Purifying: centrifuging the collected ascites to obtain supernatant, preparing protein A agarose medium, loading the supernatant into a column, diluting the ascites by 10 times with PBS, slowly loading the sample, washing the sample until an ultraviolet detector reaches the minimum value after loading, eluting with glycine elution buffer solution to obtain the required purified antibody, immediately dialyzing the purified antibody in PBS at 4 ℃ overnight, and measuring the purity, concentration and titer (the titer is represented by the maximum dilution multiple of serum with the OD value of a sample hole/the OD value of a negative hole being more than or equal to 2.1, and the negative hole is a blank control).

The simultaneous intervention of HeLa cells with 1ug/ml mouse EMC10 protein and 1ug/ml of different mouse anti-human EMC10 monoclonal antibodies (1F 12, 4B12-1, 4B12-2 and 4C 2) and detection of CREB phosphorylation levels by western blotting (earlier studies showed that EMC10 could inhibit CREB phosphorylation) resulted in the reduction of CREB phosphorylation by EMC10 protein, as shown in fig. 3, 4B12-1 and 4C2 antibodies were able to return the original reduced CREB phosphorylation to EMC10 protein intervention, indicating that the 4B12-1 and 4C2 antibodies could block the effect of EMC10 protein, whereas 1F12 and 4B12-2 could not alter the reduction of CREB phosphorylation by EMC10 protein, indicating that these two antibodies could not block the effect of EMC10 protein: 2 monoclonal antibodies of mouse anti-human EMC10 which can block biological effects of mouse EMC10 protein are screened: 4B12-1 and 4C2,1F12 were unable to block the biological effects of mouse EMC10 protein as control antibodies.

The western blotting method is as follows:

1ug/ml of mouse EMC10 protein and 1ug/ml of different mouse anti-human EMC10 monoclonal antibodies (1F 12, 4B12-1, 4B12-2 and 4C 2) were added to the culture broth, the HeLa cells were dried for 6 hours, the total cell proteins were extracted, separated by electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel, transferred to a polyvinylidene fluoride (PVDF) membrane, then incubated with rabbit anti-p-CREB monoclonal antibody (CST, cat# 9198,1:1000 dilution), rabbit anti-CREB 1 monoclonal antibody (ABclonal, cat# A10826,1:1000 dilution) and rabbit anti-alpha-Tubulin polyclonal antibody (CST, cat# 2144,1:2000 dilution), the secondary antibodies were conjugated with peroxidase (Sigma) (dilution 1:10000), and finally bands were displayed by ECL Plus (Amersham) chemiluminescence.

Monoclonal hybridoma cell strain 4C2 which secretes monoclonal antibody 4C2 (hereinafter abbreviated as 4C2 antibody) of mouse anti-human EMC 10-mouse anti-human EMC10 monoclonal antibody hybridoma cell strain 4C2 (hereinafter abbreviated as 4C2 hybridoma cell or 4C2 cell) is preserved in China general microbiological culture Collection center (CGMCC, address: north Xielu No.1, no. 3 in the region of towards the sun in Beijing city) at 18 days of the year of 2020, and the preservation number is CGMCC No.19950.

(II) sequence of murine anti-human EMC10 monoclonal antibody 4C2

1.4C2 total RNA extraction from hybridoma cells

Total RNA of the 4C2 cell samples was extracted using Trizol Reagent (thermofilter, USA), and the concentration of the total RNA samples was measured by Nanodrop to obtain 15 μg total RNA (concentration: 511.3ng/μl, volume: 30 μl, a260/a280=2.01), and 500ng was taken for agarose gel electrophoresis analysis, which showed that 28S and 18S bands were clearly visible in the total RNA samples, and that the 28S band was more than 18S in brightness, indicating that the integrity of both RNAs was good.

2. Mouse antibody fragment amplification and sequence analysis

Specific primers were designed in the constant regions of the heavy (mouse IgG1 subtype) and light (kappa) chains of the antibody, respectively, and the primer sequences were as follows: mouse IgG1 CH outer (5 'to 3'): ACAATCCCTGGGCACAAT, mouse CL-Kappa outer (5 '-3'): ACACTCATTCCTGTTGAAGCTCTTGAC. Amplification of antibodies Using 5' RACE Heavy chain fragments and light chain fragments. The amplified fragment was inserted into cloning vector pUC57 (Addgene, USA) and sequenced, and the sequencing result showed that the heavy chain variable region (V) of murine anti-human EMC10 monoclonal antibody 4C2 _H ) The coding gene sequence of (a) is SEQ ID No.3, V _H The amino acid sequence of (2) is SEQ ID No.1, wherein V _H The amino acid sequence of CDR1 of (1) is shown in positions 31-35 of SEQ ID No.1, V _H The amino acid sequence of CDR2 of (1) is shown in positions 50-68 of SEQ ID No.1, V _H The amino acid sequence of CDR3 of SEQ ID No.1 is shown in 101-103; the light chain variable region (V) of murine anti-human EMC10 monoclonal antibody 4C2 _L ) The coding gene sequence of (a) is SEQ ID No.4, V _L The amino acid sequence of (2) is SEQ ID No.2, wherein V _L The amino acid sequence of CDR1 of (2) is shown in positions 24-39 of SEQ ID No.2, V _L The amino acid sequence of CDR2 of (2) is shown as 55-61 of SEQ ID No.2, V _L The amino acid sequence of CDR3 of SEQ ID No.2 is shown at positions 94-102.

3. Eukaryotic expression vector construction

Heavy chain fragment V shown in SEQ ID No.3 _H The coding gene of (C) and the heavy chain fragment constant region of mouse IgG1 (C _H ) Is inserted into eukaryotic expression vector pAH (HAS Bind, wuhai, china) to obtain antibody heavy chain expression plasmid pAH-4C2; the light chain fragment shown in SEQ ID No.4 (V _L ) Splicing the coding gene of the mouse CL-kappa fragment (light chain fragment constant region) and inserting the fragment into a eukaryotic expression vector pAK (HAS Bind, wuhai, china) to obtain an antibody light chain expression plasmid pAK-4C2. The antibody heavy chain expression plasmid pAH-4C2 is subjected to bidirectional sequencing by adopting forward and reverse sequencing primers, and then is subjected to sequence alignment analysis; detecting the antibody light chain expression plasmid pAK-4C2 by adopting a forward sequencing primer, and then performing sequence comparison and analysis to obtain a heavy chain nucleotide sequence shown in SEQ ID No.8 (containing a coding sequence of a secretion signal peptide), and expressing a protein shown in SEQ ID No.9 (the 1 st to 21 st amino acid sequence is the secretion signal peptide, and the 22 nd to 459 nd amino acid sequence is the mouse anti-human EMC10 monoclonal antibody 4C2 heavy chain); the nucleotide sequence of the light chain is shown as SEQ ID No.10 (coding sequence containing secretion signal peptide) and expressed as SEQ ID No.11The protein shown (amino acid sequence of secretion signal peptide is 1 st to 21 st, amino acid sequence of mouse anti-human EMC10 monoclonal antibody 4C2 light chain is 22 nd to 240 th).

4. Eukaryotic expression and detection of antibodies

The two eukaryotic expression plasmids (antibody heavy chain plasmid pAH-4C2 and antibody light chain plasmid pAK-4C 2) were subjected to medium-extraction (Plasmid Midiprep kit, axyPrep, USA) and the plasmid quality was checked by agarose gel electrophoresis. 40mL of HEK 293F cells were co-transfected with antibody heavy chain plasmid pAH-4C2 and antibody light chain plasmid pAK-4C2, and after the expression was completed, the cell suspension culture supernatants were collected. To evaluate and confirm the activity of the expressed antibodies, the expression supernatants were subjected to a gradient dilution ELISA assay in synchronization with a 4C2 antibody (mouse anti-human EMC10 monoclonal antibody 4C2 secreted by a mouse anti-human EMC10 monoclonal antibody hybridoma cell line), and the results are shown in tables 1 and 2: ELISA values of approximately 30-fold dilutions of the expression supernatants were comparable to 11ng/mL of 4C2 antibody.

TABLE 1 expression supernatant gradient dilution ELISA assay

TABLE 2 gradient dilution ELISA detection of 4C2 antibody concentration

Therefore, the amino acid sequence of the heavy chain variable region of the mouse anti-human EMC10 monoclonal antibody 4C2 secreted by the mouse anti-human EMC10 monoclonal antibody hybridoma cell strain is shown as SEQ ID No.1 (the coding sequence is shown as SEQ ID No. 3), and the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 (the coding sequence is shown as SEQ ID No. 4). The heavy chain variable region and the light chain variable region are both composed of a determinant complementary region and a framework region; the determinant complementary region of the heavy chain variable region consists of CDR1 (shown in positions 31-35 of SEQ ID No.1, the coding sequence is shown in positions 91-105 of SEQ ID No. 3), CDR2 (shown in positions 50-68 of SEQ ID No.1, the coding sequence is shown in positions 148-204 of SEQ ID No. 3) and CDR3 (shown in positions 101-103 of SEQ ID No.1, the coding sequence is shown in positions 301-309 of SEQ ID No. 3); the determinant complementary region of the light chain variable region consists of CDR1 (shown as 24-39 of SEQ ID No.2, the coding sequence is shown as 70-117 of SEQ ID No. 4), CDR2 (shown as 55-61 of SEQ ID No.2, the coding sequence is shown as 163-183 of SEQ ID No. 4) and CDR3 (shown as 94-102 of SEQ ID No.2, the coding sequence is shown as 280-306 of SEQ ID No. 4). The mouse anti-human EMC10 monoclonal antibodies 4C2 in the following experiments are all the mouse anti-human EMC10 monoclonal antibodies 4C2 secreted by the mouse anti-human EMC10 monoclonal antibody hybridoma cell strain.

Antigen epitope sequence of mouse anti-human EMC10 monoclonal antibody 4C2

EMC10 protein (comprising 28-254 amino acids) from which the signal peptide is removed is divided into 5 different truncations, namely EMC10 truncations (shown as A in figure 4) from which 28-105 (delta 28-105), 66-145 (delta 66-145), 106-183 (delta 106-183), 146-225 (delta 146-225) and 184-254 amino acids (delta 184-254) are deleted, and the 4C2 antibody is utilized to perform deimmunization coprecipitation (IP) on the different truncations, and western blotting detection is performed by using an anti-Flag antibody, so that the 4C2 antibody cannot remove the EMC10 truncations (delta 146-225) IP from which 146-225 amino acids are deleted (shown as A in figure 4), which indicates that the epitope against which the 4C2 antibody aims is in the 146-225 amino acid region of the EMC10 protein; then, 3 different truncations were constructed for amino acids 146-225, namely EMC10 truncations deleted of amino acids 146-175 (delta 146-175), 171-200 (delta 171-200) and 196-225 (delta 196-225) (shown in B in FIG. 4), and the same experiment was performed to reduce the epitope again to the region of amino acids 146-175 (shown in B in FIG. 4); repeating the above study, finally determining that the epitope aimed by the 4C2 antibody is in 156-165 region (shown as C in FIG. 4), and the amino acid sequence of the EMC10 protein corresponding to the region is: VVGVSVVTHP.

EMC10 epitope acquisition experiment:

(1) The gene sequences of the wild type EMC10 (WT) with Flag tag at the C end and different truncations (as shown in figure 4) are amplified by PCR and constructed on a pLEX-MCS vector (Thermo Scientific) without tag.

(2) 293T cells were prepared in 10 cm dishes, serum-free DMEM medium (Gibco) was changed, EMC10 plasmids of the wild type and different truncations were transfected into 293T cells, and after 5 hours DMEM medium containing 10% fetal bovine serum (Gibco) was changed, and after 1 day.

(3) After 1 day more, the cells were collected with 400ul of EBC buffer (50 mM Tris-HCl pH=7.5, 120mM NaCl,0.5%NP-40), lysed, and centrifuged at 12000rpm for 10min at 4℃to collect the supernatant.

(4) 2ug of monoclonal antibody 4℃ 2 (specific preparation method is as in step one) against human EMC10 was added to the supernatant and incubated at 4℃for 4h.

(5) Protein A/G agaros (Santa Cruz Biotechnology) was added and incubated at 4℃for 1h, and the beads were washed with PBS.

(6) Adding 1X loading (Beyotime) containing SDS, boiling at 100deg.C for 5min, denaturing, and detecting wild type (i.e. EMC10 protein) and different truncated EMC10 proteins by western blot of anti-Flag-tagged antibody (Cell Signaling Technology).

2. Use of mouse anti-human EMC10 monoclonal antibodies for weight loss and improvement of obesity-related metabolic disorders

1. Effects on high fat diet induced obesity in mice

Mice with a high fat diet (60% of dietary calories from fat) at about 35 g body weight were randomized into three groups, control IgG, control 1F12 and 4C2 antibodies, 8-10 each. Each mouse of the 4C2 antibody group was given the murine anti-human EMC10 monoclonal antibody 4C2 at a dose of 3mg/kg body weight; each mouse of the control 1F12 group was given a dose of 3mg/kg body weight of the murine anti-human EMC10 monoclonal antibody 1F12; each mouse of the control IgG group was given mouse IgG at a dose of 3mg/kg body weight. The control IgG group, the 1F12 group and the 4C2 antibody group were injected 2 times per week for two weeks, and the body weights of the mice of the different groups were measured during the course, and the body weight and body weight gain change results of the mice of the different groups are shown in fig. 5 and 6, and the results show that: mice in the control IgG group (indicated by "IgG" in the figure) and the 1F12 group (indicated by "1F12" in the figure) continued to gain in weight, whereas mice in the 4C2 antibody group (indicated by "4C2" in the figure) showed a significant decrease in weight, reaching 4 grams, with a negative gain in weight, within 2 weeks, with a significant statistical difference (P <0.05, < P <0.001, < P < 0.0001) compared to the control IgG group and the 1F12 group.

Fat weight and non-fat weight of mice in the control IgG group, the 1F12 group, and the 4C2 antibody group after two weeks of injection were measured using the dual energy X-ray absorption method (DEXA), and the results are shown in fig. 7, which shows that: the fat weight of the mice was significantly reduced (P < 0.05) compared to the control IgG group (denoted by "IgG" in the figure) and the 1F12 group (denoted by "1F12" in the figure), whereas the non-fat weight was not different between the control IgG group, the 1F12 group and the 4C2 antibody group; the different tissues (heart, liver, epididymal fat, inguinal subcutaneous fat, retroperitoneal fat, mesenteric fat, brown fat, spleen, kidney, pancreas) were weighed after the mice were sacrificed and the results are shown in fig. 8, and the results show: the weights of the subcutaneous fat and liver of mice in the 4C2 antibody group (indicated by "4C2" in the figure) were significantly reduced (P < 0.05) compared to the control IgG group (indicated by "IgG" in the figure) and the 1F12 group (indicated by "1F12" in the figure), the epididymal, mesenteric and retroperitoneal fat weights were also significantly reduced, while the weights of the other tissue organs (heart, brown fat, spleen, kidney, pancreas) were not different between the control IgG, 1F12 and 4C2 antibody groups.

Mice were sacrificed 2 weeks after antibody injection and brown fat, subcutaneous fat and epididymal adipose tissue were taken, the results are shown in fig. 9, and the results show that: the volume of subcutaneous and epididymal adipocytes in the 4C2 antibody group (denoted by "4C2" in the figure) was significantly smaller than that in the control IgG group (denoted by "IgG" in the figure); the control IgG group (indicated by "IgG" in the figure) mice had a large accumulation of lipid droplets in the brown fat, while the 4C2 antibody group (indicated by "4C2" in the figure) mice had significantly reduced lipid droplets.

2. Effects on diet-induced metabolic disorders in mice

Mice with a high fat diet (60% of dietary calories from fat) at about 35 g body weight were randomized into three groups, control IgG, control 1F12 and 4C2 antibodies, 8-10 each. Each mouse of the 4C2 antibody group was given the mouse anti-human EMC10 monoclonal antibody 4C2 at a dose of 3mg/kg body weight; each mouse of the control 1F12 group was given the mouse anti-human EMC10 monoclonal antibody 1F12 at a dose of 3mg/kg body weight; each mouse of the control IgG group was given mouse IgG at a dose of 3mg/kg body weight. The control IgG group, the 1F12 group and the 4C2 antibody group were injected 2 times per week for two weeks, and the serum metabolism related index was studied, and the blood glucose (6 hours on empty stomach) and the insulin, non-esterified fatty acid, triglyceride and cholesterol in the non-empty stomach state were measured, and as a result, as shown in fig. 10, the blood glucose, non-esterified fatty acid and triglyceride of the mice in the 4C2 antibody group (indicated by "4C2" in the figure) were significantly lower than those in the control IgG group (indicated by "IgG" in the figure) and the 1F12 group (indicated by "1F12" in the figure) (< 0.05), and serum insulin and cholesterol were also significantly reduced.

3. Effects on energy metabolism in mice

Mice with a high fat diet (60% of dietary calories from fat) at about 35 g body weight were randomized into two groups, a control IgG group and a 4C2 antibody group, of 8-10 animals each. Energy metabolism of mice was studied using Oxymax indirect calorimetry system (Oxymax, columbus Instruments), mice were placed in a metabolism cage for 3 days, the first day being the adaptation phase, the second and third days being the experimental phase, the antibodies were injected once the second day, and each mouse of the 4C2 antibody group was given the anti-human EMC10 monoclonal antibody 4C2 at a dose of 3mg/kg body weight; each mouse of the control IgG group was given mouse IgG at a dose of 3mg/kg body weight. Mice were free to ingest food and water in the metabolic cages, and each day was divided into 12 hours of light/day (white area in fig. 11) and 12 hours of night (grey area in fig. 11). The system monitors the indexes of ingestion, drinking water, activities, oxygen consumption, carbon dioxide exhalation, heat production and the like of the mice in the metabolism cage in real time through an instrument of the system. As a result, as shown in fig. 11, the oxygen consumption, carbon dioxide excretion and heat production of the mice in the 4C2 antibody group (indicated by "4C2" in the figure) were all significantly higher than those in the control IgG group (P < 0.01).

The above results demonstrate that the anti-EMC 10 monoclonal antibody 4C2 can significantly reduce the weight and body fat content of obese mice and improve obesity-related glycolipid metabolic disorders by increasing energy expenditure (thermogenesis), which provides a novel therapeutic target for obesity.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

SEQUENCE LISTING

<110> university of double denier affiliated Huashan Hospital

<120> monoclonal antibodies against human EMC10 and their use in the treatment and/or prevention of obesity

<130> GNCFY200524

<160> 11

<170> PatentIn version 3.5

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Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Asp Asn Tyr Glu

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Thr His Tyr Ala Glu Ser Val Lys Gly Lys Phe Thr Ile Ser Arg Asp

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Asp Ser Lys Ser Arg Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

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Asp Thr Gly Ile Tyr Tyr Cys Thr Asp Met Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

130 135 140

Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

145 150 155 160

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

165 170 175

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

180 185 190

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

195 200 205

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

210 215 220

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

225 230 235 240

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

245 250 255

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

260 265 270

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

275 280 285

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

290 295 300

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

305 310 315 320

Met His Glu Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

325 330 335

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

340 345 350

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

355 360 365

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

370 375 380

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

385 390 395 400

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

405 410 415

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

420 425 430

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

435 440 445

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455

<210> 10

<211> 723

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

atgagggcct ggatcttctt tctcctttgc ctggccggga gggctctggc agccccgcta 60

gcagatgttt tgatgaccca aactccactc tccctgcctg tcagtcttgg agatcaagcc 120

tccatctctt gcagatctag tcagagcctt gtacacagta atggaaacac ctatttacat 180

tggtacctgc agaagccagg ccagtctcca aagctcctga tctacaaagt ttccaaccga 240

ttttctgggg tcccagtcag gttcagtggc agtggatcag ggacagattt cacactcaag 300

atcagcagag tggaggctga ggatctggga gtttatttct gctctcaaag tatacatgtt 360

ccgtggacgt tcggtggagg caccaagctg gaaatcaaac gggcagatgc tgcaccaact 420

gtatccatct tcccaccatc cagtgagcag ttaacatctg gaggtgcctc agtcgtgtgc 480

ttcttgaaca acttctaccc caaagacatc aatgtcaagt ggaagattga tggcagtgaa 540

cgacaaaatg gcgtcctgaa cagttggact gatcaggaca gcaaagacag cacctacagc 600

atgagcagca ccctcacgtt gaccaaggac gagtatgaac gacataacag ctatacctgt 660

gaggccactc acaagacatc aacttcaccc attgtcaaga gcttcaacag gaatgagtgt 720

tag 723

<210> 11

<211> 240

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu

1 5 10 15

Ala Ala Pro Leu Ala Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu

20 25 30

Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln

35 40 45

Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg

65 70 75 80

Phe Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr

100 105 110

Phe Cys Ser Gln Ser Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe

130 135 140

Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys

145 150 155 160

Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile

165 170 175

Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr

195 200 205

Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His

210 215 220

Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235 240

Claims (9)

1. A monoclonal antibody, characterized in that: the monoclonal antibody contains a peptide of the name V _H Heavy chain variable region of (2) and designated V _L Light chain variable region of (V) _H And V _L Are composed of a determinant complementary region and a framework region; the V is _H And said V _L Is composed of CDR1, CDR2 and CDR 3;

the V is _H The amino acid sequence of CDR1 of SEQ ID No.1 is shown in positions 31-35;

the V is _H The amino acid sequence of CDR2 of SEQ ID No.1 is shown at positions 50-68;

the V is _H The amino acid sequence of CDR3 of (E) is shown as SEQPositions 101 to 103 of ID No. 1;

the V is _L The amino acid sequence of CDR1 of SEQ ID No.2 is shown at positions 24-39;

the V is _L The amino acid sequence of CDR2 of SEQ ID No.2 is shown at positions 55-61;

The V is _L The amino acid sequence of CDR3 of SEQ ID No.2 is shown at positions 94-102.

2. The monoclonal antibody of claim 1, wherein: the V is _H And V _L Is derived from mice.

3. The monoclonal antibody according to claim 1 or 2, characterized in that: the V is _H The amino acid sequence of (2) is shown as SEQ ID No. 1; the V is _L The amino acid sequence of (2) is shown as SEQ ID No. 2.

4. The monoclonal antibody according to claim 1 or 2, characterized in that: the monoclonal antibody is any one of the following:

a) From said V _H And said V _L Connecting the obtained single-chain antibody;

b) Containing said V _H And said V _L Fab of (d);

c) Containing said V _H And said V _L Is a whole antibody of (a);

d) Monoclonal antibody secreted by hybridoma cell strain 4C2 with collection number of CGMCC No. 19950.

5. A biological material associated with the monoclonal antibody of any one of claims 1-4, characterized in that: the biomaterial is any one of B1) to B12):

b1 A nucleic acid molecule encoding said monoclonal antibody;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

B5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism comprising the recombinant vector of B4);

b9 A transgenic animal cell line comprising the nucleic acid molecule of B1);

b10 A transgenic animal cell line comprising the expression cassette of B2);

b11 A transgenic animal cell line comprising the recombinant vector of B3);

b12 A transgenic animal cell line comprising the recombinant vector of B4).

6. The biomaterial according to claim 5, wherein: b1 The nucleic acid molecule is a gene encoding the monoclonal antibody, which is a DNA molecule as described in a) or B) below:

a) The V is _H The coding sequence of CDR1 of SEQ ID No.3 is shown at positions 91-105, V _H The coding sequence of CDR2 of SEQ ID No.3 is shown at positions 148-204, V _H The coding sequence of CDR3 of SEQ ID No.3 is shown at positions 301-309;

the V is _L The coding sequence of CDR1 of SEQ ID No.4 is shown at positions 70-117, V _L The CDR2 encoding sequence of (2) is shown as 163-183 th bit of SEQ ID No.4, said V _L The coding sequence of CDR3 of SEQ ID No.4 is shown at positions 280-306;

B) A DNA molecule having more than 90% identity to the DNA molecule defined in a) and encoding said monoclonal antibody or antigen binding portion thereof.

7. Use of a monoclonal antibody according to any one of claims 1-4 or a biomaterial according to claim 5 or 6 for the preparation of a product for the treatment or/and prevention of obesity.

8. Use of a monoclonal antibody according to any one of claims 1-4 or a biomaterial according to claim 5 or 6 in any one of the following:

m1) use in the preparation of a product for reducing the weight of an animal;

m2) use in the preparation of a product for reducing body fat content of an animal;

m3) use in the preparation of a product for reducing visceral fat volume in an animal;

m4) in the preparation of a product for promoting thermogenesis from brown fats of an active animal.

9. Use according to claim 7 or 8, characterized in that: the product is a drug, a reagent or a kit.