KR100427645B1 - Human melanocortin-4 receptor specific antibody and the preparation method thereof - Google Patents

Abstract

The present invention relates to an antibody that specifically binds to human melanocortin-4 receptor (MC-4R) and a method for producing the antibody, the antibody of the present invention plays a pivotal role in appetite control and obesity induction It can be usefully used to search for and analyze the melanocortin-4 receptor.

Description

Human melanocortin-4 receptor specific antibody and the preparation method

The present invention relates to an antibody that specifically binds to human melanocortin-4 receptor (MC-4R) and a method for producing the antibody, the antibody of the present invention plays a pivotal role in appetite control and obesity induction It can be usefully used to search for and analyze the melanocortin-4 receptor.

Melanocortin receptor is a so-called 7-transmembrane protein that has a structure that penetrates the cell membrane seven times. It is a receptor on the cell surface having a signaling system connected with G-protein. To date, there are five types of melanocortin receptors identified in humans (see Table 1 ), and they are known to have specific tissue cell expression patterns and functions (Yeo, GSH, et al., QJ Med ., 2000 , 93, 7-14). In particular, it plays a very diverse role in terms of physiological activity. In the case of MC-1R, the color of skin and hair is determined (Robbins, LS, et al., Cell , 1993 , 72, 827-834), and MC-. 2R is expressed in the adrenal cortex and acts as a receptor for adrenocorticotropin hormone (ACTH) that regulates the secretion of steroid hormones (Clark, AJ, et al., Lancet , 1993 , 341, 461-462). It has been reported to be associated with secretion of sebaceous gland and also act as an ACTH receptor on lymphocytes (Akbulut, S., et al., Biochem. Biophys. Res. Commun ., 2001 , 281, 1086-1092). The role of MC-3R has not been clarified to date, but recent studies have shown that mice lacking MC-3R enhance adipose tissue and appetite. Indirect evidence of relevance is presented (Chen, AS, et al., Nat. Genet ., 2000 , 26, 97-102).

Type of receptor Expressed tissue MC-1R Melanocytes MC-2R Adrenal cortex, adipocytes MC-3R Hypothalamus, Thalamus, Hippocampus, Placenta, Pancreas, Stomach, Duodenum MC-4R Hypothalamus, Hypothalamus, Limbic System, Hind Brain, Brain Stem, Cortex, Spinal Cord MC-5R Brain, skeletal muscle, adipocytes, thymus spleen, testis, lung, kidney, liver, bone marrow, pituitary gland (pituitary),

In addition, MC-4R has been shown to be involved in the regulation of appetite through the analysis of mice lacking the agouti gene (Bultman, SJ, et al., Cell , 1992 , 72, 1195-1204). Agauti is a small polypeptide consisting of 131 amino acids and is known to act on MC-1R present on melanocytes by being expressed only in hair follicles under normal conditions. The natural ligand of MC-1R is known as melanocyte-stimulating hormone (α-MSH), which passes through an intermediate layer called ACTH in a precursor called pro-opiomelanocortin. It is produced through post-translational cleavage of the step. In normal mice, the action of α-MSH is neutralized by agauti peptides, resulting in an agauti phenotype that changes the hair color of the mouse. Abnormalities in energy metabolism in mice can lead to hyperphagia, weight gain, and obesity. A series of studies have shown that the antagonist-agonist interaction between endogenous ligands, called auguti-related proteins (AGRPs) and similar α-MSHs, in a natural state is similar to that of MC- It is estimated that 4R mediates signals related to appetite control in the hypothalamus (Wilson, BD, et al., Mol. Med. Today , 1999 , 5 (6), 250-256). On the other hand, reports of appetite stimulation occurred in artificially deficient MC-4R genes (Huszar, D., et al., Cell , 1997 , 88, 131-141) or with naturally mutated MC-4R. Reports that obesity has been observed in humans (Yeo, GS, et al., Nat. Genet ., 1998 , 20, 111-112) suggest that MC-4R plays a pivotal role in appetite control and obesity development. It was confirmed.

As MC-4R's role in suppressing and enhancing appetite is known, it is possible to develop an agonist for this receptor to reduce appetite or, conversely, to prepare an antagonist to enhance appetite. There have been many efforts to use it. For example, as a novel substance having such a function, a cyclic hepta-peptide cyclic hepta-peptide MT-II or an antagonist SHU9119 or HS014 has been developed. Research continues (Hurby, VJ, et al., J. Med. Chem ., 1995 , 38, 3454-3461; Kask, A., et al., Biochem. Biophys. Res. Commun ., 1998 , 248 , 245-249).

While research on MC-4R is being actively conducted as described above, there are very few reagents or techniques for detecting or analyzing the same. Investigating the expression of MC-4R using ligands of radioisotopes or other labeled α-MSH using interactions between receptor ligands is not specific for the receptors of the ligand (α-MSH is the most common Due to its affinity for MC-R), it is difficult to use in practice, and artificially manufactured MT-II, SHU9119 and HS014 do not have high specificity for each receptor of MC-R. It is true. Commercially available antibodies to date (goat anti-human MC4-R, cat # RDI-MC4RCabG; goat anti- (human / rat / mouse) MC4-R, cat # RDI-MC4RC1abG; rabbit anti-human MC4R, cat # RDI-MC4Rabr (Research Diagnostics Inc.) is also known for its western blot or immunohistochemistry because its recognized epitope sites are restricted to intracellular regions. Only available.

Thus, the present inventors first predicted the site that is expected to be exposed to the cell surface through computer modeling in order to develop a specific antibody that can detect MC-4R as it is expressed on the cell surface. The epitope was designated and the corresponding site was secured in the form of cDNA by reverse transcription PCR from a cell line expressing MC-4R, and then expressed in recombinant protein form in E. coli. The recombinant protein was then mixed with an immune enhancer (adjuvant) and injected into BALB / c mice and confirmed the production of specific antibodies by ELISA analysis. Finally, the present invention was completed by confirming that the prepared antibodies specifically recognize the MC-4R receptor through fluorescence staining using cell lines expressing or not expressing MC-4R.

SUMMARY OF THE INVENTION An object of the present invention is to provide a human melanocottin-4 receptor-specific antigen site, an antibody that specifically recognizes the same, and a method of preparing the same.

1 is a diagram showing the structure of the melanocortin-4 receptor analyzed using a computer,

FIG. 2 shows a flagellin, thiorredoxin fusion of an exoloop site consisting of 106-118 amino acids as set forth in SEQ ID NO: 1 derived from the first loop of the melanocortin-4 receptor. It is a schematic diagram showing the process of preparing a vector for expression of a protein,

3 is an electrophoresis picture of the expression of FliTrx-exoloop fusion protein analyzed by SDS-PAGE,

M: molecular weight marker,

1: non-induced cell lysate,

2: IPTG-induced cell disruption,

3: soluble fraction,

4: insoluble fraction,

5: fusion protein purified by Q-sepharose

Figure 4 shows the expression of glutathione S-transferase (GST) fusion protein of the exoloop site consisting of 106-118 amino acids as set forth in SEQ ID NO: 1 derived from the first loop of the melanocortin-4 receptor. It is a schematic diagram showing a process of manufacturing a vector for

5 is an electrophoresis picture of the expression of GST-exoloop fusion protein analyzed by SDS-PAGE,

M: size marker, 1: non-induced cell disruption,

2: IPTG-induced cell disruption, 3: water soluble fraction,

4: insoluble fraction, 5: fusion protein purified by Q-Sepharose

Figure 6 is an electrophoresis picture showing the results of reverse transcription PCR analysis of the expression of melanocytes-4 receptor in the prepared human melanocytes-4 receptor,

M: size marker, 1: HEK, 2: HEK-hMC-4R

7 is a graph showing the results of analyzing the antigen specificity of the prepared anti-MC-4R polyclonal antibody (enzyme-linked immunosorbent assay (ELISA),

Wherein the cell line with the invention that A is a 8 expressing the MC-4R -MC-4R antibody (black histogram) and a negative control wherein the antibody-human CD4 antibody were reacted (white histograms) by flow cytometry staining (flow cytometry) is a graph showing the results of the analysis,

FIG 8 B is a section of the present invention -MC-4R antibody and cell lines expressing the MC-4R (black histogram) and a negative control cell line MC-3R expression staining by flow cytometry after the reaction (white histogram) ( flow cytometry)

In order to achieve the above object, the present invention provides a human melanocottin-4 receptor-specific antigen site.

The present invention also provides a method for expressing the antigenic site in the form of recombinant protein in E. coli.

The present invention also provides an antibody specific for the human melanocottin-4 receptor and a method of preparing the same.

Hereinafter, the present invention will be described in detail.

Firstly, the present invention provides human melanocytein-4 receptor specific antigenic sites.

Since there is no information on the tertiary structure of human MC-4R or the structure of the protein on the cell membrane, the present inventors have estimated by computer modeling what regions are actually exposed outside the cell surface.

As a result, the present inventors estimated the secondary structure of the seven transmembrane domains of MC-4R and showed excellent dual immunogenicity, and at the same time, it was the most specific and structurally stable epitope for MC-4R. Loop 1 was determined (see FIG. 1 ). Specific nucleic acid and amino acid sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2 . In the present invention, 13 amino acids more appropriate as immunogens among the above sequences were selected again and designated as the MC-4R specific antigen site represented by SEQ ID NO: 3 . This epitope corresponds to the first loop 106-118, and is named MC-4R exoloop in the present invention.

The present invention also provides a method for expressing the antigenic site in the form of recombinant protein in E. coli.

The present inventors expressed the MC-4R specific antigenic site in E. coli in the form of recombinant protein. That is, after cloning the cDNA of MC-4R exoloop using the primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 , the form of the recombinant protein in E. coli using two expression vectors (pFliTrx vector and pGEX vector) system (FliTrx-exoloop and GEX-exoloop).

The present invention also provides an antibody specific for the human melanocottin-4 receptor and a method of preparing the same.

The present inventors prepared antibodies specific thereto using mice using the recombinant proteins (FliTrx-exoloop and GEX-exoloop) prepared above as immunogens. Ten days after the final immunization, mice were removed by cervical spine and killed, blood was collected, serum was isolated, and ELISA was used to confirm the titers of antibodies specific for MC-4R exoloops.

As a result, it was confirmed that the antibody of the present invention shows very high reactivity against both kinds of immunoantigens (FliTrx-exoloop and GST-exoloop), but is almost unresponsive to the GST protein expressed without the exoloop ( FIG. 6 ) . Reference).

Finally, the present inventors confirmed by immunofluorescence staining whether the antibody of the present invention can detect the expression of MC-4R in the state of being expressed on the actual cell surface.

The anti-MC-4R antibody prepared in the present invention was reacted with a cell line expressing MC-4R, and the excess antibody was washed with the cell wash solution and then treated with FITC-bound goat-anti-mouse globulin secondary antibody. After the reaction, the excess antibody was removed with the cell wash solution, and the cells were suspended in PBS solution, and the fluorescence signal was analyzed by the FACScan flow cytometer.

As a result, it was confirmed that the antibody of the present invention exhibits high specificity to the antigen reacts with MC-4R-expressing cells so strongly than when using a negative control primary antibody (refer to A in FIG. 8). In addition, the antibody of the present invention showed high reactivity with the MC-4R-expressing cell line compared with the MC-3R-expressing cell line, thus showing no cross-reaction with other MC-related receptors. (See B of FIG. 8 ).

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples.

Example 1 Determination of Human MC-4R Specific Antigen Site

Since there is no information on the tertiary structure of human MC-4R or the structure of the protein on the cell membrane, the present inventors have estimated by computer modeling what regions are actually exposed outside the cell surface. Specifically, a total of seven computer softwares (TopPred2, Stockholm University; TMpred, EMBnet-CH; TMHMM, CBS Denmark; SOSUI, Tokyo University; HMMTOP, Hungarian Academy of Science; DAS, Stockholm University; PredictProtein, Columbia University) were used. .

As a result, the present inventors estimated the secondary structure of the seven transmembrane domains of MC-4R and showed excellent dual immunogenicity, and at the same time, it was the most specific and structurally stable epitope for MC-4R. Loop 1 was determined ( FIG. 1 ). Specific nucleic acid and amino acid sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2 . In the present invention, 13 amino acids more appropriate as immunogens among the above sequences were selected again and designated as the MC-4R specific antigen site represented by SEQ ID NO: 3 . This epitope corresponds to the first loop 106-118, and is named MC-4R exoloop in the present invention.

Example 2 Expression of MC-4R Specific Antigen Site

<2-1> Expression using pFliTrx vector system

The present inventors expressed the MC-4R specific antigenic site in E. coli in the form of recombinant protein. Specifically, cDNA of MC-4R exoloop was cloned using primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 and then subcloned into pFliTrx vector of Invitrogen. The vector is a vector expressing a protein on Escherichia coli so that a desired protein is positioned between two cysteines to form a single cysteine bridge so that a natural loop can be formed. It is expressed in the form of a flagella (flagella) of Escherichia coli fused to thioredoxin and has a very good solubility and easy overexpression. CDNA corresponding to MC-4R exoloop was subcloned into the flagellin-thioredoxin site of pFliTrx vector using BstXI and XhoI ( FIG. 2 ). This expression vector (pFliTrx-hMC4R-exoloop1) was transformed into E. coli and then analyzed for protein expression by SDS-PAGE.

As a result, it was confirmed that the fusion protein of the 13mer peptide (FliTrx-exoloop) fused to flagellin-thioredoxin was excessively expressed ( FIG. 3 ). In particular, the protein was expressed in a water-soluble form. were (Fig. 3 lane 3).

The Escherichia coli transformant GI826 / pFliTrx-hMC-4R exoloop1 expressing the FliTrx-exoloop was deposited with the Korea Biotechnology Research Institute Gene Bank on July 19, 2001 (accession number: KCTC 10017BP).

<2-2> Expression using pGEX vector system

We expressed human MC-4R specific antigenic sites in E. coli using another vector system. Specifically, amplify the cDNA corresponding to the MC-4R exoloop obtained in < 2-1 , subcloning into the pcDNA4 / V5-His A vector of Invitrogen, cut it out using BstXⅠ and XhoⅠ and cut it again. GST (glutathione-S-transferase) -fusion protein expression vector was subcloned to pGEX-4T-3, from Pharmacia ( Fig. 4 ). The thus prepared vector was named pGEX-hMC4R-exoloop1 and the vector was transformed into E. coli and analyzed for protein expression by SDS-PAGE.

As a result, it was confirmed that the fusion protein of the 13mer peptide (GST-exoloop) fused with the GST protein was excessively expressed ( FIG. 5 ).

E. coli transformant TG1 / pGEX-4T-3hMC-4R exoloop1 expressing the GST-exoloop was deposited with the Korea Biotechnology Research Institute Gene Bank on July 19, 2001 (Accession Number: KCTC 10018BP).

<2-3> Purification of Expressed MC-4R Specific Antigen

MC-4R-specific antigen sites of the fusion protein forms prepared in <2-1> and <2-2> were purified and used as immunogens. Specifically, expression using the pFliTrx vector system was first transformed into E. coli GI826 using pFliTrx-hMC4R-exoloop1 for expression, followed by RM (1xM9 salts, 2%) containing 100 μg / ml ampicillin in a single colony. Casing acids, 1% glycerol, 1 mM MgCl ₂ ) medium and shaken (180 rpm) at 30 ° C. the next day, the expression IMC containing 100 μg / ml ampicillin and 100 μg / ml tryptophan (1xM9) salts, 0.2% casamino acids, 0.5% glucose, 1 mM MgCl ₂ ) diluted to 1/50 medium. Expression was induced at 37 ° C. until the media absorbed at 600 nm from 0.3 to 0.6. The cells were then collected and suspended once in 1/15 culture cell lysis buffer (50 mM Tris-HCl pH 7.0, 1 mM EDTA, 1 mM β-mercaptoethanol) and in this suspension 1 mM / ml PMSF. The suspension was disrupted and the cell wall was crushed using ultrasonic waves. The lysate was centrifuged at 13000 rpm for 20 minutes at 4 ° C, the supernatant was passed through a 0.2 μM filter, and the supernatant was bound to a DEAE-sepharose column (Pharmacia), followed by ion using 0.5 M NaCl. Purification was carried out by ion-exchange chromatography. Purity and identity of the purified FliTrx-exoloop1 fusion protein was confirmed by 12% SDS-PAGE. The confirmed fusion protein was stored at -20 ° C until dialysis and use again with PBS. Expression using the pGEX vector system was first transformed into E. coli JM109 using pGEX-hMC4R-exoloop1 for expression, followed by shaking culture (180 rpm) at 37 ° C, and then 100 μg / ml ampicillin and 20 mM glucose (glucose) the next day. It was diluted 1/50 in LB medium containing. After raising the medium at 37 ° C. until the absorbance was 0.6 at 600 nm, IPTG was added at a final concentration of 0.5 mM to induce the expression of the protein. After 3 hours, the cells were collected and suspended once in a 1/1 buffer culture STE solution (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA), centrifuged at 6000 rpm for 10 minutes, and the precipitate was separated into 1 / It was dissolved in STE buffer containing 0.1 mg / ml lysozyme and 0.2 mM PMSF in 25 culture and left in ice for 15 minutes. 5 mM DTT and 1.5% sarcosyl were then added to the cell suspension to disrupt the cell wall by sonication. The lysate was centrifuged at 13000 rpm for 20 minutes at 4 ° C., and then the supernatant was passed through a 0.2 μM filter. The supernatant was bound to a glutathione Sepharose-4B affinity column (Pharmacia). The column was washed with PBS and eluted by adding 10 mM reduced glutathione (Amresco) to elution buffer (1 mM EDTA, 0.02% Triton X-100, 50 mM Tris-HCl pH 8.0). Purity and identity of the purified GST-exoloop1 fusion protein was confirmed by 15% SDS-PAGE. The confirmed fusion protein was stored at -20 ° C until dialysis was again used with PBS.

Example 3 Preparation of Antibodies Specific to MC-4R Exoloop

The present inventors prepared an antibody specific thereto using a mouse using the MC-4R exoloop prepared in Example 2 as an immunogen. Specifically, 13mer peptides in the form of FliTrx fusion proteins were injected into 6-8 week old BALB / c mice. In the first immunization, a total of 40 µg of purified protein was dissolved in 100 µl of phosphate-buffered saline (PBS) and mixed with the same amount of complete stimulant (complete Freund's adjuvant), followed by incomplete Freund's. adjuvant) was intraperitoneally injected at intervals of one week up to the fourth. Ten days after the final immunization, mice were removed by cervical spine and killed, blood was collected, serum was isolated, and ELISA was used to confirm the titers of antibodies specific for MC-4R exoloops.

As a result, it was confirmed that the antibody of the present invention shows very high reactivity against both kinds of immunoantigens (FliTrx-exoloop and GST-exoloop), but is almost unresponsive to the GST protein expressed without the exoloop ( FIG. 6 ) . ). Thus, the antibodies of the present invention are specific for MC-4R, and proved particularly successful in producing antibodies against the desired epitopes.

Experimental Example 1 Immunofluorescence Staining Using Antibodies Specific to MC-4R Exoloop

It was confirmed by immunofluorescence staining whether the antibody of the present invention can detect the expression of MC-4R in the state of being expressed on the actual cell surface. Specifically, the anti-MC-4R antibody prepared in the present invention was diluted 1: 100 in PBS and then reacted with HEK-293, a cell line expressing MC-4R as a primary antibody, at 4 ° C. for 30 minutes. Wash the excess antibody with cell wash solution (PBS / 0.1% BSA / 0.02% Na-azide), and then treat FITC-bound goat-anti-mouse globulin secondary antibody for 30 minutes and react with cell wash solution. The antibody was removed. Finally, the cells were suspended in 250 μl of PBS solution and analyzed for fluorescence using a FACScan flow cytometer.

As a result, it was confirmed that the antibody of the present invention reacted with MC-4R expressing cells very strongly compared to when using the negative control primary antibody (black histogram) (white histogram) ( Fig. 8 of the present invention). A ). In addition, it was confirmed that the antibody of the present invention showed high reactivity with the cell line expressing MC-4R (black histogram) compared with the cell line expressing MC-3R (white histogram) and cross-reacted with other MC related receptors. It was shown that it reacts only with MC-4R, not visible ( B of FIG. 8 ).

The present invention relates to an antibody that specifically binds to human melanocortin-4 receptor (MC-4R) and a method for producing the antibody, the antibody of the present invention plays a pivotal role in appetite control and obesity induction It can be usefully used to search for and analyze the melanocortin-4 receptor.

Claims (11)

Epitopes of human melanocytein-4 receptor consisting of the amino acid sequence of SEQ ID NO: 3 . An expression vector comprising a DNA encoding the epitope of claim 1. The expression vector according to claim 2, wherein the DNA consists of the nucleotide sequence set forth in SEQ ID NO: 4 . The expression vector according to claim 2, which is pFliTrx-hMC4R-exoloop 1. The expression vector according to claim 2, which is pGEX-hMC4R-exoloop 1. E. coli transformants (KCTC 10017BP and 10018BP) into which the vector of any one of claims 3 to 5 is introduced. The method for producing the epitope of claim 1, wherein the transformant of claim 6 is cultured and the epitope of claim 1 is recovered from the culture. An antibody against the epitope of claim 1. The polyclonal antibody according to claim 8, which is obtained from the serum of a mouse by injecting the epitope of claim 1 into a mouse. delete delete