Peptide concatemer inducing production of antibody against apolipoprotein b-100, vaccine for obesity treatment, method for preparing concatemer, polynucleotide, expressing vector

FIELD: biotechnology, immunology, biochemistry, medicine.

SUBSTANCE: invention proposes peptide concatemer inducing production of antibodies against apolipoprotein B-100 that inhibit lipase effect and inhibit binding LDL with LDL receptors. This concatemer consists of amino acid sequence of peptide repeating four times. Amino acid sequence is given in the invention description. Also, invention describes a concatemer-base vaccine used in treatment and prophylaxis of obesity and a method for preparing concatemer in E. coli cells using a vector. Invention discloses a polynucleotide encoding concatemer and expressing vector comprising the indicated polynucleotide. Using the invention provides inhibition of obesity.

EFFECT: valuable medicinal properties of concatemer and vaccine.

7 cl, 16 dwg, 1 tbl, 6 ex

 

The technical field

The present invention relates to a vaccine for the treatment of obesity. More specifically, the present invention relates to a vaccine, which includes mimetic peptide epitope of apolipoprotein b-100, its concatemer or modified peptides.

Background of the invention

The serum lipids include cholesterol, triglycerides (TG), free fatty acids, phospholipids and the like, and are in the blood stream in the form of lipoprotein, which is a complex of lipid and apolipoprotein.

Of these lipoproteins, low-density lipoprotein (LDL) is the main carrier of TG and cholesterol. Significantly increased the number of patients suffering from arteriosclerosis, a disease coronary artery disease or myocardial infarction, linked to elevated LDL-cholesterol in the blood, due to a change in diet or other factors.

Consequently, there have been various studies to reduce LDL-cholesterol and determine the cause of the above diseases to treat patients suffering from the above diseases.

LDL-cholesterol, the main etiological factor for disease in adults, associated with lipid metabolism, can turn under the action of macrophages in high-density lipoprotein (HDL). In addition, LDL-cholesterol can also revertase another substance or to turn into bile acids in the liver (Brown M.S. and Goldstein J.L., 1983, Annu. Rev. Biochem., 52:223-261).

Apolipoprotein B-100 is the major protein of LDL and is also in the lipoprotein very low density (VLDL) and chylomicrons. LDL-cholesterol in the blood can be removed by phagocytosis involving macrophages when stimulated the formation of antibodies that recognizes apolipoprotein B-100, as apolipoprotein B-100 leads to the fact that LDL particles are associated with LDL-receptors on the cell surface (Dalum I. et al., 1997, Mol. Immunol., 34(16-17): 1113-20).

When a macromolecule such as an antibody, binds to apolipoprotein B-100, which is located on the surface of LDL, lipase such as lipoproteinlipase cannot hydrolyze TG and the like, due to steric inconsistencies caused by the fact that the macromolecule is associated with apolipoprotein B-100. Therefore, the formation of free fatty acids, the main factor for the occurrence of obesity, it is possible to inhibit use of antibodies that can communicate with apolipoprotein B-100.

Recently, several studies have attempted to reduce the level of LDL-cholesterol and suppress the occurrence of arteriosclerosis with use of the vaccine in different animal models, such as a mouse or rabbit. For example, C.R.Alving reported that cholesterol can be modified metabolites or its Oka the population and that the modified cholesterol in some cases there may be a strong antigenic determinant (C.R. Alving et al., 1989, Biochem. Soc. Trans., 17(4): 637-9; C.R. Alving et al., 1996, J. Lab. Clin. Med., 127: 40-49; C.R. Alving et al., 1996, Curr. Top. Environ. Immunol., 210: 181-6).

In addition, it was reported that serum are endogenous antibodies to cholesterol (J.T. Wu, L.L., 1997, Clin. Lab. Med., 17(3): 595-604, Review). It was also reported that in the experiment in which rabbits were induced arteriosclerosis and hypercholesterolemia when fed cholesterol-feed, the manifestation of hypercholesterolemia and atherosclerosis in rabbits immunized with an introduction containing cholesterol liposomes were suppressed or significantly decreased compared with the control group.

This antibody induced by the vaccine cholesterol is immunoglobulin M (IgM), which is associated with VLDL, lipoprotein, intermediate density (IDL), and LDL. Based on the foregoing, it is believed that it was possible to develop a vaccine for the treatment or prophylaxis of hyperlipidemia or atherosclerosis caused by high cholesterol (Bailey J.M., 1994, Science, 264: 1067-1068; Palinski W. et al., 1995, Proc. Natl. Acad. Sci. U.S.A., 92(3): 821-5: R. Wu et al., 1999, Hypertension, 33(1): 53-9).

Applicants have found that obesity can be effectively prevented mimetic peptide epitope of apolipoprotein b-100, and based on the foregoing, has developed a vaccine for the treatment of obesity.

Disclosure of invention

Therefore, the aim of the present invention is obespechivayuschego peptides for epitope of apolipoprotein b-100, his concatemer and modified peptides.

Another purpose of this invention is the provision of a method of obtaining the above mimetic peptides for epitope of apolipoprotein b-100, concatemer and modified peptides.

Another purpose of this invention is the provision of a vaccine for treatment or prevention of obesity, which includes the above mimetic peptides for epitope of apolipoprotein b-100, its concatemer and modified peptides.

The purpose of the present invention is achieved by providing mimetic peptides for epitope of apolipoprotein b-100, concatemer and modified peptides.

In the present invention was used library of peptides for screening phage epitope of human apolipoprotein b-100, associated with monoclonal antibodies (MabB23). Subjected to the screening of the above peptides represented mimetic peptides structurally similar to the antigenic determinant, which can be recognized by antibodies, and data mimetic peptides were synthesized according to the amino acid sequences were subjected to a screening of peptides.

The library of peptides is a way of finding the three-dimensional shape of antigenic determinants. That is, the DNA fragments that encode arbitrarily sequenced peptide is IDA, insert into DNA, which encodes the minor envelope protein of the phage, and then inserted into RF (reading frame) DNA and transformed into E. coli for expression. Expressed on the surface of E. coli peptides are subjected to interaction with the antigen in order to select peptides structurally similar to the antigenic determinant.

In order to get anticigarette, mice subjected to immunization with the introduction of the above mimetic peptides. It was proved that the thus obtained anticavity recognizes the original apolipoprotein B-100 and simultaneously mimetic peptides and LDL (Identification of Antigenic Determinants for the Murine Monoclonal Antibodies Against Apolipoprotein A-1 and Apolipoprotein B-100 by using Phage-displayed Random Peptide library, Chi-Hoon Kim, Hanyang Univ., 1997).

Mimetic peptides for epitope of apolipoprotein b-100 of the present invention can be selected from the peptides of sequences of SEQ.ID. No. 1, SEQ.ID. No. 2, SEQ.ID. No. 3, or combinations thereof.

Mimetic peptides of the present invention can be used in the form of concatemer in order to improve their antigenic determinant. As implementation of the present invention can bind two or more mimetic peptide with each other. Desirable concatemer, consisting of three (3) fifteen (15) peptides. More preferably concatemer of the present invention includes four (4) peptide sequence SEQ.I. No. 1.

"Concatemer" above mimetic peptide of the present invention refers to a polymer in which the ends of the above mimetic peptides are linked to each other.

"Modified peptide" above mimetic peptide of the present invention relates to variants of the mimetic peptides that can be recognized by monoclonal or polyclonal antibodies to apolipoprotein B-100. Such options include substitutions, deletions, additions, and chemical substitution of one or more amino acids in the peptide mimetic of the present invention.

Another purpose of this invention is the provision of a method of obtaining mimetic peptide, its concatemer and modified peptides, which includes: i) the stage of insert DNA, which encodes the above mimetic peptide, its concatemer or modified peptide into a vector; (ii) transformation of the above vector into cells of the host and then incubation and (iii) the extraction of the above mimetic peptide, its concatemer or modified peptides from host cells.

Preparation of the vaccine can be performed in any conventional way with mimetic peptide, its concatemers or modified peptides of the present invention. Preferably, the method receiving the Oia of the above drug composition, in which the active compound is mixed or diluted immune adjuvant, drug to enhance immunity, carrier, excipient or diluent, was selected from the group consisting of tablets, pills, granules, powder, pills, suspensions, emulsions, liquid, syrup, aerosol, soft or hard gelatin capsules, sterile solution for injection, sterile powder and the like.

Immune adjuvant that can be used in the compositions of the present invention, is a view of proteins containing the epitope T-cell (e.g., a surface protein of hepatitis b virus), an inert carrier, such as an aluminium salt, bentonite, latex, acrylic particle, and the like; hydrophobic antigen (e.g., lipid), emulsions water-in-oil and oil-water, education depot (e.g., polysaccharide), an activator of T cells, such as PPD, polyadenine, polibrasil and the like; an activator of b cells (for example, b-cell mitogen), surface-active agent such as saponin, lysolecithin, retinal, Quil A (quil A), a liposome, and the like; a substance to enhance the activity of macrophages and activators of the alternative pathway of complement, such as inulin, zymosan, endotoxin, levamisol, C. parvum, and the like.

"The carrier protein" in the present invention means a pharmaceutically acceptable substance, such as protein is whether aluminium salt which can transport mimetic peptide, its concatemer and modified peptides of the present invention, in the blood stream.

Suitable carriers, excipients or diluents in the composition of the present invention can be used is a salt of aluminum, Phenoxyethanol, water, saline, lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the composition according to the present invention may further include a filler, articulately agent, lubricant, humectant, flavor, emulsifier and antiseptic.

The composition according to the present invention can be prepared in the usual way well known in this field, for the induction of an immune response in a mammal with one (1) or more of the introductions.

A vaccine for the treatment of obesity according to the present inventionyou can enter in various ways, such as oral, transdermal, intradermal, intravenous or intramuscular administration, preferably intradermal injection.

The effective dose of the vaccine of the present invention is 0.1-10 µg (active peptide) per kg of body weight, preferably 0.5 to 1.0 microgram per kg is, however, a real dose of the active substance vaccines can be defined depending on several factors, such as immune status, route of administration, the patient's condition, age, gender, body weight, and the like. Therefore, within the indicated doses in no way limit the scope of the present invention.

Major pharmaceutical effects of the vaccine of the present invention is the prevention or treatment of obesity by means of the mechanism by which antibodies induced mimetic peptide, its concatemers or modified peptides, bind to the epitope of apolipoprotein b-100 on the surface of LDL and thereby sterically hinder and inhibit the lipase, which is involved in the formation of fatty acids, which is the main etiological factor of obesity.

In addition, the vaccine of the present invention has an effect to suppress hyperlipidemia mechanism by which LDL is detected and can be easily removed by macrophage through opsonization, caused by human antibodies induced mimetic peptide, its concatemers or modified peptides and conjugated epitope of apolipoprotein b-100 on the surface of LDL.

Other pharmaceutical effect of the composition of the present invention is the prevention or treatment of obesity by suppressing the accumulation of lipids, such as cholesterol free fatty acids in the cell by m is charisma, when the human antibodies, induced mimetic peptide, its concatemers or modified peptides, bind to the epitope of apolipoprotein b-100 on the surface of LDL and thereby inhibit the specific binding of LDL to LDL-receptor located on the cell surface.

Brief description of figures

The above objectives and other advantages of the present invention will become better understood when a detailed description of its preferred embodiment with reference to the accompanying figures, in which:

In figures 1A-1d presents the structure and composition of the vector for expression of the mimetic peptide of the present invention. In Fig. 1A shows the structure of the leader of the cassette of Fig. 1d shows the structure of the cassette LB, figure 1C shows the structure of the cassette BL and figure 1d shows the structure of the expression vector rvh.

The figure 2 presents the means of obtaining the vector rvh and rvh for the expression of the mimetic peptide of the present invention.

The figure 3 presents the results of polyacrylamide gel electrophoresis (PAGE), conducted to identify the cassette LB.

The figure 4 presents the results PAGE, conducted to identify the cassette BL included in the plasmid pBlue-BL.

The figure 5 presents the results PAGE, conducted to confirm n the Board and the number of copies of the DNA inserted into plasmid pBX1 and pBX3.

The figure 6 presents the results of Western blotting performed to identify the expressed peptide PBl4.

The figure 7 presents the results PAGE with the addition of sodium dodecyl sulfate (SDS), were conducted to confirm the purified peptide PBl4.

The figure 8 presents the results of Western blotting performed to confirm the reactivity of the purified peptide PBl4the anti-PBl4-serum.

The figure 9 presents the results of an ELISA conducted to determine the avidity mouse antibodies induced by peptide PBl4.

The figure 10 presents a graph which shows the inhibitory effect PBl4to increase the body weight of the mouse.

In figures 11a and 11b presents graphs showing the change in body weight of mice depending on the vaccine PBl4according to the present invention through 20 weeks after administration of the drug, which can disturb the function of the hypothalamus.

The figure 12 presents a graph that represents the influence on the concentration of lipids in blood serum with a vaccine PBl4.

Best mode for carrying out the present invention

In the following the present invention will be described in detail. However, the present invention is explained below, before whom tableno only to illustrate implementation of the present invention and is not intended to limit the scope of the present invention.

Example 1: synthesis and annealing of the oligonucleotide

Oligonucleotides were obtained by chemical synthesis in Genemed Synthesis (San Francisco, CA, USA) according to the sequence provided by the present applicants. For phosphorylation of the 5'-end of oligonucleotides, 50 μl of 100 pmol/μl of oligonucleotide were incubated with 10 μl of 10 mm ATP, 3 μl of 10 U/µl T4 polynucleotide kinase (Takara, Otsu, Japan) and 7 ál of 10X buffer for kinase within two (2) hours at 37°C.

Each aliquot sample volume of 10 μl of the above-mentioned phosphorylated oligonucleotides were mixed together, heated at 80°C for 5 min and then slowly cooled to room temperature, spending thus annealing with obtaining specific conjugation of complementary circuits.

Example 2: ligation

The mixture for ligation were prepared by mixing 1 µl of the DNA vector, 5 µl of the DNA insert, 1 ál of DNA T4 ligase (NEB, Beverly, MA, USA), 1 µl 10X buffer for enzyme reactions (NEB, Beverly, MA) and 2 μl of distilled water, then incubated at 16°With during the night.

Example 3: constructing expressing vector rvh for the expression of the mimetic peptide of apolipoprotein b-100

Stage 1: planning vector

A plasmid vector for expression of the mimetic peptide typically includes a leader tape and one or more genes peptide PBl. As shown in Figo is e 1, plasmid rvh, which includes one (1) PBl gene, was obtained by cloning the top of the cassette (Fig. 1A) in the website for polychloropene plasmid pQE30 (Qiagen, Hilden, Germany). The obtained plasmid was digested HindIII and SalI and the small fragment was replaced by a cassette LB (Fig. 1b) to obtain plasmid rvh, which is suitable for convenient insertion of multiple quantities of cassettes BL (Fig. 1C).

Meanwhile, plasmid pBluescript II SK+ was digested SalI and XhoI and ligated with cassettes BL, where was derived single-multiple cartridge BL. Selected putative repeats genes peptide PBl in the plasmid pBlue-BL, cut and was subcloned into the pBX1 (Fig. 1d).

Stage 2: obtaining a vector for the expression of monopeptide PBl

Leader tape were prepared by annealing oligonucleotides with sequences SEQ.ID. No. 10 and SEQ.ID. No. 11, was synthesized in the same manner as in examples 1 and 2. Then the vector QE30 (Qiagen, Hilden, Germany), which was split SalI and BamHI, ligated with the above-mentioned leader tape to obtain the plasmid pQE-leader. As a result of the above expression vector pQE30 additionally included six histidine residues at the N-end of the expressed protein for convenient protein purification. The above leader tape designed with the inclusion of site recognition (DDDDKI; SEQ.ID. No. 12) for enterokinase to reduce to a minimum the additional amino acids.

P. the method of example 1 was synthesized four of the oligonucleotide, sequence which presents SEQ.ID. No. 4-7, fosforilirovanii and then annealed respectively with complementary oligonucleotides for synthesis cassettes LB, presented in figure 1b (SEQ.ID. No. 13 and 14). 40 μl of the annealed oligonucleotides were mixed with 3 ál 1 U/ál DNA ligase T4, 5 ál 10X buffer for the enzyme, and 2 ál of distilled water to obtain a mixture for ligation. Then the mixture for ligation was incubated overnight for binding of oligonucleotides to each other.

After completion of the reaction, the reaction mixture was applied onto a 20% polyacrylamide gel and electrophoresis was performed. The cartridge LB (oligonucleotides size 52 BP) (Fig. 3) identified during the application of gel ethidium bromide (EtBr).

In figure 3 the strip M is the speed DNA fragment size of 20 BP, lane 1 represents the reaction solution. The cartridge LB was extracted from the gel using a set of extraction from gel with QIAEX II (Qiagen, Hilden, Germany).

The above plasmid pQE30-leader were digested HindIII and SalI and then ligated with the above-mentioned cassette LB, following the method in example 2, to obtain the vector for expression of the mimetic peptide PBl. Received expressing vector was called rvh and a peptide expressed by a vector, called peptide PBl1(see Fig. 2).

Stage 3: obtain a vector for the expression of concatemer peptide PBl 1

According to the method of example 1 was synthesized four of the oligonucleotide, the sequence of which is presented SEQ.ID. No. 4, 5, 8, and 9, were fosforilirovanii and then annealed respectively with complementary oligonucleotides for synthesis cassette BL in figure 1C (SEQ.ID. No. 15 and 16). Then these oligonucleotides were ligated with each other by way of stage 2 and then put on 20% polyacrylamide gel electrophoresis. Identified oligonucleotides size 55 BP (leader tape) staining of the gel with EtBr. The cartridge BL extracted from the gel using a set of extraction from gel with QIAEX II and were digested SalI and XhoI.

Meanwhile, plasmid pBluescript II SK (Stratagene, La Jolla, CA, USA) were digested SalI and XhoI. The vector was subjected to electrophoresis in 0.8% of agarose gel and extracted using a kit for extraction from gel with QIAEX II (Qiagen, Hilden, Germany).

Spent the same ligation reaction as in example 2, to obtain the plasmid pBlue-BL using 5 µl of DNA cassettes BL and 1 μl of the above cut DNA vector.

pBlue-BL were digested SalI and XhoI and was extracted with cassette BL. Plasmid rvh was obtained by insertion of this cassette BL in the SalI site of the vector rvh obtained in stage 2. In addition, the received vectors rvh and rvh prepared by changing the number of cassettes BL, which was inserted into the SalI site of the vector rvh, with two (2) to three (3) (see Fig. 2).

The peptides expressed by the vectors Pb is 2, rvh and rvh, represented concatemer, which included two (2) or four (4) peptide PBl. They were named respectively PBl2,PBl3and PBl4.

Stage 4: identification insert

Cell hosts (E. coli M15 [pREP4]; Qiagen, Hilden, Germany) was transformed with the plasmid pBlue-BL and sown in a Cup with 1% agar and then incubated for 16 h at 37°that they may form colonies of E.coli. One of the colonies formed on the agar plate was assessed, was made in 10 ml of LB medium and incubated with shaking at 37°With over sixteen (16) hours and then was isolated plasmid using the system for DNA purification (Wizard PLUS SV DNA miniprep DNA purification system; Promega, Madison, Wl, USA). The plasmids isolated from transformed E. coli were incubated with restrictase SalI and XhoI for cleavage at 37°within one (1) h and were analyzed by 20% PAGE (Fig. 4). In figure 4, the strip M is the speed DNA fragment size of 20 BP, lane 1 represents the oligonucleotide product obtained in stage 3, lane 2 is DNA cassette BL allocated 20% of the PAGE to the stage 3, and lane 3 represents the recombinant plasmid pBlue-BL-treated restrictase. As shown in figure 4, it was confirmed that the plasmid pBlue-BL contained cartridge BL.

E. coli (M15[pREP4]) transformed with plasmid rvh or rvh and DNA plasmids were isolated, as explained above, to confirm the numbers and what iently inserts into the DNA cassette. Selected plasmids were digested with restrictase SalI and HindIII and analyzed 20% PAGE (Fig. 5). In figure 5, the strip M is the speed DNA fragment size of 20 BP, lanes 1 and 3 represent plasmid rvh containing cassette LB, but not BL, lane 2 represents a plasmid carrying one cassette LB and two BL with the desired direction. On the other hand, the strip 4 is a plasmid having at least one cassette LB and two BL, however, in the opposite direction. As shown in figure 5, on how many tapes In cassettes BL or LB) was inserted and in which direction they were inserted into the vector rvh, you can get a map of the impact of restricted.

In addition, it was confirmed that the DNA sequence cassettes In included in a plasmid, which was obtained from transformed E. coli is identical to the desired sequences. Plasmids were obtained using the set Wizard PLUS DNA miniprep and sequenced using the kit for DNA sequencing Sequennase (Ver. 2.1) (Amersham, Cleveland, UK).

Example 4: expression of peptide PBl4in E. coli and the selection

Phase 1: confirmation of expression of peptide PBl4

To confirm the expression of the peptide PBl4three types of transformed E. coli M15[pREP4] were cultured in broth LB agar containing ampicillin and kanamycin. One type of E. coli M15[pREP4] was transformed with plasmid rvh, the other is false t who was informirovali pQE30, and another was the untransformed E. coli M15[pREP4]. Each of the resulting colonies was transferred to solid media, respectively, in liquid LB medium that contained 100 μl/ml ampicillin and 25 μl/ml kanamycin, and incubated overnight. The cultures were incubated at 37°within one (1) hour with shaking until the optical density at 600 nm reached the limits of 0.5-0.7. Then the culture medium was made 1 mm isopropylthio-β-galactopyranoside (IPTG) to increase expression of recombinant protein and was further cultured at 37°within five (5) hours were Collected 1 ml of culture medium and centrifuged at 14000 rpm for 2 min to precipitate bacterial cells. The cell precipitate obtained by centrifugation, suspended in 50 µl of 2X SDS solution [100 mm Tris-Cl, pH 6.8, 20% glycerol (wt./vol.), 4% SDS (wt./about), 2% 2-mercaptoethanol, 0.001% of Bromphenol blue] for carrying out SDS-PAGE. The suspended solution was heated at 95°within five (5) minutes, and then 10 μl of the solution was introduced into a hole filled gel and electrophoresis was performed at 20 mA for five (5) h (Mighty Small II, Hoefer, USA). The concentration of acrylamide in the concentrating gel and the separating gel, are used, respectively, was equal to 5% and 15%, and used as a standard protein marker size pre-painted one hundred the dart SeeBlue (250 kDa - 4 kDa; NOVEX, San Diego, CA, USA) and standard wide limit Mark12 (200 kDa to 2.5 kDa). After electrophoresis the gel was stained with Kumasi brilliant blue R-250 for 1 h and the dye was removed decolorizing solution (5% methanol and 7% acetic acid) within ten (10) hours

To confirm that the expressed protein was represented by a peptide PBl4proteins in the gel for electrophoresis were subjected to Western-blotting using rabbit anti-RV antibodies (Fig. 6). Anticigarette was obtained by immunization with egg white, conjuncion with peptide PBl obtained by chemical synthesis Bio-Synthesis, Inc. (Lewisville, TX, USA). In figure 6, the strip M is the label of the pre-painted standard SeeBlue, lane 1 represents the medium used for incubation of E. coli M15[pREP4], which is not transformed, the band 2 is the medium used for incubation of E. coli M15[pREP4], which transformed vector QE30, band 3 is the medium used for incubation of E. coli M15[pREP4], which transformed vector rvh.

As shown in figure 6, only transformed rvh E.coli expressed recombinant peptide RBl4with the specific immunity against murine anti-PBl-serum.

Stage 2: determination of the solubility of the expressed peptide

E. coli M15[pREP4], which is as transformed vector rvh, incubated in the same way as in stage 1. Took 10 ml of culture medium and centrifuged to collect cells. The cell precipitate obtained by centrifugation, suspended in 5 ml of solution for lysis of the cells (300 mm NaCl, 50 mm NaH2PO4, 10 mm imidazole, pH 8.0) to obtain the natural protein from the cells. After cooling the solution with the suspended sediment was treated with ultrasound at 20 cycles for cell lysis. The supernatant was obtained by centrifugation at 4°S, 10000 rpm for 30 min the solution was mixed with the same volume of 2X SDS solution and perform SDS-PAGE in the same way as described in stage 1. Then, each solution was warming at 95°C for 5 min In the SDS-PAGE confirmed that the peptide PBl4you can select and clear from the soluble extract And included in the insoluble crude extract of C.

Stage 3: purification of peptide PBl4

Stage 3-1: affinity chromatography

For purification of recombinant peptide derived in stage 1, used resin Ni-NTA purification of His-tagged proteins. Affinity chromatography using the force of attraction between saturated in resin Ni+and histidine residues at the end of the expressed protein is a well-known convenient method of purification protein of interest.

First of all, Ecoli M15[pREP4], which transformed rvh, made in 1 l culture medium LB and incubated at 37°before the optical density at 600 nm was higher to 0.6. The ratio of culture medium LB and vector rvh was fifty (50) to one (1). IPTG was added to a final concentration of 1 mm and again incubated for five (5) hours After incubation of the cell precipitate was obtained by centrifugation of culture medium at 6000g for 30 min and the precipitate was stored at -70°With during the night. Sediment, thawed on ice, suspended in a solution for dissolving (300 mm NaCl, 50 mm NaH2PO4, 10 mm imidazole, pH 8.0), using a 5 ml solution for dissolving 1 g of sediment. Cells were literally by sonication in the same way as in stage 2, and then centrifuged at room temperature at 10000g for 30 min in the same buffer (8M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0), and the precipitate was added to the cell debris to resuspendable and to denature proteins in them, and a solution of suspended sediment was treated with ultrasound for more protein could be dissolved in the buffer. The suspension was centrifuged at 8000 rpm for 30 min to remove cellular debris, which was not solubilities in 8M urea. To 4 ml of the above supernatant was added to 1 ml of resin Ni-NTA at 4°and who was shaking at 200 rpm for 2 h to capture proteins, including His-tag.

The supernatant containing the protein complex/Ni-NTA carefully applied to the chromatographic column (dimensions: 2 cm (inner diameter) × 2.7 cm (height)). After sedimentation of the resin when the lid allow excess buffer. The column was washed with 20 ml of buffer to create pH (8M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0) and then another 20 ml of buffer (8M urea, 0.1 m NaH2PO4, 0.01 M Tris-HCl, pH 6.3) for leaching of proteins that were not specifically linked to the resin Ni-NTA. Target proteins containing a His-tag, was suirable two (2) times 5 ml of buffer with low pH (8M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 5,9), then four (4) times 5 ml of strongly acidic buffer (8M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 4.5) and then used SDS-PAGE to confirm buervenich target proteins using 15% acrylamide gel (Fig. 7). In figure 7, the strip M is a pre-stained marker size SeeBlue, and lane 7 represents the purified peptide PBl4.

The above purified proteins were dialyzed against PBS (8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l NaH2PO4, 0.24 g/l KH2PO4to restore their original conformation. Used tubes for dialysis were to limit the molecular weight of 3500 Da. During the first dialysis for 5 h used 3 liters of PBS, containing the his 2M urea, and then used two (2) times during the night 5 l PBS without urea.

Stage 3-2: librianna chromatography

Hydrophobic chromatography was carried out to increase the purity of the peptide PBl4who got on stage 3-1.

Ammonium sulfate was gradually added to a final concentration of 20%, to the solution containing the peptide PBl4that was suirable with resin Ni-NTA on stage 3-1, and then the pH was brought to 7.0. The solution was kept for three hours or more, after being completely melted 10% of ammonium sulfate, and then the solution was applied on the column with phenyltetrazol [content: fenilefrina resin for rapid elution (Pharmacia, Sweden); the size of the column: 1 cm (inner diameter) × 3 cm (height)].

Each faction has suirable from the column by passing an eluent (8M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 6.3) through the column with a speed of eluent 0.5 ml/min converts the gradient of ammonium sulfate from 10% to 0%, were applied to the gel for SDS-PAGE. Collected fraction containing the peptide PBl4and were dialyzed in buffer solution to be desalted, and at the same time remove the urea, which was used as a denaturing agent.

Stage 3-3: remove the His-tag

2M urea was added to the buffer solution (50 mm NaCl, 20 mm Tris-HCl, 2 mm CaCl2, pH 7.4), which was suitable Claudine denaturing agent and imidazole, etc. of purified His-tagged protein and the activation of enterokinase. Cialisovernight peptide PBl4obtained in stage 3-2, again were dialyzed using the specified containing urea buffer for desalting of peptide PBl4and during which the concentration of urea is gradually reduced by repeated dialysis against buffer with reduced levels of urea. To the solution containing the peptide PBl4in which the buffer was replaced with the specified second buffer, was added 3 u/ml of enterokinase and incubated at 23°C. the Solution was collected every hour and then analyzed using SDS-PAGE to verify the removal of the His-tag of the His-tagged peptide PBl(PBl4+his).

Stage 3-4: ion-exchange chromatography

Unwanted proteins and peptides, which were formed as a result of processing enterokinase were removed by ion-exchange chromatography.

The solution containing the peptide PBl4-hiswho got on stage 3-3, were dialyzed in buffer for dialysis (2M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 7.0) and buffer was largely shared. The solution, which were dialyzed, were applied to the resin DEAE-separate (Phamacia, Uppsala, Sweden). Then the column was balanced buffer for equilibration (50 mm sodium phosphate buffer, 2M urea, pH 7.0) and the peptide was suirable in the concentration gradient of NaCl from 0 on the 1M using a different buffer (50 mm sodium phosphate buffer, 2M urea, 1M NaCl)(speed eluent: 0.5 ml/min). Received each faction and accumulated containing the target protein fraction. The presence of the peptide PBl4-hisconfirmed by SDS-PAGE after concentrating compartments.

Stage 4: quantitative analysis of PBl4

The purified peptide PBl4that was received in the same way as in stage 3, were analyzed quantitatively by colorimetric method using reagent ICA for microanalysis (Pierce, Rockford, USA).

Stage 5: confirmation of the properties of recombinant peptidePBl4

The purity of the peptides PBl4that were identified in stage 3, and their immunogenicity against antisera, which were obtained using a synthetic peptide PBl4as antigen, was determined by Western blotting with ECL (Amersham, Cleveland, UK). After SDS-PAGE (example 2, step 1), the gel is incubated with the PVDF membrane in buffer (0.3% of Tris, 1.5% glycine, 20% methanol) at a constant voltage of 60 V for three (3) hours for the transfer of protein from gel to PVDF membrane. Then batirovna the membrane was incubated with 5 ml of a blocking solution (TBS, pH 7.5, 5% skimmed milk powder (wt./vol.), 0.02% tween-20) for 1.5 h and then three times washed with TTBS (buffered by Tricom saline containing 0.1% Tween-20), respectively, for 15 min, 5 min and 5 min EN is vyvorotka against peptide PBl (see stage 1 in example 2) was diluted in TTBS solution in a ratio of one (1) to five thousand (5000) and then incubated with the membrane for 1.5 hours To confirm the purity of the peptide PBl4used anticigarette against peptide PBl4(example 3). After washing the gel with TTBS three times for 15 min, 5 min and 5 min, the membrane was incubated for 1.5 h at room temperature with a solution, in which conjugated with alkaline phosphatase F(ab)'2-goat antimachine IgG (H+L) (Zymed, San Fransisco, CA) was diluted in TTBS solution in a ratio of one (1) to one thousand (1000). The membrane was again washed three times with TTBS and then stained by adding BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/microcinema of tetrazole (Sigma)). A solution of BCIP/NBT was removed using TTBS solution after dyeing. In the Western blotting downregulation of the peptide PBl4can be recognized using anti-PBl4-serum.

In the case of ECL, the PVDF membrane (Gelman Science, BioTraceRused instead of nitrocellulose membrane. In addition, the first antibodies used in the ratio of one (1) to ten thousand (10000) and HRP-conjugated rabbit antimachine IgG (Pierce, Rockford, IL, USA) was used as second antibody in a ratio of one (1) to ten thousand (10000). 1 ml of solution a reagent for Western blotting ECL+Plus (Amersham) per 25 ml of solution used in the color reaction. When the color is sufficiently developed, the membrane was inserted into the film cassette, respectively for 5, 10, 20 and 30 seconds the La effect film, to the bands on the gel could be detected (Fig 8). In figure 8, the strip M is ECL detection label (Gibco BRL), and band 1 represents the peptide PBl4. As follows from Fig. 8, the downregulation of the peptide PBl4can be recognized using anti-PBl4-serum.

In addition, Western blotting, in which the peptide PBl4using polyclonal antibodies isolated from rabbit serum, when applied column with protein G (Bio-Rad, USA) gave the same results.

Example 5: receiving mouse anti-peptide PBl4antibodies

Used in this case, the peptide PBl4represented the peptide PBl4-hisfrom which has been deleted his-tag at the stage 3-3 example 2.

Stage 1: ligation of peptide PBl4and OVA

As of carrier protein ovalbumin (OVA) was added to a purified on stage 3 in example 2, the peptide PBl4in a molar ratio of one (1) to ten (10), and were incubated for 1 h at 4°C. To a solution of the peptide PBl4-ovalbumin was added 2% (vol./about.) glutaraldehyde in the same volume and were incubated for 1 h with constant shaking. Then to the reaction mixture were added glycine, while the final concentration became equal to 0.2 M) to stop the reaction.

After the reaction remaining in the reaction mixture of glutaric the Alda is ID-glycine was removed by dialysis using a membrane for dialysis (MWCO 12000-14000 (Spectrum R,Dominguez, CA, USA).

Stage 2: immunization of mice

The peptide, which linked the OVA in stage 1, was concentrated and used for immunization of mice. The amount of antigen, which was injected into mice, was 5 g, which was equal to the number of peptide PBl4before linking with OVA. The antigen emulsified in the same amount of adjuvant, was injected into mice intraperitoneally in a volume of 0.2 ml.

Complete adjuvant's adjuvant (CFA) was used as adjuvant for the first injection and incomplete adjuvant's adjuvant (IFA) was used as adjuvant in the booster immunization with two (2) times two(2)-week intervals. Control mice were injected BSA (bovine serum albumin).

Five (5) days after the last injection mice were collected 1 ml of blood by puncture of the heart, and the blood was rolled for 30 min at 37°C. Then, the blood was centrifuged for 30 min at 4°, 2500g and the clot was removed from the blood. The supernatant (serum) was incubated over night at 4°With full concentration remaining coagulated substances blood and centrifuged for 20 min at 10000g. The obtained supernatant was poured in several tubes. The serum, which was used in the experiment were stored at 4°the rest was stored at -20°C.

Stage 3: determination of the avidity of anti-PBl4-antic the l indirect ELISA

The avidity of the antibodies was determined using serum obtained in stage 2. 100 μl of peptide PBl4was introduced into each well of 96-well plate to micrometrology (Flacon: Pro-link) and he was left alone at 4°C for 6 h or more and then three (3) times washed with TTBS (buffered by Tricom saline containing 0.05% tween-20). To each well was added 200 μl of a blocking solution (1% BSA in TTBS) and incubated at 37°within one (1) h and then washed three (3) times with TTBS. 100 μl of selected serum diluted in blocking solution in a ratio of one (1) to 102-105was added to the reaction solution and incubated at 37°C for 1 h and then washed three (3) times in 200 μl of TTBS. 100 μl of HRP-conjugated anti-rabbit IgG antibody (Pierce, Rockford, IL), diluted in blocking solution in a ratio of one (1) to 103was added to the reaction solution and incubated at 37°C for 1 h and washed three (3) times in 200 μl of TTBS. A solution of the set of substrates of HRP (Bio-Rad) was mixed with a solution In a ratio of nine (9) to one (1). 100 μl of the mixture was added to the reaction solution and stained for 30 min and then was determined by optical absorption of the reaction mixture at 405 nm, as in the case of ELISA (EL312e, Bio-Tek Ins.)(Fig. 9). In figure 9 it is shown that murine anti-Christ. ate, specific to the peptide PBl4can be used for Western blotting and ELISA when thousandth (1) breeding (3,0 on the X-axis in the figure).

Example 6: effect of the vaccine PBl4anti-obesity using the model in mice

Stage 1: induction of obesity in mice

In this case, used mice ICK at the age of 5 weeks (Korean center for conducting experiments on animals, Seoul, Korea). Mice were grown in the nursery, in which the temperature was maintained at 17-25°and fed mixed feed (Sam Yang Feed Ltd., Seoul, Korea [ingredients: water 11,8% or more protein 20,0% or more, crude lipids 3.0% or more, crude fiber 10.0 percent or lower, crude ash 10,0% or lower, calcium 0.6% or below and phosphorus 0.4% or more]. For the induction of obesity mice were administered Goldilocks (GTG). GTG has the ability to induce desensitization of ventro-medial nuclei of the hypothalamus (VMH). Therefore, mice that were injected GTG, did not feel satiety, and they always had the desire to eat. Used in this case GTG is a very unstable compound, which easily decomposes in water or in the presence of moisture. Therefore, 100 mg GTG (Sigma, Inc.) was diluted in 1 ml of sesame oil (Sigma, Inc.) and used by the method of Brecher et al. (Brechere G. and Waxler S.H. Proc. Soc. Exp. Biol. Med., 70: 498-501 (1949)) to introduce the desired number of GTG.

Mice were distributed for the formation of the op is based (twenty (20) mice) and control group (4 mice) and the animals of the experimental group was injected 25 ál GTG, at that time, as the control group did not enter.

To experience determined the body weight of mice in the experimental group, and were selected and used in the experience of the mouse, in which the deviation in body weight was negligible. Values of body weight, determined in mice after one (1) week after the introduction of the GTG, were in the range of 26.5-29,5,

Seven mice in the group with the introduction of the GTG was induced obesity, whereas the rest do not. Mice lacking induced obesity, the newly introduced GTG, and in this case, all mice were induced obesity.

All mice with induced obesity was divided into three (3) groups. One week after the second injection GTG mice of the experimental group 1, consisting of seven (7) mice were injected peptide PBl4obtained in stage 2 of example 3. In addition, mice (experimental group 2, consisting of seven (7) mice) another group of three was introduced instead of ovalbumin peptide PBl4as a false experience and the vaccine was not introduced to the other group (experimental group 3, consisting of six (6) mice with induced obesity. On the other hand, 0.2 ml PBC injected control group for comparison with experimental groups in order to confirm the validity of the vaccine of the present invention.

In addition, the feed used in this case, mixed with egg yolk and dried at 50°to improve potrebi the deposits of cholesterol thus to maintain the level of cholesterol in the serum of mice could be increased. Food was provided in sufficient quantity to cause disease associated with cholesterol. Each day was determined by body weight in mice.

As shown in figure 10, the body weight of mice that were administered the vaccine in experimental group 1increased from 27.7±0.4 g to 52.2±1.7 g 12 weeks after the introduction of the GTG. The data give grounds to conclude that no significant difference in the increase of body weight in mice of the experimental group 1 and control groupwas absent. However, the body weight of mice from the experimental group 2which was administered ovalbumin after the occurrence of obesity and experimental group 3after induction of obesity vaccine not have introduced increased steadily from 28,3±0.5 g to 68,9±2,8, Therefore, it has been shown that obesity can be suppressed by the introduction of vaccines based on peptide PBl4.

In figure 10 G1 and G2 represent the time for the GTG and V1, V2 and V3 represent the time of the introduction of vaccines based on peptide PBl4.

The figure 11 presents the appearance of mice with induced obesity. The mouse at the age of 20 weeks of the experimental group 1 (Fig. 11a: normal mouse) was compared with the mouse at the age of 20 weeks from the experimental group 3 (Fig. 1b: the mouse obese). As shown in Fig. 11, it was confirmed that the vaccine according to the present invention is effective for suppressing obesity.

Stage 2: determining the level of cholesterol in the blood

After the first injection GTG compared the cholesterol level in the blood of mice at the age of 12 weeks from the control group with those of the mouse at the age of 12 weeks, which was introduced GTG, from the experimental group 1 and 2. The concentration of total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol were determined by enzymatic method using sets Cholestezyme-V Triglyzyme-V, HDL-C555 (Shin Yang Chemicals, Seoul, Korea) and LDL-EX (Denka Bio-Research co., Ltd., Tokyo, Japan). In each experiment according to the optical density calibration curve built using standard calibrator D (Denka Bio-Research co., Ltd., Tokyo, Japan) to reduce the error of the experience. Of interest is the optical density was determined by a calibration curve to determine the concentration and composition of lipids, the data obtained are presented in table 1 and on figure 12.

As shown in table 1 and on figure 12, in the induction of obesity has been shown that no significant difference in the composition of the cholesterol in mice in the experimental groups and control was absent, while in General, the concentration in blood total cholesterol, HDL-C and LDL-C was slightly increased.

Industrially applicable shall be

The vaccine of the present invention, which contains mimetic peptides for epitope of apolipoprotein b-100, its concatemer and modified peptides can suppress the occurrence of obesity without causing autoimmune reactions in the body.

Consequently, circulatory disease associated with LDL, can be treated with the vaccine of the present invention is more efficient than temporary and expensive conventional methods, which inhibited the enzyme associated with metabolism of cholesterol.

Although the present invention has been shown and described in detail when referring to specific examples, specialists in this area, obviously, it is clear that it is possible to make various changes in form and detail without departing from the essence and scope of the present invention defined by the attached claims.

1. Concatemer peptide, inducing the production of antibodies against apolipoprotein b-100, which inhibit the action of lipase and prevent the binding of LDL to the LDL receptor, where the specified concatemer includes four (4) peptide, amino acid sequence of each of which are represented in SEQ.ID. No. 1.

2. A vaccine for treatment or prevention of obesity, which includes concatemer according to claim 1.

3. The vaccine according to claim 2, where the specified vaccine administered by intradermal injection.

4. Vacci the as according to claim 2, where the type of a specified vaccine selected from the group consisting of tablets, pills, granules, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile solution for injection and sterile powder.

5. The method of producing concatemer according to claim 1, which includes

i) stage of the insertion polynucleotide, coderush genetic information concatemer according to claim 1, in vector;

ii) transformation of E. coli with the vector obtained in stage i), and incubation of the transformed E. coli;

iii) the extraction of concatemer according to claim 1 from E. coli, incubated at stage ii).

6. Polynucleotide encoding concatemer according to claim 1.

Abstractrule vector, which includes polynucleotide encoding concatemer according to claim 1.



 

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