CN112546201A

CN112546201A - Application of artificial lipoprotein particle apoA-IV fat body in treatment and/or prevention of diabetes

Info

Publication number: CN112546201A
Application number: CN201910916045.6A
Authority: CN
Inventors: 刘平生; 曹震; 周畅; 徐式孟
Original assignee: Institute of Biophysics of CAS
Current assignee: Weikang Pingsheng (Beijing) Biotechnology Co.,Ltd.
Priority date: 2019-09-26
Filing date: 2019-09-26
Publication date: 2021-03-26

Abstract

The invention discloses application of an artificial lipoprotein particle apoA-IV fat body in treatment and/or prevention of diabetes. The artificial lipoprotein particle apoA-IV fat body disclosed by the invention is obtained by recruiting apolipoprotein apoA-IV by fat body. The artificial lipoprotein particle apoA-IV fat body can obviously improve the sugar tolerance level of animals, and the effect of the particle is higher than that of free protein apoA-IV, so that the effect is better. Therefore, the artificial lipoprotein particle apoA-IV fat body and the preparation method thereof have wide and huge application prospects in treatment and/or prevention of animal diabetes, improvement of glucose tolerance level of animals, and food and medical treatment.

Description

Application of artificial lipoprotein particle apoA-IV fat body in treatment and/or prevention of diabetes

Technical Field

The invention relates to the application of an artificial lipoprotein particle apoA-IV fat body in the treatment and/or prevention of diabetes in the field of biotechnology.

Background

Two structures of neutral lipid wrapped by a single-layer phospholipid membrane exist in human bodies, one is lipid drop widely existing in animals, plants and microorganisms, and the lipid drop is an organelle and consists of a neutral lipid core, a single-layer phospholipid membrane and related proteins. Many human diseases, particularly metabolic diseases such as fatty liver, atherosclerosis and diabetes, are closely related to the formation and dynamic changes of lipid droplets. The other is lipoprotein particles which are mainly present in blood and extracellular fluid and transport hydrophobic lipids, different apolipoproteins are on the surfaces of different lipoprotein particles, the types and proportions of the lipoprotein particles have very important influence on human health, and the lipoprotein particles are closely related to metabolic diseases such as fatty liver, atherosclerosis, diabetes and the like. Although the structures of lipoprotein particles and lipid droplets are very similar, the distribution of the two in vivo is different, and the protein types on the surface are different: lipid droplets are present in cells, while lipoprotein particles are mainly present in blood; the major protein on the lipid droplets is the lipid droplet intrinsic/structural protein, while the major protein on the lipoprotein particle is the apolipoprotein. Among them, Apolipoprotein a-IV (apoA-IV) is a member of Apolipoprotein family a, is mainly expressed and secreted in the small intestine, and is mainly present in the serum in the form of Chylomicron (CM), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL) in the blood.

Disclosure of Invention

The technical problem to be solved by the invention is how to develop safe and effective medicaments for treating and/or preventing diabetes mellitus of animals and improving the sugar tolerance level of the animals.

In order to solve the technical problems, the invention firstly provides the application of lipoprotein particle apoA-IV fat body in preparing products for treating and/or preventing diabetes or products for improving the sugar tolerance level;

the lipoprotein particle apoA-IV adipose body is obtained by recruiting apolipoprotein apoA-IV by adipose body.

In the above application, the apolipoprotein apoA-IV can be A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 2;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

A3) The protein can be specifically a protein shown as a sequence 3 or a sequence 5 in a sequence table.

In order to facilitate the purification of the protein of A1), the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in sequence 2 of the sequence listing is labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 2 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in sequence 1 encodes the protein shown in sequence 2.

The apolipoprotein apoA-IV can be obtained by expression of prokaryotic cells or eukaryotic cells. The prokaryotic cell can be virus or bacteria, and the eukaryotic cell can be yeast cell, insect cell or mammalian cell.

In the above application, the lipoprotein particle apoA-IV fat body can be obtained by recruiting apolipoprotein apoA-IV by fat body.

In the above application, the diabetes may be mammalian diabetes, and the sugar tolerance level may be a mammalian sugar tolerance level. In one embodiment of the invention, the mammal is a C57BL/6J mouse.

The lipoprotein particles apoA-IV fat body also belong to the protection scope of the invention.

The invention also provides a preparation method of the lipoprotein particle apoA-IV fat body, which comprises the following steps: the lipoprotein particle apoA-IV adipose body is obtained by recruiting apolipoprotein apoA-IV from the adipose body.

The invention also provides a kit of parts, which may consist of an adipose body and the apolipoprotein apoA-IV, or an adipose body and a biological material related to the apolipoprotein apoA-IV;

the biomaterial is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding an apolipoprotein apoA-IV;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

In the kit, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 1 in the sequence table;

b12) DNA molecule shown in sequence 1 in the sequence table;

b13) a cDNA molecule or a DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b11) or b12) and codes the apolipoprotein apoA-IV;

b14) hybridizes with the nucleotide sequence defined by b11) or b12) or b13) under strict conditions and encodes a cDNA molecule or a DNA molecule of the apolipoprotein apoA-IV.

The kit can be used for preparing lipoprotein particle apoA-IV fat body, or preparing products for treating and/or preventing diabetes, or preparing products for improving sugar tolerance level.

Wherein 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.

The nucleotide sequence encoding apolipoprotein apoA-IV of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the apolipoprotein apoA-IV isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the apolipoprotein apoA-IV and have the apolipoprotein apoA-IV function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. 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 assess the identity between related sequences.

In the above kit, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS,hybridization and washing of membranes were carried out at 65 ℃.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above kit, the expression cassette containing a nucleic acid molecule encoding apolipoprotein apoA-IV (apolipoprotein apoA-IV gene expression cassette) described in B2) refers to a DNA capable of expressing apolipoprotein apoA-IV in a host cell, and the DNA may include not only a promoter for initiating transcription of the apolipoprotein apoA-IV gene but also a terminator for terminating transcription of the apolipoprotein apoA-IV gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the apolipoprotein apoA-IV gene expression cassette can be constructed by using the existing expression vector.

In the kit, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid can be specifically a vector pET28a-SMT 3.

B3) The recombinant vector can be pET28a-SMT 3-apoA-IV. The pET28a-SMT 3-apoA-IV is a recombinant vector obtained by replacing a DNA fragment between BamHI and Hind III recognition sequences of a vector pET28a-SMT3 with a DNA fragment shown in a sequence 1.

In the kit, the microorganism may be yeast, bacteria, algae or fungi. The bacterium can be Escherichia coli, such as Escherichia coli E.

The application of the kit in the preparation of a product for treating and/or preventing diabetes or a product for improving the sugar tolerance level also belongs to the protection scope of the invention.

In the above application, the diabetes may be mammalian diabetes, and the sugar tolerance level may be a mammalian sugar tolerance level. The mammal may be a human or a mouse. In one embodiment of the invention, the mammal is a C57BL/6J mouse.

Hereinbefore, the fat body may be prepared according to a process comprising the steps of:

a1) and (3) vortexing the phospholipid and the neutral lipid in a buffer solution to realize reaction of the phospholipid and the neutral lipid, then centrifuging, collecting an upper liquid phase, and separating the upper liquid phase to obtain the fat body.

The "separation of fat bodies from the upper liquid phase" may comprise the steps of:

a2) purifying the upper liquid phase for more than two times; the method of each purification may be: mixing the upper liquid phase with the buffer solution uniformly, then layering the mixture, and collecting the upper liquid phase;

a3) mixing the upper liquid phase obtained in step a2) with the buffer solution, then separating the upper liquid phase from the buffer solution, and collecting the lower liquid phase which contains fat bodies.

The buffer may be buffer B.

The solute of the buffer B and the concentration thereof in the buffer can be as follows: 15 mM-25 mM HEPES, 80 mM-120 mM KCl, 1.5-2.5 mM MgCl₂(ii) a The solvent may be deionized water; the pH can be 7.2 to 7.6.

The solute of the buffer solution B and the concentration thereof in the buffer solution can be specifically as follows: 20mM HEPES, 100mM KCl, 2mM MgCl₂(ii) a The solvent can be deionized water; the pH may be specifically 7.4.

In the step a1), the parameters of the vortex may be: the total time is 3-5 min;

in step a1), the parameters of the centrifugation may be: 18000-22000g, 3-7 min.

In the step a1), the parameters of the vortex may specifically be: the total time is 4 min.

In the step a1), the centrifugation parameters may specifically be: 20000g, 5 min.

In the step a2), the upper-layer liquid phase is uniformly mixed with the buffer solution for more than the times of the purification of the upper-layer liquid phase for more than two times, and no precipitate is formed after the layering.

In step a2), the "layering" is achieved by centrifugation, and the parameters of the centrifugation may be: 18000-22000g, 3-7 min.

In the step a2), the "layering" is realized by centrifugation, and the parameters of the centrifugation may specifically be: 20000g, 5 min.

In step a3), the "layering" is achieved by centrifugation, and the parameters of the centrifugation may be: 800-1200g, 3-7 min.

In the step a3), the "layering" is realized by centrifugation, and the parameters of the centrifugation may specifically be: 1000g, 5 min.

The phospholipid is b1), b2) or b 3):

b1)1, 2-bis- (9Z-octadecenoyl) -sn-glycerol-3-phosphocholine (DOPC);

b2)1, 2-bis- (9Z-octadecenoyl) -sn-glycerol-3-phosphocholine (DOPC) and 1, 2-bis- (9Z-octadecenoyl) -sn-glycerol-3-phosphoethanolamine (DOPE);

b3)1, 2-bis- (9Z-octadecenoyl) -sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioctadecylsn-glycero-3-phosphocholine (DSPC).

The neutral lipid is c1) or c 2):

c1) a triglyceride;

c2) cholesterol Oleate (CO) and triglycerides.

In the b2), the mass ratio of the 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphocholine to the 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphoethanolamine can be 1: 0.01-2;

in the b3), the mass ratio of 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphorylcholine to 1, 2-dioctadecylsn-glycero-3-phosphorylcholine can be 1: 0.01-2;

in the c2), the mass ratio of the triglyceride to the cholesterol oleate can be 1-5: 1.

in the b2), the mass ratio of 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphocholine to 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphoethanolamine can be specifically 2:1, 1:1 or 1: 2;

in the b3), the mass ratio of 1, 2-bis- (9Z-octadecenoyl) -sn-glycero-3-phosphocholine to 1, 2-dioctadecylsn-glycero-3-phosphocholine can be specifically 2:1, 1:1 or 1: 2;

in the c2), the mass ratio of the triglyceride to the cholesterol oleate can be specifically 5:1, 4:1, 3:1 or 2: 1.

The preparation method of the Triglyceride (TAG) can be as follows: (1) taking 1 dead SD rat, taking subcutaneous fat and omentum majus fat, and cutting into pieces; (2) placing the crushed tissue obtained in the step (1) into a centrifuge tube, adding lipid extraction solution A (chloroform: deionized water is 1:1, v/v), violently swirling for 1 minute, and then centrifuging for 10 minutes at 8000 g; (3) taking the lower organic phase obtained in the step (2), placing the lower organic phase in a new centrifugal tube, and repeatedly extracting the lower organic phase until the lower organic phase is clear according to the extraction method in the step (2) if the lower organic phase is turbid; (4) drying the lower organic phase obtained in the step (3) under high-purity nitrogen (if the lower organic phase becomes turbid in the drying process, repeatedly extracting according to the extraction method in the step (2)); (5) and (4) drying the lower organic phase obtained in the step (4) under high-purity nitrogen (weighing the organic phase for 3 times continuously without changing the mass), and obtaining the triglyceride as a product.

The triglyceride may be Triolein (TO).

The Triolein (TO) may be specifically a product from Sigma company under the catalogue number 92860.

The mass ratio of the phospholipid and the neutral lipid may be any one of (d1) to (d 6):

(d1)0.25～3:5；

(d2)3:5；

(d3)2:5；

(d4)1:5；

(d5)1:10；

(d6)1:20。

in the present invention, the product may be a medicament.

The invention uses the artificially constructed fat body as a new nano-drug carrier carrying apolipoprotein apoA-IV, and the formed artificial lipoprotein particle apoA-IV fat body has the remarkable advantages that: (1) the apolipoprotein apoA-IV mainly exists in a form of lipoprotein particles in vivo, so that the artificially constructed particles can effectively simulate the lipoprotein particles in vivo, are closer to the physiological state, and are safer and more effective. (2) The exogenously given lipoprotein particle apoA-IV fat body can obviously improve the sugar tolerance level of animals, and the effect of the particle is higher than that of free protein apoA-IV, and the effect is better. Therefore, the artificial lipoprotein particle apoA-IV fat body and the preparation method thereof have wide and huge application prospects in treatment and/or prevention of animal diabetes, improvement of glucose tolerance level of animals, and food and medical treatment.

Drawings

FIG. 1 is a schematic diagram of vector construction.

FIG. 2 shows the results of the identification of recombinant proteins apoA-IV derived from prokaryotic systems. a is the silver staining result and b is the Westernblot result.

FIG. 3 shows the results of identifying His-tagged recombinant proteins apoA-IV derived from eukaryotic systems. a is the R250 staining result and b is the WB result. Lane 1 is a commercial BSA standard, lane 2 is a purified reduced recombinant protein apoA-IV, and lane 3 is a purified non-reduced recombinant protein apoA-IV.

Fig. 4 is a detection of fat bodies.

A: the upper image shows the differential interference contrast optical microscopy results, the lower image shows the fluorescence microscopy results, and the scale bar shows 1 μm for the morphology of fat bodies.

B: the size of the fat body, the upper graph is the distribution of the size of the fat body, and the lower graph is the size of the fat body.

C: lipid composition of fat body.

FIG. 5 shows the results of the detection of the artificial lipoprotein particle apoA-IV fat body construction.

A: silver staining results after incubation of protein with adipose bodies. Each lane is, from left to right, a protein molecular weight standard, HIS-SMT3-GFP, upper adipose body 2, lower solution 2, HIS-SMT 3-apoA-IV-GFP, upper adipose body 1, and lower solution 1.

B: fluorescence microscopy of the resulting fat after incubation of the protein with the fat. a1-a3 is the result of the incubation of HIS-SMT3-GFP with adipose bodies, b1-b3 is the result of the incubation of HIS-SMT 3-apoA-IV-GFP with adipose bodies, b2 white arrows indicate the circles formed by HIS-SMT 3-apoA-IV-GFP, the scale bar is 1 μm, and the solid white boxes at the lower left corners of a3 and b3 are enlarged pictures where the dashed white boxes are located.

C: silver staining results of adipose bodies obtained after incubation of different concentrations of HIS-SMT 3-apoA-IV-GFP with adipose bodies.

Lanes

1, 2, 3, 4, 5, 6 are HIS-SMT 3-apoA-IV-GFP at 0.02, 0.05, 0.1, 0.2, 0.3, 0.5 μ g/μ l, respectively. The upper graph shows the silver staining results of the incubation of proteins with different concentrations with the same amount of fat, and the lower graph shows the WB results of the incubation of proteins with different concentrations with the same amount of fat, wherein the primary antibody is Anti-His antibody (Imagen Biosciences), and the secondary antibody is goat Anti-mouse IgG/horseradish peroxidase (China fir Jinqiao).

FIG. 6 is an analysis of the properties of the apoA-IV artificial lipoprotein particles in a body. a: silver staining analysis of the concentration of apoA-IV recruited to the body of fat, arrows indicate the particles of artificial lipoprotein apoA-IV body of fat, and the solution is free apoA-IV protein that was not recruited to the body of fat. b: SDS-PAGE (denaturing gel) analysis of apoA-IV on adipose bodies (adiposomes) alone, free apoA-IV and artificial lipoprotein particles apoA-IV, top panel is silver stained and bottom panel is WB results. c: Native-PAGE (non-denaturing gel) analysis of apoA-IV on fat bodies (adiposomes) alone, free apoA-IV and artificial lipoprotein particles apoA-IV, top panel silver staining results and bottom panel WB results. The apoA-IV protein used is free apoA-IV protein of prokaryotic origin without any tag, i.e.apoA-IV (B).

FIG. 7 the artificial lipoprotein particle apoA-IV adipose bodies significantly increased the glucose tolerance level in mice. FIG. A shows that the glucose tolerance level of mice is remarkably improved by an artificial nano lipoprotein particle apoA-IV (B) fat body constructed by prokaryotically derived apoA-IV (B). a: silver staining analysis the concentration of apoA-IV (B) recruited to the body of fat: 0.7. mu.g/10. mu.l,

lanes

1, 2, 3, 4 represent non-fat bound apoA-IV protein at concentrations of 0.125, 0.25, 0.5, 1. mu.g/10. mu.l, respectively, and the arrows indicate the artificial lipoprotein particles apoA-IV (B) fat bodies, and the solution is free apoA-IV (B) that has not been recruited to the fat bodies. b: blood glucose values at different time points after glucose injection, c: area under the glucose curve after injection of different samples. Each group of 3 mice was injected with 100. mu.l of Saline, 100. mu.l of apoA-IV (B) (final concentration of 0.5mg/kg), 100. mu.l of fat body, and 100. mu.l of apoA-IV (B) fat body (final concentration of 0.5mg/kg, in terms of apoA-IV (B)), respectively. And the figure B shows that the sugar tolerance level of mice is obviously improved by the artificial nano lipoprotein particle apoA-IV (C) fat body constructed by the apoA-IV (C) of eukaryotic origin. a: silver staining analysis the concentration of apoA-IV (C) recruited to the body of fat: 0.8. mu.g/10. mu.l,

lanes

1, 2, 3, 4 represent non-fat bound apoA-IV protein at concentrations of 0, 0.125, 0.25, 0.5, 1, 2. mu.g/10. mu.l, respectively, and the arrows indicate the fat mass of the artificial lipoprotein particles apoA-IV (C), the solution being free apoA-IV (C) that was not recruited to the fat mass. b: blood glucose values at different time points after glucose injection, c: area under the glucose curve after injection of different samples. Each group of 4 mice was injected with 100. mu.l of Saline, 100. mu.l of apoA-IV (C) (final concentration of 0.5mg/kg, based on apoA-IV (C)), 100. mu.l of adipose bodies, and 100. mu.l of apoA-IV (C) adipose bodies (final concentration of 0.5mg/kg), respectively. Statistical analysis, data shown as Means + -SEM, and two sets of significance analyses were Student's t-test (two-tailed). Represents P ≦ 0.05, and represents P ≦ 0.01. The apoA-IV protein used is free apoA-IV protein of prokaryotic origin without any tag, i.e.apoA-IV (B).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The vector pET28a is a product of Novagen company, and the vector pET28a-SMT3 is obtained by modification on the basis of the vector pET28 a. The specific methods for obtaining the vector pET28a-SMT3 and the protein Ulp1 are described in the literature: hu et al, research associations for the substrate binding domain of Hsp70 toner Ssa1 from Saccharomyces cerevisiae, Biomol NMR Assign.2015Oct; 9(2) 329-32(https:// doi.org/10.1007/s 12104-015-. The vector pET28a-SMT3 is described in this document as pET28a plasmid ligation an N-terminal 6XHis tag and a SUMO tag.

Coli E.coli Rosetta is a product of TIANGEN Corp.

The formula of the buffer solution A is as follows: 500mM Tris,1.5M sodium chloride, 40% glycerol (v/v), pH 7.8 with hydrochloric acid.

2xSample Buffer formula: 100mM Tris, 8% SDS (m/v), 20% glycerol (v/v), 0.2% bromophenol blue (m/v), 200mM DTT.

Imidazole at 40mM was added to buffer A to give a solution having an imidazole concentration of 40 mM.

The imidazole concentration of 500mM was 500mM when imidazole was added to buffer A.

The JG-1A high-pressure cell disruptor is a product of Guangzhou energy-gathering biotechnology limited company.

The Chelating Sepharose Fast Flow chelated with nickel ions is a product of GE Healthcare, and the column is a product of Thermo.

2-bis- (9Z-octadecenoyl) -sn-glycero-3-phosphocholine (DOPC) is a product of Avanti corporation.

The formula of the buffer solution B is as follows: 20mM HEPES, 100mM potassium chloride, 2mM magnesium chloride, pH 7.4 with hydrochloric acid.

C57BL/6J mice: beijing Wittiulihua laboratory animal technology Co.

Blood glucose meter and blood glucose test strip: roche.

Saline (Saline): chemical agents of the national drug group, ltd.

Glucose, isopropyl thiogalactoside (IPTG), imidazole, DTT, SDS, bromophenol blue, Saline (salene), sodium chloride, glycerol: chemical agents of the national drug group, ltd.

HEPES, Triglyceride (TAG): Sigma-Aldrich. LipidTox Green and LipidTox Red: invitrogen corporation.

The OptimaTM MAX ultracentrifuge is a product of Beckman, the FV1000 laser confocal microscope is a product of Olympus, and the dynamic light scattering instrument is a product of Beckman.

Example 1 preparation of apolipoprotein apoA-IV (apoA4)

One) preparation of recombinant protein apoA-IV from prokaryotic system

1. Vector construction

The DNA fragment between the recognition sequences of restriction endonucleases BamH I and Hind III of a vector pET28a-SMT3 is replaced by apoA-IV gene shown in a sequence 1 in a sequence table to obtain a recombinant vector, the obtained recombinant vector with a correct sequence is marked as pET28a-SMT 3-apoA-IV, pET28a-SMT 3-apoA-IV can express a fusion protein formed by apoA-IV protein (the amino acid sequence is a sequence 2 in the sequence table) and SMT3 and 6XHis, the fusion protein is marked as HIS-SMT 3-apoA-IV, and the expression of HIS-SMT 3-apoA-IV is driven by a T7 promoter.

The vector construction is schematically shown in FIG. 1, and the inserted apoA-IV gene sequence is marked by a dashed rectangle.

2. Protein purification

And (3) introducing the constructed recombinant vector pET28a-SMT 3-apoA-IV into Escherichia coli E.coli Rosetta to obtain a recombinant bacterium, and marking the obtained recombinant bacterium as E-pET28a-SMT 3-apoA-IV.

E-pET28a-SMT 3-apoA-IV is inoculated in LB liquid medium containing kanamycin, the mixture is placed at 37 ℃ and cultured in a shaker at 200rpm, when the concentration of bacterial liquid reaches OD 600-0.6, isopropyl thiogalactoside (IPTG) is added into a culture system, the final concentration of the IPTG in the culture system is 0.4mM, and then the culture system is induced and cultured for 24 hours at 16 ℃. Collecting induced culture bacteria liquid, using buffer solution A to resuspend the bacteria liquid, then using a JG-1A high-pressure cell crusher to crush to obtain bacteria lysate, carrying out ultracentrifugation on the obtained cell lysate under the centrifugation condition of 30000g for 60min, and collecting supernatant. 50. mu.l of the supernatant was added to an equal volume of 2XSample Buffer, and the resulting mixture was used as sample 1 (supernatant fraction). The remaining supernatant was incubated with a nickel ion-chelated Chelating Sepharose Fast Flow, incubated at 4 ℃ for 2 hours, transferred to a 4ml column, and the effluent (i.e., effluent) was collected, 50. mu.l of the effluent was added to an equal volume of 2XSample Buffer, and the resulting mixture was used as sample 2 (effluent fraction). The nonspecific band was washed with the packing in the 40mM imidazole resuspension column, and the wash effluent was collected, 50. mu.l of which was added to an equal volume of 2XSample Buffer, and the resulting mixture was used as sample 3(40mM fraction). The target protein was eluted by using 500mM imidazole as a packing in a resuspension column, and the eluted fraction was collected, 50. mu.l of which was added to an equal volume of 2XSample Buffer, and the resulting mixture was used as sample 4(500mM fraction). And finally, adding Ulp1 into the eluent for enzyme digestion of the SMT3 label of the nitrogen end of the target protein, adding the enzyme digestion product into the column again for reverse hanging to remove the SMT3 label, and collecting the effluent liquid to obtain the recombinant protein apoA-IV solution. 50. mu.l of the recombinant protein apoA-IV solution was added to 2 xSampleBuffer, and the resulting mixture was used as sample 5 (apoA-IV fraction). The above samples 1 to 5 were analyzed by SDS-PAGE, followed by silver staining and immunoblotting (Westernblot, WB) analysis for identification. The primary antibody used in Westernblot was Anti-His antiboy (Imagen Biosciences) and the secondary antibody was goat Anti-mouse IgG/horseradish peroxidase (China fir Jinqiao).

The results are shown in FIG. 2, in which FIG. a shows silver staining results and FIG. b shows WB results. There are many non-specific bands in lanes of

samples

1 and 2, and the target fusion protein is mainly in sample 1, the target fusion protein is enriched in lanes of

samples

3 and 4 at the position of molecular weight of 72kDa, and the recombinant protein apoA-IV labeled with SMT3 is enriched in lanes of sample 5 at the position of molecular weight of 45 kDa. The result shows that the recombinant protein apoA-IV derived from a prokaryotic system is successfully obtained by the method and is abbreviated as apoA-IV (B).

II) preparation of recombinant protein apoA-IV from eukaryotic system

The purified recombinant protein apoA-IV (the amino acid sequence of which is the sequence 3 in a sequence table) with a His label from a eukaryotic system is obtained by using HEK293 cells by Tortoise Tech Biotech (Nanjing) Ltd, and the gene sequence of the apoA-IV is the sequence 1 in the sequence table. The results are shown in FIG. 3, where a shows the results of R250 staining and b shows the results of Westernblot. The primary antibody used in Westernblot was Anti-His antiboy (Imagen Biosciences) and the secondary antibody was goat Anti-mouse IgG/horseradish peroxidase (Kyowa Kimura). Lane 1 is a commercial Bovine Serum Albumin (BSA) standard, lane 2 is a purified reduced recombinant protein apoA-IV, and lane 3 is a purified non-reduced recombinant protein apoA-IV. The result shows that the eukaryotic system-derived His-tagged recombinant protein apoA-IV is successfully obtained, and the purity of the protein apoA-IV reaches over 90 percent, which is called apoA-IV (C) for short.

Example 2 preparation of Artificial lipoprotein particles apoA-IV adipose bodies

Preparation of fat body

Preparing fat body by vortex and two-step centrifugation, comprising the following steps:

(1) 80 μ l of 2-bis- (9Z-octadecenoyl) -sn-glycerol-3-phosphocholine solution (containing 2mg DOPC) was added to a microcentrifuge tube, and the solvent was blown dry with high purity nitrogen.

(2) After step (1) is completed, 100. mu.l of buffer B and 5mg of Triglyceride (TAG) are added to the microcentrifuge tube, vortexed for 4min (vortexed for 10s, stopped for 10s) to obtain a milky-white lipid mixture 1 (i.e., the initial preparation fraction), and the lipid mixture 1 is centrifuged at 20000g for 5min (18000-22000 g for 3-7min in practical applications). After centrifugation, the bottom of the microcentrifuge tube is a sediment component 1, and a liquid phase system is layered into two layers (the upper layer is a white band 1, and the part below the white band 1 is a solution 1).

(3) After completion of step (2), the solution 1 and the precipitated fraction 1 were discarded by aspiration, the white band 1 was retained, 100. mu.l of buffer B was added, and vortexed to give a milky white lipid mixture 2.

(4) And (3) uniformly mixing the lipid mixture 2 obtained in the step (3) in a vortex manner, centrifuging for 5min at 1000g (in practical application, centrifuging for 3-7min at 800-1200 g), layering the liquid phase system after centrifugation to form two layers (the upper layer is a white band 2, and the part below the white band 2 is the solution 2), and collecting the solution 2, namely the fat body.

The structure of the fat body is observed by FV1000 laser confocal microscopy, the size of the fat body is measured by a dynamic light scattering instrument, and the lipid composition of the fat body is analyzed by thin layer chromatography. The results are shown in fig. 4, panel a showing the morphology of fat bodies, left panel FV1000 confocal laser microscopy, right panel differential interference phase contrast optical microscopy at a scale bar of 5 μm, Green showing constructed fat bodies stained with the neutral lipid-specific dye LipidTox Green. The graph B shows the size of the fat body, the upper graph shows the distribution of the size of the fat body, the lower graph shows the size of the fat body, and the average size of the fat body is about 150.6 nm. Panel C is the lipid composition of the adipose body, consisting of TAG and DOPC.

Secondly, construction of the artificial lipoprotein particle apoA-IV fat body

1. Preparation of HIS-SMT 3-apoA-IV-GFP

The DNA fragment between the recognition sequences of restriction endonucleases BamH I and Hind III of a vector pET28a-SMT3 is replaced by a DNA fragment shown in a sequence 4 in a sequence table (namely, a fusion gene of apoA-IV and GFP), the obtained recombinant vector is marked as pET28a-SMT 3-apoA-IV-GFP, pET28a-SMT 3-apoA-IV-GFP can express a fusion protein formed by apoA-IV, GFP, SMT3 and 6XHis, and the fusion protein is marked as HIS-SMT 3-apoA-IV-GFP. The amino acid sequence from apoA-IV to GFP in the fusion protein is a sequence 5 in a sequence table.

The recombinant vector pET28a-SMT 3-apoA-IV was replaced with pET28a-SMT 3-apoA-IV-GFP by the method of step one in example 1, the protein used was not digested with Ulp1, contained a tag, and the other steps were not changed, and the protein was purified by expression to prepare a fusion protein HIS-SMT 3-apoA-IV-GFP with 6XHis and SMT3 tag.

2. Preparation of HIS-SMT3-GFP

The DNA fragment between the recognition sequences of the restriction endonucleases BamHI and Hind III of the vector pET28a-SMT3 is replaced by a GFP gene shown in the 1147-th 1866 th site of the sequence 4 in the sequence table, the obtained recombinant vector is marked as pET28a-SMT3-GFP, pET28a-SMT3-GFP can express a fusion protein formed by GFP and SMT3 and 6XHis, and the fusion protein is marked as HIS-SMT 3-GFP. The amino acid sequence of GFP in the fusion protein is 383-rd-E621 th site of a sequence 5 in a sequence table.

According to the method of step 2 in the first step of the example 1, the recombinant vector pET28a-SMT 3-apoA-IV is replaced by pET28a-SMT3-GFP, the protein used is not subjected to Ulp1 enzyme digestion, contains a tag, and is not subjected to other steps, and the protein is expressed and purified to prepare the fusion protein HIS-SMT3-GFP with 6XHis and SMT3 tags.

3. Construction of Artificial lipoprotein particle apoA-IV adipose body

Taking 70 μ l of the fat prepared in the first step, adding protein HIS-SMT 3-apoA-IV-GFP to the fat to obtain a mixed system, standing the mixed system at room temperature for 1 hour, and gently swirling the tube every 10 min. Then the lipid droplets were washed for a total of three times, the specific method was as follows: centrifuging the mixture for 5min at room temperature for 20000g to form two layers (the upper layer is fat 1), pumping out the lower layer solution 1, reserving the upper layer, and re-suspending the upper layer fat 1 with 100 μ l buffer solution B to obtain a fat suspension, namely the artificial lipoprotein particle apoA-IV fat.

According to the method, HIS-SMT 3-apoA-IV-GFP is replaced by HIS-SMT3-GFP, and other steps are not changed, so that the artificial lipoprotein particle GFP fat body is obtained. In the process, two layers of the liquid phase system are respectively an upper layer fatty body 2 and a lower layer solution 2.

The HIS-SMT 3-apoA-IV-GFP was replaced with the apoA-IV (B) and apoA-IV (C) samples of example 1, respectively, and the other steps were not changed, and the resulting artificial lipoprotein particles were designated as the artificial lipoprotein particle apoA-IV (B) fat body and the artificial lipoprotein particle apoA-IV (C) fat body, respectively, according to the above method. In the process, two layers of the liquid phase system are respectively an upper layer fatty body 2 and a lower layer solution 2.

Then, the HIS-SMT 3-apoA-IV-GFP protein, the upper layer fat body 1, the lower layer solution 1, the HIS-SMT3-GFP protein, the upper layer fat body 2 and the lower layer solution 2 are respectively taken for analysis.

The results are shown in FIG. 5, in which panel A shows the results of silver staining after incubation of HIS-SMT3-GFP and HIS-SMT 3-apoA-IV-GFP with fat bodies, and the lanes in which the component of fat bodies incubated with the protein HIS-SMT3-GFP (i.e., upper fat body 2) is present have no bands, while the lanes in which the component of fat bodies incubated with the protein HIS-SMT 3-apoA-IV-GFP (i.e., upper fat body 1) is present have significant bands, indicating that the protein HIS-SMT3-GFP cannot be recruited to fat bodies, and the protein HIS-SMT 3-apoA-IV-GFP can be specifically recruited to fat bodies.

Panel B is fluorescence microscopy results of adipose bodies obtained after incubation of HIS-SMT3-GFP and HIS-SMT 3-apoA-IV-GFP with adipose bodies. FIGS. a1-a3 are the results of incubation of HIS-SMT3-GFP with adipose bodies, a1 adipose bodies stained Red with the neutral lipid-specific dye LipidTox Red, no GFP signal detected in a2, a3 is a picture of the fusion of a1 and a2, and Red is the constructed adipose bodies. Fluorescence microscopy results further indicated that protein GFP could not be recruited to adipose bodies. FIGS. b1-b3 show the results of incubation of HIS-SMT 3-apoA-IV-GFP with fat body, b1 shows the form of fat body, b2 shows the signal of HIS-SMT 3-apoA-IV-GFP, white arrows indicate the circle formed by HIS-SMT 3-apoA-IV-GFP, b3 shows the fusion of b1 and b2, the solid white box at the bottom left is an enlarged picture of the dashed white box, and green fluorescence signal shows that HIS-SMT 3-apoA-IV-GFP can be well localized on the surface of red signal fat body, further indicating that protein apoA-IV can be specifically recruited to fat body.

The above results indicate that the fat body can specifically recruit apolipoprotein apoA-IV, the structure of which is very similar to that of natural lipoprotein particles.

In order to measure the saturated concentration of HIS-SMT 3-apoA-IV-GFP bound to adipose bodies, experiments were performed using HIS-SMT 3-apoA-IV-GFP at different concentrations and the same amount of adipose bodies according to the above-described method for preparing apoA-IV adipose bodies as artificial lipoprotein particles, and the resulting incubated adipose bodies were subjected to SDS-PAGE silver staining.

Panel C shows the silver staining results of different concentrations of HIS-SMT 3-apoA-IV-GFP in fat bodies incubated with equal volumes and amounts of fat bodies (volume 70. mu.l, concentration calculated as OD600, OD600 of 3.4). The HIS-SMT 3-apoA-IV-GFP in the reaction systems of

lanes

1, 2, 3, 4, 5 and 6 are respectively 0.02, 0.05, 0.1, 0.2, 0.3 and 0.5 mu g/mu l, the bands appearing in

lanes

1, 2, 3 and 4 become thicker gradually with the increase of the protein concentration, the bands appearing in

lanes

5 and 6 have no obvious change in thickness, and the results show that the concentration of the HIS-SMT 3-apoA-IV-GFP recruited in fat body reaches saturation when the protein concentration reaches 0.3 mu g/mu l.

4. Characteristics and advantages of the Artificial lipoprotein particle apoA-IV fat body

The characteristics of the artificial lipoprotein particle apoA-IV fat body were examined, and the results are shown in FIG. 6. FIG. a is the silver staining result of the apoA-IV concentration in the apoA-IV fat body of the artificial lipoprotein particle obtained in step 3,

lanes

1, 2, 3, 4, 5, 6, 7 are apoA-IV protein not bound to fat body (free apoA-IV protein derived from prokaryotic system without any tag, i.e., apoA-IV (B)), the concentrations are 0, 0.0625, 0.125, 0.25, 0.5, 1, 2. mu.g/10. mu.l, respectively, and the arrow indicates apoA-IV bound to fat body (i.e., the apoA-IV fat body of the artificial lipoprotein particle obtained in step 3), and the amount of apoA-IV carried by the apoA-IV fat body of the artificial lipoprotein particle is 1.2. mu.g/10. mu.l as analyzed by ImageJ and EXCEL software.

The silver staining band gray levels of apoA-IV protein of different concentrations were as follows:

an equation of the protein concentration and the silver staining stripe gray scale is obtained according to the contents in the table: y is 96748x +42715, the silver staining band gray scale of apoA-IV in the apoA-IV fat body of the artificial lipoprotein particle is 161247, and the concentration of apoA-IV protein in the apoA-IV fat body of the artificial lipoprotein particle is 1.2 mu g/10 mu l according to the equation.

Panel B shows the results of SDS-PAGE (denaturing gel) silver staining and Westernblot of adipose bodies, apoA-IV (i.e.apoA-IV (B)) and artificial lipoprotein particle apoA-IV adipose bodies, with only a single band in the lane where the apoA-IV and artificial lipoprotein particle apoA-IV adipose body components are located. FIG. c shows results of Native-PAGE (non-denaturing gel) silver staining and Westernblot of fat bodies, apoA-IV and artificial lipoprotein particles, the apoA-IV fraction in lanes showing two bands, the upper band being dimeric apoA-IV and the lower band being monomeric apoA-IV, and the artificial lipoprotein particle apoA-IV fat fraction in lanes showing only a single band and being monomeric apoA-IV. It was shown that the adipose bodies selectively recruited monomeric apoA-IV.

The primary antibody used in Westernblot was apoA-IV antibody (WuhanyunconTech Co., Ltd.) and the secondary antibody was goat anti-rabbit IgG/horseradish peroxidase (China fir Jinqiao).

Example 3 Artificial lipoprotein particle apoA-IV adipose bodies significantly increased the glucose tolerance level in mice

The effects of the artificial lipoprotein particle apoA-IV (B) fat body and the artificial lipoprotein particle apoA-IV (C) fat body obtained in example 2 were examined in the tolerance to sugar in mice, C57BL/6J mice, 13 weeks old.

Mice were first starved for 16h and then separately injected tail vein with equal volumes of the following samples: 100. mu.l of physiological Saline (Saline), 0.5mg/kg of body weight of apoA-IV (B) in example 1, 0.5mg/kg of body weight of apoA-IV (C), 100. mu.l of fat body (OD600 of 3.4) obtained in the first step of example 2, 0.5mg/kg of body weight of apoA-IV (B) as an injection amount, and 0.5mg/kg of body weight of apoA-IV (C) as an injection amount, of artificial lipoprotein particles apoA-IV (C) as an injection amount.

Half an hour after injecting the above samples, respectively intraperitoneally injecting 150 μ l of glucose solution (glucose dissolved by deionized water) with an injection amount of 2g glucose/kg body weight, then measuring blood glucose levels after 0, 15, 30, 60, and 120min of glucose injection by a blood glucose meter, respectively, and detecting and comparing the effects of the above samples on improving glucose tolerance level of mice.

Silver staining of the apoA-IV concentration in the fat body of the artificial lipoprotein particle apoA-IV (B) the results of silver staining are shown in FIG. 7, wherein

lanes

1, 2, 3, 4 show the apoA-IV protein not bound to the fat body (free apoA-IV protein derived from prokaryotic system without any tag, i.e., apoA-IV (B)) at concentrations of 0.125, 0.25, 0.5, 1. mu.g/10. mu.l, respectively, and the arrow indicates the fat body of the artificial lipoprotein particle apoA-IV (B), and the apoA-IV (B) concentration carried by the fat body of the artificial lipoprotein particle apoA-IV (B) is 0.7. mu.g/10. mu.l, which was obtained by the same calculation method as 4 in step two of example 2.

The blood glucose values at different time points after glucose injection are shown in table 1.

TABLE 1 blood glucose test results (mM) at different time points after glucose injection

In table 1, 3 mice were injected for each sample, and the smaller the P value in the significance analysis, the more significant.

The results show that the fat body treatment group could not improve the glucose tolerance level of mice relative to the saline treatment group, while both the free protein apoA-iv (B) treatment group and the artificial lipoprotein particle apoA-iv (B) fat body treatment group could significantly improve the glucose tolerance level of mice, and comparing the areas under the curves of the groups, the artificial lipoprotein particle apoA-iv (B) fat body treatment group was more significant (P0.0017) compared with the free protein apoA-iv (B) treatment group (P0.0132).

Silver staining of the apoA-IV concentration on the fat body of the artificial lipoprotein particle apoA-IV (C) the results of silver staining are shown in FIG. 7B,

lanes

1, 2, 3, 4, 5, 6, which are apoA-IV protein not bound to the fat body (free apoA-IV protein derived from prokaryotic system without any tag, i.e., apoA-IV (B)) at concentrations of 0, 0.125, 0.25, 0.5, 1, 2. mu.g/10. mu.l, respectively, and the arrow indicates the fat body of the artificial lipoprotein particle apoA-IV (C), and the apoA-IV (C) concentration carried by the fat body of the artificial lipoprotein particle apoA-IV (C) obtained by the same calculation method as 4 in step two of example 2 is 0.8. mu.g/10. mu.l.

The blood glucose values at different time points after glucose injection are shown in table 2.

TABLE 2 blood glucose test results (mM) at different time points after glucose injection

In table 2, 4 mice were injected for each sample, and the smaller the P value in the significance analysis, the more significant.

The results show that the fat body treatment group can not improve the glucose tolerance level of mice relative to the physiological saline treatment group, while the free protein apoA-IV (C) treatment group and the artificial lipoprotein particle apoA-IV (C) fat body treatment group can both significantly improve the glucose tolerance level of mice, and compared with the area under the curve of each group, the artificial lipoprotein particle apoA-IV (C) fat body treatment group has higher significance (P is 0.0096), and has better effect relative to the free protein apoA-IV (C) treatment group (P is 0.0288).

<110> institute of biophysics of Chinese academy of sciences

<120> use of artificial lipoprotein particle apoA-IV fat body for treating and/or preventing diabetes

<160> 5

<170> PatentIn version 3.5

<210> 1

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Pro Leu Ala Thr Asn Leu Lys Asp Lys Phe Asn Arg Asn Met Glu Glu

165 170 175

Leu Lys Gly His Leu Thr Pro Arg Ala Asn Glu Leu Lys Ala Thr Ile

180 185 190

Asp Gln Asn Leu Glu Asp Leu Arg Arg Ser Leu Ala Pro Leu Thr Val

195 200 205

Gly Val Gln Glu Lys Leu Asn His Gln Met Glu Gly Leu Ala Phe Gln

210 215 220

Met Lys Lys Asn Ala Glu Glu Leu Gln Thr Lys Val Ser Ala Lys Ile

225 230 235 240

Asp Gln Leu Gln Lys Asn Leu Ala Pro Leu Val Glu Asp Val Gln Ser

245 250 255

Lys Val Lys Gly Asn Thr Glu Gly Leu Gln Lys Ser Leu Glu Asp Leu

260 265 270

Asn Arg Gln Leu Glu Gln Gln Val Glu Glu Phe Arg Arg Thr Val Glu

275 280 285

Pro Met Gly Glu Met Phe Asn Lys Ala Leu Val Gln Gln Leu Glu Gln

290 295 300

Phe Arg Gln Gln Leu Gly Pro Asn Ser Gly Glu Val Glu Ser His Leu

305 310 315 320

Ser Phe Leu Glu Lys Ser Leu Arg Glu Lys Val Asn Ser Phe Met Ser

325 330 335

Thr Leu Glu Lys Lys Gly Ser Pro Asp Gln Pro Gln Ala Leu Pro Leu

340 345 350

Pro Glu Gln Ala Gln Glu Gln Ala Gln Glu Gln Ala Gln Glu Gln Val

355 360 365

Gln Pro Lys Pro Leu Glu Ser Arg Asp Pro Pro Val Ala Thr Met Val

370 375 380

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

385 390 395 400

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

405 410 415

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr

420 425 430

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr

435 440 445

Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His

450 455 460

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

465 470 475 480

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

485 490 495

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

500 505 510

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr

515 520 525

Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile

530 535 540

Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln

545 550 555 560

Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val

565 570 575

Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys

580 585 590

Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr

595 600 605

Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

610 615 620

Claims

1. Use of lipoprotein particles apoA-IV fat bodies for the manufacture of a product for the treatment and/or prophylaxis of diabetes or for the manufacture of a product for increasing the level of glucose tolerance;

2. Use according to claim 1, characterized in that:

the apolipoprotein apoA-IV is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 2;

3. Use according to claim 2, characterized in that: A3) the protein is shown as a sequence 3 or a sequence 5 in a sequence table.

4. Use according to any one of claims 1 to 3, characterized in that: the lipoprotein particle apoA-IV adipose body is obtained by recruiting apolipoprotein apoA-IV monomer by adipose body.

5. The lipoprotein particle apoA-IV fatty body of any one of claims 1-4.

6. A process for the preparation of the lipoprotein particles apoA-IV fatty bodies of any of claims 1-4 comprising: the lipoprotein particle apoA-IV adipose body is obtained by recruiting apolipoprotein apoA-IV from the adipose body.

7. A kit consisting of an adipose body and apolipoprotein apoA-IV, or an adipose body and a biological material related to apolipoprotein apoA-IV;

the biomaterial is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding an apolipoprotein apoA-IV;

B2) an expression cassette comprising the nucleic acid molecule of B1);

8. The kit of claim 7, wherein: the apolipoprotein apoA-IV is an apolipoprotein apoA-IV monomer;

B1) the nucleic acid molecule is b11) or b12) or b13) or b14) as follows:

b12) DNA molecule shown in sequence 1 in the sequence table;

9. Use of a kit according to claim 7 or 8 for the preparation of a product for the treatment and/or prophylaxis of diabetes or for the preparation of a product for increasing the level of glucose tolerance.