WO2006011491A1 - Plasmid vector, transgenic animal, method of screening drug and drug - Google Patents

Abstract

[PROBLEMS] To prepare a mouse suffering from no obesity even in feeding with a high-caloric feed, and to provide a vector capable of allowing overexpression of adiponectin in the liver, a transgenic animal with the use of this vector, a method of screening a remedy for adiponectinemia, a preventive or a remedy for an increase in fat mass and/or body weight gain, and so on. [MEANS FOR SOLVING PROBLEMS] A plasmid vector containing a human serum amyloid P component (SAP) promoter and an adiponectin gene; a transgenic mouse having the same transferred thereinto; a screening method by using the same; and a preventive or a remedy for an increase in fat mass and/or body weight gain which contains an adiponectin gene or adiponectin.

Description

 Specification

 Plasmid vector, transgenic animal, medicine screening method and medicine

 Technical field

 [0001] The present invention relates to a vector that can be used as a model animal that can be used as a disease state model or a disease prevention constitution model, a vector that can produce a large amount of a target substance at a specific site, and the vector And screening methods for drugs such as anti-obesity drugs such as hyperadiponectin, adipose tissue and adipocyte growth inhibitors, weight gain inhibitors, life-prolonging drugs, etc. The present invention relates to a preventive or therapeutic agent for an increase in fat mass such as an increase in fat cells and Z or weight gain caused by sucrose diet intake.

 Background art

 Obesity / diabetes / hyperlipidemia / hypertension is caused by visceral fat hypertrophy / accumulation, etc., and is a disease that is a cause of shortening of life. These include intake of high-calorie diet and lack of exercise. It is a type of lifestyle-related disease caused by complicated involvement of environmental factors, and there is a strong need for treatment methods.

 [0003] Recent advances in genetic analysis technology have revealed the existence of adipocyte force-in, a site force-in specifically expressed in adipocytes. Among them, adiponectin, which has 244 amino acid strength, Abundant force in normal human blood is lacking in patients with obesity and type 2 diabetes, and its deficiency causes insulin resistance, causing not only obesity and type 2 diabetes but also arteriosclerosis It has become clear that this is also the cause.

 [0004] In an attempt to confirm that adiponectin can cause pathogenesis such as obesity and diabetes, an animal model lacking the adiponectin gene has been created.

 Patent Document 1: Japanese Patent Laid-Open No. 2003-339272

[0005] However, the model animal has been confirmed to exhibit insulin resistance and intimal thickening, and it has been suggested that adiponectin deficiency may cause obesity, diabetes, and arteriosclerosis. The fact of weight gain caused by its deficiency has been confirmed Not in. Therefore, whether or not supplementation of deficient adiponectin can directly prevent weight gain has been proven or suggested.

 [0006] By the way, it is known that hyperadiponectinemia occurs in patients with anorexia nervosa, and if hyperadiponectinemia can be corrected, there is a possibility of treating anorexia nervosa. is there.

[0007] In addition, since adiponectin expression is considered to involve several steps before the anti-obesity effect is exerted, substances that exhibit certain behavior due to adiponectin expression are It is likely to be useful as an obesity drug.

[0008] The present inventors use a transgenic animal that expresses adiponectin in a larger amount than usual in order to examine whether adiponectin is capable of preventing weight gain due to, for example, ingestion of high-calorie food or the like. Was considered suitable.

[0009] However, adiponectin is a substance that exists in a large amount in the blood of normal animals.

It is also considered that the expression level in the cell has reached the limit level required in the living body, so that the high adiponectin expression state exceeding the normal level is maintained for a long period of time, and the sustained high adiponectin state is maintained. It was thought that it was almost impossible to search for substances that behave.

 Disclosure of the invention

 Problems to be solved by the invention

[0010] The authors, etc. ii, Human Serum Amyloid P component promoter-rabbit β-globin intron-Human adiponectin cDNA Has successfully produced a transgenic animal with a higher blood adiponectin level than normal, and the transgenic mouse has a reduced blood fat even under normal diet. Even under high sucrose diet, fat accumulation such as increase in fat mass including increase of fat cells in subcutaneous fat and visceral fat does not occur, and even under the same condition, although it was raised under high calorie diet As a result of discovering that the life span is longer than that of bred wild-type mice and supporting the role of adiponectin in preventing diseases such as obesity and diabetes, the present invention It is those that Tsu, it is an object of the present invention, a large amount of issued adiponectin in the liver Provided vectors, transgenic animals using such vectors, screening methods for anti-adiponectinemia or anti-obesity drugs, fat accumulation suppression and Z or weight gain suppression due to increased adipocytes, etc. There is.

Means for solving the problem

The above object is achieved by the following invention.

(First invention)

A plasmid vector containing a liver-specific expression promoter and adiponectin gene.

(Second invention)

It is a plasmid vector containing a promoter and adiponectin gene derived from a gene of a protein specifically produced in the liver.

(Third invention)

Human serum amyloid P component (hereinafter referred to as “SAP”) A plasmid vector containing a promoter and adiponectin gene

((Fourth invention)

The plasmid vector according to any one of the above, wherein the adiponectin is human adiponectin.

(Fifth invention)

A plasmid vector containing a liver-specific expression promoter and a target gene. (Sixth invention)

A transgenic animal into which the promoter and adiponectin gene described in any of the above are introduced.

(Seventh invention)

A transgeneic rodent animal into which the promoter and adiponectin gene described above are introduced.

(Eighth invention)

Tran into which the promoter and adiponectin gene described above are introduced This is a screening method for a therapeutic agent for hyperadiponectinemia using a sujienic animal.

 (Ninth invention)

An anti-obesity drug screening method comprising the following steps (1) to (3):

 (1) A step of analyzing the gene expression state of the transgenic animal into which the promoter and adiponectin gene described in any of the above are introduced.

 (2) A step of selecting a substance exhibiting a certain behavior caused by adiponectin expression based on the result of analysis!

 (3) A step of selecting one of the following (i) to (iv) as an anti-obesity drug.

 (i) Substance selected in (2)

 (ii) Substance capable of controlling in vivo expression of (i)

 (iii) Substances that have structural or functional similarities to (i) or (ii)

 (iv) a gene capable of expressing any of (i) to (iii)

(Tenth invention)

A preventive or therapeutic agent for fat increase and Z or weight gain, characterized by comprising an adiponectin gene or adiponectin.

(Eleventh invention)

It is a preventive or therapeutic agent for fat mass increase and Z or weight gain caused by high calorie and Z or high sucrose diet, characterized by containing adiponectin gene or adiponectin.

(Twelfth invention)

A preventive or therapeutic agent for increasing fat mass and Z or weight gain, comprising the plasmid vector according to any one of the above first to fourth.

The invention's effect

The first to fourth inventions are useful for the production of transgenic animals that express adiponectin at high concentrations, and as gene therapy drugs for diseases caused by adiponectin deficiency (obesity, diabetes, arteriosclerosis, etc.) Can be used. The fifth invention is useful for preparing a transgenic animal that expresses a target gene at a high concentration in a liver-specific manner, and is useful as a gene therapy drug that expresses in a liver-specific manner. Sixth force By the transgenic animal of the seventh invention, the physiological significance of adiponectin of the following 1) to 4) is clarified, and adiponectin or its agonist is effective for lifestyle-related diseases such as diabetes and hypertrophy. Can know the possibility of being a therapeutic drug

1) Discover new phenotypes other than obesity suppression, weight loss, insulin resistance improvement and arteriosclerosis suppression, and discover new effects of adiponectin.

 2) Clarify the new effects and mechanisms of adiponectin, especially in the liver.

 3) It is possible to know the reaction or side effects caused by a high concentration of adiponectinemia.

4) Prolonging the lifespan of wild-type mice bred on high-calorie diets that are originally short-lived will help to elucidate adiponectin protection against long-calorie and Z- or high-sugar diets.

According to the eighth invention, it becomes possible to screen for therapeutic agents for hyperadiponectinemia. The ninth invention makes it possible to screen for anti-obesity drugs.

According to the tenth and eleventh inventions, it is possible to prevent or treat the increase in fat mass and the increase in Z or weight due to high calorie and Z or high sucrose diet intake. According to the twelfth invention, adiponectin can be surely expressed at a high concentration.

FIG. 1 is a graph showing blood adiponectin concentration (average) in a group of transgenic mice of the present invention. Wild represents a wild-type mouse, and Linel represents a transgenic mouse (Tg mouse).

 FIG. 2 is a graph showing the state of weight gain (average) when a high-calorie diet is administered to the transgenic mouse (Tg mouse) (male) group of the present invention.

FIG. 3 is a graph showing the increase in body weight when the (male) group and normal food are administered to the transgenic mice (Tg mice) of the present invention. FIG. 4 is a graph showing the state of weight gain (average) when a high-calorie diet was administered to the transgenic mouse (Tg mouse) (female) group of the present invention.

 FIG. 5 is a diagram showing the state of weight gain (average) when normal food is administered to the group of transgenic mice (Tg mice) (female) of the present invention.

 FIG. 6 is a view showing the blood fat (neutral fat, cholesterol) reducing effect of the transgenic mouse (Tg mouse) (male) of the present invention under normal feeding.

FIG. 7 is a graph showing the visceral fat and subcutaneous fat reducing effects of the transgenic mouse (Tg mouse) (male) of the present invention under a high calorie high sucrose diet.

 FIG. 8 is a graph showing the life prolonging effect of the transgenic mouse (Tg mouse) (male) of the present invention under a high calorie high sucrose diet.

 [FIG. 9] A diagram showing the size of subcutaneous adipocytes of wild-type mice under a high-calorie high-sucrose diet (HE staining, 100 times).

 FIG. 10 is a view showing the size of subcutaneous adipocytes of a transgenic mouse (Tg mouse) of the present invention under a high calorie high sucrose diet (HE staining, 100 times).

 [Fig. 11] A diagram showing the visceral fat cell size of wild-type mice under a high-calorie high-sucrose diet (HE staining, 100 times).

 FIG. 12 is a view showing the visceral fat cell size of a transgenic mouse (Tg mouse) of the present invention under a high-calorie high-sucrose diet (HE staining, 100 times).

 FIG. 13 is a diagram showing the size of subcutaneous adipocytes of wild-type mice under normal diet (HE staining, 100 times).

 FIG. 14 is a view showing the size of subcutaneous fat cells of a transgenic mouse (Tg mouse) of the present invention under normal diet (HE staining, 100 times).

 FIG. 15 is a diagram showing the visceral fat cell size of wild-type mice under normal diet (HE staining, 100 times).

 FIG. 16 is a view showing the visceral fat cell size of the transgenic mouse (Tg mouse) of the present invention under normal diet (HE staining, 100 times).

BEST MODE FOR CARRYING OUT THE INVENTION

As described above, adiponectin is a substance that exists in a large amount in the blood of normal animals. Human adiponectin is expressed by a known human adiponectin gene described later. The adiponectin used in the present invention includes proteins extracted from yeast and Escherichia coli by genetic recombination, proteins produced by chemical synthesis, and the like in addition to natural extracted proteins from animals including humans.

 [0015] Further, the adiponectin used in the present invention includes a variant in which a part of the amino acid sequence is deleted, substituted or added as long as it has a function substantially equivalent to that of adiponectin. The function substantially equivalent to adiponectin refers to a function capable of preventing obesity, diabetes, arteriosclerosis and the like by gene administration or administration as an expressed polypeptide to adiponectin gene-deficient mice of Patent Document 1, for example.

 [0016] In the adiponectin gene referred to in the present invention, as long as the expressed protein can express a protein having substantially the same function as the above-mentioned adiponectin, a part of the gene sequence is deleted or other Also included are those that have been replaced by bases or have other base sequences added to the structural gene or at the 5, 5 or 3, end. The human adiponectin gene sequence is represented by, for example, NCBI ACCESSION No. D45371.

 [0017] These genes can also be synthesized based on cDNA produced from human adiponectin mRNA using reverse transcriptase or the like.

 [0018] In the present invention, when producing a transgenic animal, in order to make the gene expression more reliable and confirm its in vivo effect, it is derived from the same animal as the transgenic animal. It is preferred to use the adiponectin or adiponectin gene. On the other hand, when selecting an adiponectin gene derived from a species different from that of the animal to be introduced, it is preferable in that the behavior of the adiponectin can be searched by distinguishing from the adiponectin originally possessed by the target animal.

When adiponectin or adiponectin gene is used as the medicament of the present invention, it is derived from humans, and it is produced by genetic recombination in Escherichia coli and yeast, and a synthetic protein consisting of the same or similar sequence. This is preferable because it has less influence on the living body and can surely exert the original function of adiponectin. <Plasmid vector of the present invention>

 The plasmid vector of the present invention contains a liver-specific expression promoter and adiponectin gene as its constituent elements.

 [0020] (Liver-specific expression promoter)

 Examples of liver-specific expression promoters include promoters derived from genes of proteins specifically produced in the liver, such as the SAP promoter (Iwanaga T, et al. Liver—specinc and high-level expression of human serum amyloid P component gene in transgenic mice. Dev Genet. 1989; 10 (5): 365-71.).

 [0021] The plasmid used in the plasmid vector of the present invention is not particularly limited. For example, those having a large gene expression level in eukaryotes are preferred. It preferably contains a poly A sequence that is useful for fishing. Specific examples include pSG5 plasmid vector (Stratagene). In addition, it is preferable to use a part of this pSG5 also in the plasmid vector of the fifth invention other than the first to fourth cases.

 [0022] (Vector manufacturing method)

 The plasmid vector of the present invention can be produced according to a conventional method (Current Protocols in Molecular Biology Frederick M. Ausubel (Author) (1988/11/14) John Wiley & Sons Inc).

 [Use of the plasmid vector of the present invention]

 The plasmid vector of the present invention can be used for production of a transgenic animal as a disease state model.

 <Other plasmid vector of the present invention>

 In addition, other plasmid vectors of the present invention include those containing a gene of the target protein instead of the adiponectin gene. This plasmid vector can efficiently express the target protein in the liver.

 <Transgenic animal of the present invention>

 (Type of animal)

As animals used in the present invention, rodents are preferred, such as mice, rats, Forces such as Muster Among others, C57BL / 6N mice (B6 mice), Balb / c mice and the like are preferably used, and C57BL / 6N mice are particularly preferable.

 In the following description of the present invention, there are cases where the introduction rodent is a mouse as an example. However, the present invention is not limited to the mouse.

 [0027] (Method for producing transgenic animals)

 The transgenic animal of the present invention can be produced, for example, by transplanting an embryo obtained by the following method to a borrowed animal. However, methods that are generally used for gene transfer are not limited to these methods.

 [0028] (Method for producing embryo for production of transgenic animals)

 For example, a gene fragment such as a plasmid vector containing a specific promoter and adiponectin gene obtained as described above is injected into a pronuclear embryo of a mouse fertilized egg by, for example, microphone injection or the like. Confirmation of the recombinant gene injection can be confirmed by PCR using a DNAesay kit [Qiagen, Germ any] extracted from tissues such as the tail of a generated mouse (Funda).

 [0029] However, it is preferable to use a transgenic animal that stably expresses the adiponectin gene. Therefore, the above-mentioned animal is founded as a founder and crossed with a wild-type mouse. It is preferable to select those that highly express adiponectin.

 [0030] Examples of screening methods include PCR analysis using tail genomic DNA, ELISA analysis of adiponectin in serum, and Western blotting analysis of adiponectin in liver tissue.

 [0031] The amount of the gene fragment containing the promoter and the adiponectin gene can be appropriately adjusted according to the degree of the desired disease, etc., and is not particularly limited.

[0032] (Application)

The transgenic animal of the present invention can be used to verify the role of adiponectin in obesity, diabetes, etc., in addition to the angular force different from that of adiponectin-deficient mice. It can also be used for screening of drugs such as anti-obesity drugs. <Screening method for therapeutic agent for hyperadiponectinemia of the present invention>

 The screening method of the present invention can be carried out by ELISA analysis of adiponectin in serum, Western blotting analysis of adiponectin in liver tissue before and after administration of a test substance.

[0034] In the screening method of the present invention, in addition to so-called primary screening for selecting a desired preventive or therapeutic agent from a plurality of candidates, the effect of preventing or treating a test substance is confirmed. Secondary screening (confirmation test) is also included.

[0035] In addition, since it is known that hyperadiponectinemia occurs in patients with anorexia nervosa, the screening method for a therapeutic agent for hyperadiponectinemia is to prevent and improve anorexia nervosa. 'Therapeutic agent screening methods are also included.

<Screening method for anti-obesity drug of the present invention>

 The antiobesity drug screening method of the present invention can be carried out, for example, as follows.

 First stage: The gene expression state of a transgenic animal of the present invention that expresses adiponectin at a high concentration and a wild type that shows a general expression level of adiponectin are compared.

 Second stage: Based on the results of the analysis, a substance that exhibits a certain behavior due to the expression of adiponectin is selected. Substances that exhibit a certain behavior include substances that have been newly expressed in the body, substances that have increased in expression, substances that have decreased in expression, and substances that have disappeared. Third stage: Select one of the following (i) to (iv) as an anti-obesity drug.

 (i) Substance selected in the second stage

 (ii) Substance capable of controlling in vivo expression of (i)

 (iii) Substances that have structural or functional similarities to (i) or (ii)

 (iv) a gene capable of expressing any of (i) to (iii)

[0037] In the first stage, normal diet may be used. However, for example, when fed with high calorie and Z or high sucrose diet, the gene expression state between wild type and transgenic animals This is preferable because the difference is considered to be clear. According to the examples described below, wild type and trance are more likely to be fed with high-calorie and Z- or high-sugar diets than with normal diets. This is apparent from the remarkable difference in the size of fat cells from that of the animal.

 [0038] A high-calorie diet is one that contains more fat than a normal diet. For example, a fat amount of 30% by weight or more is preferred, and more preferably 40% by weight or more. Specific examples include high-calorie diets for breeding mice (7.5% carbohydrate, 24.5% protein, 60% fat, oriental yeast KK, F2HFD2).

 [0039] A high sucrose diet is one that contains more sugar than a normal diet, but when used in the first stage, the higher the sugar ratio, the easier it is to obtain a difference from the regular diet in comparative experiments. Therefore, for example, the sucrose content is preferably 15% by weight or more, more preferably 20% by weight or more.

 [0040] In the third stage (ii), “(i) substance capable of controlling in vivo expression” means that (i) is newly increased in expression or expression level due to adiponectin!] In the case of such a substance, “(i) substance capable of controlling in vivo expression” is a substance that promotes the expression of (i). When (i) is a substance whose expression is reduced or eliminated due to adiponectin, “(i) substance capable of controlling in vivo expression” is a substance that suppresses the expression of (i).

 [0041] In the third stage (iii), “a substance having structural or functional commonality with (i) or (ii)” means, for example, (i) or (ii) a force protein Examples include a protein having the same enzyme activity as the protein, and a substance having the same basic skeleton when (ii) is a low molecular weight compound.

 [0042] The effect of the selected substance is, for example, administered to a wild-type animal or an animal with adiponectin deficiency, followed by administration of high calorie and Z or high sucrose diet, and the body weight is measured over time. By using the fourth stage, the effect as an anti-obesity drug can be confirmed.

 [0043] By selecting an adiponectin gene derived from a species different from the animal to be introduced as the adiponectin gene used in the transgenic animal of the present invention, the target animal can be distinguished from the original adiponectin and searched. .

[0044] It should be noted that this screening method also confirms the prophylactic or therapeutic effect of a test substance in addition to the so-called primary screening for selecting a desired prophylactic or therapeutic agent from a plurality of candidates. A secondary screening (confirmation test) is included to confirm.

 <Medication of the present invention>

 Adiponectin or a gene thereof can be used as a preventive or therapeutic agent for fat mass increase and Z or weight gain. Among them, it is useful as a preventive or therapeutic agent for fat mass increase and Z or weight gain caused by high calorie and Z or high sucrose diet. The increase in fat mass includes an increase in the number of fat cells in addition to an increase in the size of fat cells.

[0046] In addition to adiponectin, which is an active ingredient, the pharmaceutical (prophylactic or therapeutic agent) of the present invention may contain other ingredients as long as the effect thereof is not inhibited. For example, it is pharmaceutically acceptable. As carriers, excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption enhancers, humectants, adsorbents, fillers , And can be formulated by known methods using additives such as bulking agents, moisturizers, and preservatives. Examples of excipients include organic excipients and inorganic excipients.

 [0047] Further, when the active ingredient used in the medicament (preventive or therapeutic agent) of the present invention is a gene for adiponectin, it can be used as a solvent such as water and other injections as long as the effect is not inhibited. Commonly used components and the like can be included.

 [0048] Furthermore, the medicament of the present invention may be combined with conventionally known medicaments of the same purpose.

 [0049] As the administration form of the medicament of the present invention, when the active ingredient is adiponectin, in addition to oral administration, intravenous administration such as intravenous injection, intramuscular administration, transdermal administration, intradermal administration, subcutaneous administration, abdominal administration Intracavitary administration, rectal administration, mucosal administration, inhalation, etc. may be mentioned, but intravenous administration such as intravenous injection is preferable in terms of maintaining a constant blood clot concentration.

 [0050] Examples of dosage forms for oral administration include forms such as tablets, capsules, granules, powders, pills, troches, or syrups.

[0051] The content of the active ingredient in the preparation of adiponectin used as the medicament of the present invention varies depending on the dosage form, cannot be generally limited, and is within the range where various dosage forms are possible. For example, in the case of a liquid preparation, 0.0001 to 10 (wZv%), preferably <0.001 to 5 (wZv%), particularly in the case of an injection, 0.0002 to 0. 2 (w / v%), preferably 0.001 to 0.1 (w / v%), 0.01 to 50 (wZw%) for solid agents, preferred Further, the force that can be prepared as 0.02 to 20 (wZw%) or the like is not necessarily limited to this range.

 [0052] The dosage of the medicament of the present invention varies depending on the route of administration, symptoms, age, body weight, form of the preventive or therapeutic agent, etc. For example, a subject who needs to treat the active ingredient (adiponectin) in the medicament. 0.005 to 500 mg per kg body weight, preferably 0.1 to: LOOmg, but for adults, the lower limit is 0. Olmg (preferably 0.1 mg), the upper limit is 20 g (preferably 2000 mg , More preferably 500 mg, and even more preferably lOO mg), and it is desirable to administer in one or several divided doses according to the symptoms.

 [0053] As a gene form when the gene encoding adiponectin is used as a drug in gene therapy, DNA, RNA, plasmid, virus vector, etc. can be used. It may be a real chain. Thus, even when used as a drug in gene therapy, a modified pSG5 plasmid containing a SAP promoter, a rabbit j8 globulin intron, a poly A sequence, etc. as used in the preparation of the above-described transgenic animal. Vectors are preferred.

 [0054] In the case of using a plasmid, a method of directly administering an expression plasmid into a muscle (DNA vaccine method), a ribosome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electopore position method, etc. The ribosome method is preferred.

 [0055] When viral vectors are used (Nikkei Science, April 1994, 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 (15), (1994 ), And references cited therein), etc., can be carried out by incorporating the gene of interest into the virus. Examples of the virus used for the virus vector include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, poliovirus, simbis virus and other DNA viruses or RNA viruses. Among viruses, retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are preferred, and adenoviruses are particularly preferred.

[0056] In order to actually act a gene as a medicine, in addition to the "in vivo method" in which the gene is directly introduced into the body, the gene is introduced into cells collected from humans, and then the gene is introduced. There is an “ex vivo method” in which the cells are returned to the body [Nikkei Science, April 1994, 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 (15), (199 4), and their cited references, etc.] are preferred in that the in vivo method is less expensive and simple, and the ex vivo method is more efficient in introducing genes into cells. Is preferred because it is good.

 [0057] When administered by the "in vivo method", an appropriate route of administration can be selected according to the disease or symptom to be treated. Examples of the administration route include vein, artery, subcutaneous, intradermal, intramuscular and the like.

 [0058] When administered by the "in vivo method", for example, it can be in the form of a preparation such as a liquid. In general, it is possible to add various components that are commonly used in injections and the like as required by the form of injections containing genes.

 [0059] In addition, a ribosome or membrane fusion ribosome containing a gene (such as Sendai virus (HVJ) ribosome) can be used as a form of a ribosome preparation such as a suspension, a freezing agent, or a centrifugal concentrated freezing agent.

 [0060] Further, when the gene for adiponectin is used for gene therapy, the content of the gene in the preparation can be appropriately adjusted depending on the disease to be treated, target organ, patient age, body weight, etc. The normal gene is 0. OOOlmg ~: L00mg, preferably 0.001mg ~ lOmg, and it is appropriate to administer this once every several days to several months.

 Example

 [0061] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0062] Example 1

 (1) Production of transgenic mice

In order to express adiponectin in the mouse liver at a higher concentration, a vector containing SAP-promoter rabbit j8-globulin intron-human adiponectin cDNA-polyadenylation signal was constructed. For this purpose, we used pSG5 vector (Stratagene), which is an expression vector with early SV40 promoter ^ —— rabbit 13-globulin intron polyadenylation signal. Transfer pSG5 vector to SCSI 10 cell The pSG5 vector was increased with the Qiagen plasmid kit.

 The SAP promoter was used to selectively express adiponectin in the liver. Based on reports of overexpression of leptin and BNP in the liver using this promoter, PCR was performed by amplifying -677 bp to -9 bp upstream of the human SAP gene and adding Sal 1 and Cla 1 cleavage sites at both ends. The product was incorporated into the TA easy vector. By digesting this integrated TA easy vector with Sal 1 and Cla 1 restriction enzymes and ligating the excised PCR product to the Sal 1 and Cla 1 sites of the pSG5 vector, the early SV40 promoter in the pSG5 vector is obtained. Replaced with SAP promoter. In this way, an expression vector (hSAP_pSG5) containing a human SAP promoter rabbit j8-globulin intron-polyadenylation signal that can be expressed specifically in the liver was constructed. Using a cDNA library of mouse adipose tissue, primers were prepared to create BamHl and EcoRl sites at both ends, and the full length of human adiponectin cDNA was amplified by PCR. A PCR product with BamHl and EcoRl cleavage sites at both ends was incorporated into the TA easy vector. This integrated TA eas y vector was digested with BamHl and EcoRl restriction enzymes, and the excised product was ligated between BamHl and EcoRl present in the cloning site of the hSAP-pSG5 expression vector.

[0063] As a result, a vector containing a human SAP promoter, one rabbit β globulin intron, human adiponectin cDNA polyadenylation signal was constructed. The vector prepared above was cleaved with Sal 1, the vector-derived sequence was removed by agarose gel electrophoresis, and the DNA fragment (about 2 kb) was purified by Gene Clean II kit.

 [0064] Using the DNA fragment of the purified vector, a transgenic mouse was prepared according to a conventional method. As the mouse, C57BL / 6N mouse was used.

 [0065] (2) Confirmation of adiponectin expression

 When the adiponectin concentration in the blood of this mouse was measured by ELISA, as shown in FIG. 1, the amount of adiponectin in one group was 5 to 10 times the normal amount. The blood concentration shown in Fig. 1 is the average value of each group (8 animals in each group).

Although not shown in the figure, a group showing 3 to 5 times the adiponectin concentration was also obtained. That is, in the method for producing a transgenic animal of the present invention, various adiponectin genesis are generated. Transgenic animals with current capabilities can be obtained.

 Since these transgenic mice express a high concentration of adiponectin immediately after birth, they are suitable for investigating the effects of hyperadiponectinemia over a long period of time.

 In addition, this adiponectin is expressed specifically in the liver and is suitable for examining the direct influence of adiponectin on the liver.

 In addition, since human adiponectin is expressed, its function can be evaluated in distinction from mouse-derived adiponectin.

 [0066] (3) Confirmation of obesity prevention effect by adiponectin

 In each of this group of mice and wild type mice, (g) 80 g of high calorie diet for breeding mice (7.5% carbohydrate, 24.5% protein, 60% fat, Oriental yeast KK, F2HFD2) was mixed with 20 g of sucrose. I started to give it a high-calorie diet or (2) a regular diet from the 8th week of life. Each group had 8 animals. The same experiment was conducted for both males and females.

 [0067] As can be seen from FIG. 2 to FIG. 5, wild type mice (wild) showed a significant weight gain, whereas the transgenic mice of the present invention fed a high-calorie diet until 15 weeks of age. Nevertheless, he gained power without any weight gain.

 Even if this cause of lifestyle-related diseases in humans, such as continuous intake of high-calorie diet, it is thought that obesity was suppressed by high expression of adiponectin.

 That is, it was confirmed that the transgenic animal of the present invention is useful for elucidating the mechanism for preventing and improving obesity of adiponectin under the administration of a high-calorie diet.

[0068] Although not shown, in the case of the mouse group having a blood adiponectin concentration of 3 to 5 times described in (2) above, it is not as wild as the group showing 5 to L0 times. It was confirmed that the increase in body weight was suppressed compared to the mold.

[0069] Therefore, the plasmid vector of the present invention is also useful for treatment of adiponectin deficiency, obesity 'diabetes' prevention of arteriosclerosis' and the like.

[0070] Example 2

(Confirmation of blood fat reduction effect by adiponectin under normal diet) For each group of transgenic mice and wild-type mice prepared as in Example 1.

The normal diet started to be applied after weaning when it became available. Each group had 8 animals. In each group, the experiment was conducted with males.

[0071] Fig. 6 shows the results of measurement of blood fat (neutral fat and cholesterol) after breeding these mice up to 20 weeks of age.

[0072] As shown in FIG. 6, it was proved that Tg mice were low in lipids in blood compared with wild-type mice under normal diet. By investigating the mechanism of action of this Tg mouse or adiponectin and developing its agonist, it can be a therapeutic agent for hyperlipidemia

'I have sex.

[0073] Example 3

 (Confirmation of the effect of adiponectin in reducing visceral fat and subcutaneous fat under high calorie high sucrose diets)

 [0074] A high-calorie high-sucrose diet of the same kind as in Example 1 was started to be given to each group of transgenic mice and wild-type mice prepared as in Example 1 from the 8th week after birth. There were 6 animals in each group. In each group, the experiment was conducted with males.

 [0075] Fig. 7 shows the results of measurement of visceral fat and subcutaneous fat after these mice were raised to 20 weeks of age after birth.

 [0076] As can be seen from FIG. 7, it was proved that Tg mice were not only those with visceral fat mass but also those with low subcutaneous fat mass compared to wild-type mice under the high calorie high sugar diet. By investigating the mechanism of action of this Tg mouse or adiponectin and developing its drug, it may be a therapeutic drug that reduces fat mass and improves metabolic syndrome (metabolic syndrome).

 [0077] Visceral fat (wild-type mouse n = 6, 9304.4 ± 1251.1 gZ mouse 100 g # 11 Tg mouse n = 6, 22 71.9 ± 571.2 gZ mouse 100 g)

 Subcutaneous fat (wild-type mouse n = 6, 6580.6 ± 3254.7gZ mouse 100g # 11 Tg mouse n = 6, 17 05.0 ± 326.0gZ mouse 100g)

 Significant differences were visceral fat, P = 0.0001, subcutaneous fat, P = 0.005.

[0078] Example 4 (Confirmation of life-prolonging effect of adiponectin under high calorie and Z or high sucrose diet)

[0079] A high-calorie high-sucrose diet of the same kind as in Example 1 was started to be given to each group of transgenic mice and wild-type mice prepared as in Example 1 from the 8th week after birth. There were 12 animals in each group. In each group, the experiment was conducted with males.

 [0080] Fig. 8 shows the results of measuring the lifespan of these mice.

 [0081] As can be seen from Fig. 8, in the high calorie high sucrose diet, wild type mice almost finished their life at 80 weeks of age. All the cases proved to be long-lived mice that survived at least 95 weeks of age. By investigating the mechanism of action of this Tg mouse or adiponectin on the lifespan, and inventing the agonist, it has the potential to become a drug that prolongs the lifespan.

 [0082] Example 5

 (Confirmation of the effect of adiponectin to suppress adipocyte expansion under high calorie and Z or high sucrose diet)

 [0083] (1) Inhibition of the expansion of subcutaneous fat cells with a high-calorie high-sugar diet

 Each group of transgenic mice and wild type mice prepared as in Example 1 began to receive a high calorie high sucrose diet from the 8th week of life. There were 6 animals in each group. In each group, the experiment was conducted with males.

 [0084] After these mice were raised to 20 weeks of age, subcutaneous adipose tissue of the whole mouse was collected under ether anesthesia. After measuring the weight, a part of it was fixed in formalin. The results of observing the size of subcutaneous fat are shown in FIGS. 9 and 10 (100-fold HE staining).

 [0085] Fig. 9) Wild mice (5) Subcutaneous adipose tissue

 (Fig. 10) # 11 Tg mouse subcutaneous adipose tissue

 [0086] As shown in Fig. 9-10, under high calorie high sucrose diet, fat cell size in subcutaneous adipose tissue is # 11 Tg mice compared to wild mice. Obviously small.

 [0087] (2) Effect of inhibiting the expansion of visceral fat cells with a high calorie high sucrose diet

In order to observe the visceral fat of the mice reared in (1), visceral adipose tissue present in the upper part of the mouse accessory testicle, around the kidney, and around the intestine was collected under ether anesthesia. After measuring the weight, a part of it was fixed in formalin. The result of observing the size of visceral fat Shown in Figures 11 and 12 (100x HE staining).

[0088] Fig. 11) Wild mice (5) Visceral adipose tissue

 (Figure 12) # 11 Tg mouse Visceral adipose tissue

[0089] As shown in Figs. 11-12, the fat cell size in the visceral adipose tissue is clearly higher in the # 11 Tg mouse than in the wild mouse under the high calorie high sugar diet feeding condition. small.

[0090] (3) Reference: Size of subcutaneous fat cells in normal diet

 For each group of transgenic mice and wild-type mice prepared as in Example 1.

I started to feed normal food from weaning. Each group had 8 animals. In each group, male experiments were performed.

 [0091] After raising these mice up to 16 weeks of age, subcutaneous adipose tissue of the whole mouse was collected under ether anesthesia. After measuring the weight, a part of it was fixed in formalin. The results of observing the size of subdermal fat are shown in FIGS. 13 and 14 (100-fold HE staining).

 [0092] Fig. 13) Wild mice (5) Subcutaneous adipose tissue

 (Fig. 14) # 11 Tg mouse subcutaneous adipose tissue

 [0093] As can be seen from Figs. 13 and 14, the size of adipocytes in subcutaneous adipose tissue is comparable to that of wild mice under the conditions of normal feeding.

 [0094] (4) Reference: Visceral fat cell size in normal diet

 In order to observe the visceral fat of the mice bred in (3), visceral adipose tissue present in the upper part of the mouse accessory testicle, around the kidney, and around the intestine was collected under ether anesthesia. After measuring the weight, a part of it was fixed in formalin.

 [0095] The results of observing the size of visceral fat are shown in Figs. 15 and 16 (100-fold HE staining).

 [0096] Fig. 15) Wild mice (5) Visceral adipose tissue

 (Fig. 16) # 11 Tg mouse Visceral adipose tissue

 [0097] As can be seen from Figs. 15 and 16, the size of adipocytes in visceral fat thread and tissue is comparable or slightly higher in the # 11 Tg mice than in the wild mice under normal feeding conditions. small.

 Industrial applicability

[0098] A vector containing the adiponectin gene of the present invention can be used to produce a disease model animal. Transgenic animals using this vector are also adiponectin It is useful for examining the role of obesity in obesity, and is useful for screening for a therapeutic agent for hyperadiponectinemia, a preventive agent for anorexia nervosa, and an anti-obesity agent.

Furthermore, the plasmid vector of the present invention is a vector capable of producing a large amount of the target product in the liver.

And the pharmaceutical of this invention can prevent the increase in the amount of fat resulting from high calories and Z or high sucrose, etc., and can prevent or treat weight gain.

Claims

The scope of the claims  [1] A plasmid vector containing a liver-specific expression promoter and an adiponectin gene [2] A plasmid vector containing a promoter and adiponectin gene derived from a gene of a protein specifically produced in the liver. [3] Human serum amyloid A plasmid vector containing a P component promoter and an adiponectin gene.  [4] The plasmid vector according to any one of claims 1 to 3, wherein the adiponectin is human adiponectin.  [5] A plasmid vector containing a liver-specific expression promoter and a target gene.  [6] A transgenic animal into which the promoter according to any one of claims 1 to 5 and the adiponectin gene are introduced. [7] A transgenic rodent animal into which the promoter according to any one of claims 1 to 5 and the adiponectin gene are introduced. [8] A method for screening a therapeutic agent for hyperadiponectinemia, using a transgenic animal into which the promoter according to any one of claims 1 to 4 and the adiponectin gene are introduced.  [9] A method for screening an anti-obesity drug, comprising the following steps (1) to (3):  (1) A step of analyzing the gene expression state of a transgenic animal into which the promoter and adiponectin gene according to any one of claims 1 to 4 have been introduced.  (2) A step of selecting a substance exhibiting a certain behavior caused by adiponectin expression based on the result of analysis!  (3) A step of selecting one of the following (i) to (iv) as an anti-obesity drug.  (i) Substance selected in (2)  (ii) Substance capable of controlling in vivo expression of (i)  (iii) Substances that have structural or functional similarities to (i) or (ii)  (iv) a gene capable of expressing any of (i) to (iii) [10] Adiponectin gene or fat content characterized by containing adiponectin Preventive or therapeutic agent for increase and z or weight gain. [11] A preventive or therapeutic agent for fat increase and Z or weight gain caused by a high calorie and Z or high sucrose diet, comprising an adiponectin gene or adiponectin.  [12] The preventive or therapeutic agent according to claim 10 or 11, comprising the plasmid vector according to any one of claims 1 to 3.