Model for promoting mouse obesity through OGDHL, modeling method and application

Abstract

The invention discloses a model for promoting mouse obesity through OGDHL. The model is obtained by inducing systematic high expression of mouse OGDHL through an adenovirus overexpression technology. The invention further discloses a modeling method and application of the model for promoting mouse obesity through OGDHL, the model building process is simple and convenient, the success rate is high, the result is stable and reliable, the time for feeding experimental animals is saved, the consequence of model building failure is avoided, and the method is suitable for popularization and application. The animal model established by the invention has important practical application value in research on etiology, pathology, drug screening, clinical diagnosis and treatment and the like of atherosclerosis, and can be widely popularized.

Description

Technical Field

[0001] The present invention belongs to the field of basic medical technology, and specifically relates to a mouse obesity model promoted by OGDHL, and also relates to a modeling method and application of the mouse obesity model promoted by OGDHL. Background Art

[0002] Adipose tissue contains a large number of fat cells and is one of the most important metabolic organs in the human body. Its morphological characteristics include single or aggregated spherical or hemispherical fat masses, surrounded by a certain number of blood vessels, nerves and connective tissue support. Adipose tissue plays an important regulatory role in the human body, and changes in its morphology and function are closely related to obesity, metabolic syndrome, cardiovascular disease and other diseases.

[0003] Oxoglutarate dehydrogenase-like (OGDHL) is the major rate-limiting subunit of OGDHC. Downregulation of OGDHL inhibits the key mitochondrial multienzyme OGDHC activity, leading to αKG accumulation, reversing the oxidative decarboxylation of isocitrate dehydrogenase (IDH) in the TCA cycle for glucose and glutamate degradation, affecting cell cycle arrest, apoptosis, energy metabolism, and leading to citrate accumulation and lipid synthesis.

[0004] However, there is no existing technology to establish an animal model to analyze the effect of OGDHL overexpression on lipid metabolism under high-fat diet (HFD) conditions. At the same time, in the existing technology, it usually takes 12 weeks to intervene in experimental animals in the construction of experimental models of small and medium-sized animals, which consumes a lot of time and manpower. Summary of the invention

[0005] The first object of the present invention is to provide a mouse obesity model promoted by OGDHL, which is helpful for studying the effect of OGDHL overexpression on lipid metabolism under high-fat diet conditions and shortens the time for establishing the animal model.

[0006] The second object of the present invention is to provide a method for modeling a mouse obesity model promoted by OGDHL.

[0007] The third object of the present invention is to provide an application of promoting mouse obesity model by OGDHL The first technical solution adopted by the present invention is: a method for modeling a mouse obesity model promoted by OGDHL, wherein the animal model is obtained by inducing systemic high expression of OGDHL in mice using adenovirus overexpression technology.

[0008] The second technical solution adopted by the present invention is: a method for modeling a mouse obesity model by promoting OGDHL, which is specifically carried out according to the following steps: Step 1, construction of adenovirus OGDHL vector and cell infection; Step 2: The adenovirus OGDHL vector obtained in step 1 was injected into mice through the tail vein. After injection, the mice were fed with HFD for 6 weeks to obtain an animal model.

[0009] Furthermore, the specific steps of constructing the adenovirus expression vector in step 1 are as follows: Step 1.1, using Hind Ⅲ and Sal Ⅰ to double-digest mouse OGDHL cDNA plasmid, electroeluting to recover OGDHL cDNA fragment, the nucleotide sequence of cDNA fragment is shown in SEQ ID NO:1, using Hind Ⅲ and Sal Ⅰ to double-digest pCMV-OGDHL plasmid, electroeluting to recover pAdTrack-CMV Hind Ⅲ and Sal Ⅰ double-digested linear fragment, then ligating OGDHL cDNA fragment and pAdTrack-CMV Hind Ⅲ and Sal Ⅰ double-digested linear fragment by T4 DNA ligase, and transforming the ligated product into E. coli In DH5α competent cells, the pAdTrack-OGDHL recombinant plasmid was extracted by alkaline lysis after culture; Step 1.2: The pAdTrack-OGDHL recombinant plasmid obtained in step 1.1 was digested with PemⅠ, and the linear fragment was recovered by electroelution. The pAdTrack-OGDHL PemⅠ linear fragment was electroporated into the pAdEasy-1 containing E. coli BJ5183 electrocompetent cells, the transformation products were cultured and the pAdEasy-OGDHL recombinant plasmid was extracted by alkaline lysis method; Step 1.3, using Pac I to digest the pAdEasy-OGDHL obtained in step 1.2 to obtain a Pac I linearized recombinant plasmid pAdEasy-OGDHL fragment, using Pac I enzyme to linearize the recombinant plasmid pAdEasy-OGDHL fragment to transfect HEK293 cells, culturing the cells for 7 to 10 days after transfection, observing cell growth and green fluorescent protein expression every day, and when the green fluorescent protein expression in the cells is "comet-shaped" and cytopathic effect occurs, collecting the HEK293 cells and vortexing to lyse the cells, centrifuging after lysis, collecting the supernatant, which is the Ad-OGDHL virus solution, and storing the Ad-OGDHL virus solution at -80°C for later use; Step 1.4, on the day of infection, the Ad-OGDHL virus solution obtained in step 1.3 was added to 1.5 mL of DEME complete culture medium and cultured for 90 minutes, and then 3.5 mL of DMEM complete culture medium was added for culture. When the cells showed obvious cytopathic effect and were accompanied by shedding, the virus was collected and the infection with Ad-OGDHL virus solution was repeated 4 times. The total amount of the obtained virus was collected to obtain the adenovirus expression vector Ad-OGDHL of the present invention.

[0010] Furthermore, the HEK293 cells were cultured in a culture dish one day before transfection in step 1.3 to ensure that the cell density of the cells during transfection was 30%-50%.

[0011] Furthermore, one day before infection in step 1.4, HEK293 cells were cultured in a culture dish to ensure that the cell density was 75%-85% during infection.

[0012] The third technical solution adopted by the present invention is: application of the OGDHL-promoting mouse obesity model in promoting mouse obesity.

[0013] The fourth technical solution adopted by the present invention is: using OGDHL to promote the use of mouse obesity model in promoting HFD-induced lipid accumulation.

[0014] The fifth technical solution adopted by the present invention is: using OGDHL to promote the use of mouse obesity model in inducing the production of mouse plasma adipokines.

[0015] The beneficial effects of the present invention are: 1. The model of the present invention promotes weight gain in mice through overexpression of OGDHL In order to explore the effect of OGDHL on HFD-induced BAT lipid accumulation, the present invention established an animal model by tail vein injection. Ad-OGDHL and Ad-GFP virus particles were injected respectively, and the weight changes were observed. The results showed that the weight of the Ad-OGDHL group increased significantly compared with the control group. Western blotting confirmed that the expression level of Ad-OGDHL in BAT increased by 1.5 times.

[0016] 2. The model of the present invention promotes lipid accumulation in mice through overexpression of OGDHL In order to determine the cause of the weight gain in mice, the present invention separated the epididymal adipose tissue (eWAT), inguinal adipose tissue (iWAT) and BAT of mice, and the results showed that the weight of eWAT, iWAT and BAT in the Ad-OGDHL group was significantly increased compared with the GFP control group. However, after overexpression of OGDHL, the weight of the spleen and kidney decreased significantly. The liver weight did not change between the GFP group and the OGDHL group. The results of H&E staining of pathological sections of eWAT, iWAT and BAT showed that after administration of Ad-OGDHL, the fat droplets were larger than those in the control group.

[0017] 3. The model of the present invention affects the production of plasma adipokines in HFD-induced mice through overexpression of OGDHL In order to explore the molecular mechanism of OGDHL overexpression on adipocyte-related hormone secretion, the present invention uses Western blotting to detect the expression of adiponectin, leptin, FGF21, and FABP4 proteins in plasma. It is worth noting that compared with the GFP group, the expression of plasma FGF21 protein was significantly reduced and the expression of plasma adiponectin protein was significantly enhanced after Ad-OGDHL administration. However, compared with the control group, the expression of plasma leptin and FABP4 proteins in Ad-OGDHL did not change. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the process of the present invention.

[0019] Figure 2 This is a schematic diagram of the weight change of mice injected with Ad-OGDHL or Ad-GFP virus particles in Example 2 of the present invention; Figure 3 This is a schematic diagram of OGDHL promoting HFD-induced lipid accumulation in the present invention; Figure 4 This is a diagram showing the morphology of fat cells in the adipose tissue of the present invention; Figure 5 This is a diagram showing the identification of plasma adipokines in HFD-induced mice of the present invention. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] The present invention provides a mouse obesity model promoted by OGDHL, wherein the animal model is obtained by inducing systemic high expression of OGDHL in mice using adenovirus overexpression technology.

[0022] like Figure 1 As shown, please follow the steps below: Step 1, construction of adenovirus OGDHL vector and cell infection; Step 1.1, using Hind Ⅲ and Sal Ⅰ to double-digest mouse OGDHL cDNA plasmid, electroeluting to recover OGDHL cDNA fragment, the nucleotide sequence of cDNA fragment is shown in SEQ ID NO:1, using Hind Ⅲ and Sal Ⅰ to double-digest pCMV-OGDHL plasmid, electroeluting to recover pAdTrack-CMV Hind Ⅲ and Sal Ⅰ double-digested linear fragment, then ligating OGDHL cDNA fragment and pAdTrack-CMV Hind Ⅲ and Sal Ⅰ double-digested linear fragment by T4 DNA ligase, and transforming the ligated product into E. coli In DH5α competent cells, the pAdTrack-OGDHL recombinant plasmid was extracted by alkaline lysis after culture; Step 1.2: The pAdTrack-OGDHL recombinant plasmid obtained in step 1.1 was digested with PemⅠ, and the linear fragment was recovered by electroelution. The pAdTrack-OGDHL PemⅠ linear fragment was electroporated into the pAdEasy-1 containing E. coli BJ5183 electrocompetent cells, the transformation products were cultured and the pAdEasy-OGDHL recombinant plasmid was extracted by alkaline lysis method; Step 1.3, using Pac I to digest the pAdEasy-OGDHL obtained in step 1.2 to obtain a Pac I linearized recombinant plasmid pAdEasy-OGDHL fragment, using Pac I enzyme to linearize the recombinant plasmid pAdEasy-OGDHL fragment to transfect HEK293 cells, culturing the cells for 7 to 10 days after transfection, observing cell growth and green fluorescent protein expression every day, and when the green fluorescent protein expression in the cells is "comet-shaped" and cytopathic effect occurs, collecting the HEK293 cells and vortexing to lyse the cells, centrifuging after lysis, collecting the supernatant, which is the Ad-OGDHL virus solution, and storing the Ad-OGDHL virus solution at -80°C for later use; One day before transfection in step 1.3, place HEK293 cells in a culture dish and culture them to ensure that the cell density of the cells is 30%-50% during transfection; Step 1.4: On the day of infection, the Ad-OGDHL virus solution obtained in step 1.3 was added to 1.5 mL of DEME complete culture medium and cultured for 90 min, and then 3.5 mL of DMEM complete culture medium was added for culture. When the cells showed obvious cytopathic effect and were accompanied by shedding, the virus was collected and the cells were repeatedly infected with the Ad-OGDHL virus solution 4 times. The total amount of the obtained virus was collected to obtain the adenovirus expression vector Ad-OGDHL of the present invention. One day before infection in step 1.4, place HEK293 cells in a culture dish and culture them to ensure that the cell density is 75%-85% during infection; Step 2: The adenovirus OGDHL vector obtained in step 1 was injected into mice through the tail vein. After injection, the mice were fed with HFD for 6 weeks to obtain an animal model.

[0023] The present invention also provides the use of the OGDHL-promoting mouse obesity model in promoting mouse obesity.

[0024] The present invention also provides the use of OGDHL to promote the mouse obesity model in promoting HFD-induced lipid accumulation.

[0025] The present invention also provides the use of OGDHL to promote the mouse obesity model in inducing the production of mouse plasma adipokines.

[0026] The present invention will be further described below by way of examples and drawings.

[0027] Example 1 The method for modeling mouse obesity by promoting OGDHL includes the following steps: Step 1: Selection of experimental animals; Step 2, construction of adenovirus OGDHL vector and cell infection; Step 3: Adipose tissue morphology analysis; Step 4: Protein extraction and Western blotting analysis.

[0028] Step 1 is as follows: male mice, 8 weeks old, weighing 20-22 grams, were selected as experimental animals and purchased from the Experimental Animal Center of Xi'an Jiaotong University. The experimental animals were kept in clean, specific pathogen-free conditions. The experimental animal licenses used were: SCXK (Shaanxi Huaxi Pharmaceutical Co., Ltd.); 2007-2005), SYXK (Shaanxi Huaxi Pharmaceutical Co., Ltd.); 2003-0026); Step 2 specifically includes the following steps: Step 2.1, the selected experimental animals were randomly divided into two groups, Ad-GFP and Ad-OGDHL, with 6 animals in each group, and 1×1011 Ad-OGDHL and Ad-GFP virus particles were used as controls. The virus particles were injected into the tail vein of mice once every three weeks for a total of five times. After the injection of adenovirus, the mice were fed with HFD; Step 2.2: After constructing the recombinant adenovirus vector encoding OGDHL (Ad-OGDHL), the OGDHL cDNA was subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid was transformed into BJ5183 competent cells, the recombinant adenovirus was packaged and amplified in 293 cells, and after purification, the virus titer was detected by TCID50.

[0029] Step 3 specifically included: the adipose tissue of the experimental animals was removed and fixed in 10% formalin buffer, the fixed specimens were processed into frozen blocks, sectioned according to standard procedures, and stained with hematoxylin-eosin (H&E), and the sections were quantitatively sliced ​​and photographed under a microscope equipped with a digital camera (Nikon, Tokyo, Japan). The diameter and area of ​​adipocytes were measured using Image J software. The results of H&E staining of pathological sections of eWAT, iWAT, and BAT showed that after the administration of Ad-OGDHL, the lipid droplets were larger than those in the control group.

[0030] Step 4 specifically includes: the adipose tissue and liver of the experimental animals are homogenized in a refrigerated lysis buffer containing 1% protease inhibitor and phosphorylase inhibitor, and then clarified by centrifugation. The protein content is determined using a BCA protein concentration assay kit. After protein denaturation, the expression of the target protein is detected by western blotting. Briefly, 30 μg of protein samples were subjected to 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA), and stained with antibodies against OGDHL 1:1000 (Cat. No. ABIN2781696, Antibodies), adiponectin 1:1000(Cat. No. AF1119, R&D), fibroblast growth factor 21(FGF21) 1:1000 (Cat. No. AF3057, R&D), fatty acid binding protein 4 (FABP4) 1:1000 (Cat. No. AF1443, R&D), uncoupled protein 1 (UCP-1) 1:1000 (Cat. No. MAB6158, R&D), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) 1:2000 (Cat. No. CY6630, Abways), adiposetriglyceride lipase (ATGL) 1:2000 (Cat. No. T58408S, Abmart), perilipin 1 1:5000 (Cat. No.CY6765, Abways), perilipin 2 1:2000 (Cat. No. CY6811, Abways), leptin 1:1000 (Cat. No. AF498, R&D), β-actin 1:1000 (Cat. No.SC69879, Santa Cruz). Incubate overnight at 4°C according to the manufacturer's instructions. After washing with tris-buffered saline-tween-20, sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature.After washing with tris-buffered saline, the antigen-antibody complex was visualized using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA). β-Actin was used as a loading control.

[0031] Example 2 Through the modeling method and application of OGDHL promoting obesity in mice, adenovirus overexpression technology was used to analyze the effect of OGDHL overexpression on lipid metabolism under high-fat diet (HFD) conditions. Animals were randomly divided into two groups of adenovirus-green fluorescent protein (Ad-GFP) or Ad-OGDHL, with 1×1011 Ad-OGDHL or Ad-GFP virus particles as controls. Ad-GFP or Ad-OGDHL was injected into the tail vein once every three weeks, for a total of five injections. After adenovirus injection, the mice were fed HFD. The weight changes were observed. Samples were collected, quantitatively sliced ​​under a microscope, and photographed under a microscope equipped with a digital camera (Nikon, Tokyo, Japan). Image J software was used to measure the diameter and area of ​​adipocytes. Western blotting was used to detect the molecular mechanism of OGDHL overexpression in plasma on the secretion of adipocyte-related hormones, and finally statistical analysis was performed. The experimental process of the present invention has uniform sampling and high credibility of the results. Specifically comprising the following steps: Step 1: Selection of experimental animals Male, 8 weeks old, weighing 20-22 g, 6 animals per group, were purchased from the Experimental Animal Center of Xi'an Jiaotong University. The experimental animals were housed under clean, specific pathogen-free conditions, and the experimental animal licenses used were: SCXK (Shaanxi Huaxi Pharmaceutical Co., Ltd.; 2007-2005), SYXK (Shaanxi Huaxi Pharmaceutical Co., Ltd.; 2003-0026). Each cage could accommodate up to three animals. The animals were housed at 25 °C and 50-80% relative humidity under normal light cycles.

[0032] Step 2: Construction of adenovirus OGDHL vector and cell infection Animals were randomly divided into two groups: adenovirus-green fluorescent protein (Ad-GFP) or Ad-OGDHL, with 1×1011 Ad-OGDHL or Ad-GFP virus particles as control. These particles were injected into the tail vein of mice. Ad-GFP or Ad-OGDHL was injected once every three weeks for a total of five injections. After adenovirus injection, mice were fed HFD.

[0033] Mice were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg / kg body weight). Animal experiments were performed in strict accordance with the Animal Experiment Guide of Xi'an Medical University, which was adapted from the Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, 2011). All animal experiments were approved by the Experimental Animal Management Committee of Xi'an Medical University.

[0034] like Figure 1 As shown, Ad-OGDHL or Ad-GFP virus particles were injected respectively, and the weight changes were observed. The results showed that the weight of the Ad-OGDHL group increased significantly compared with the control group. Western blotting confirmed that the expression level of Ad-OGDHL in BAT increased by 1.5 times.

[0035] A recombinant adenovirus vector encoding OGDHL (Ad-OGDHL) was constructed. The OGDHL cDNA was subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid was transformed into BJ5183 competent cells. The recombinant adenovirus was packaged and amplified in 293 cells. After purification, the virus titer was detected by TCID50. An empty adenovirus vector (Ad-GFP) was constructed as a control.

[0036] Step 3: Adipose tissue morphology analysis Adipose tissue was excised and fixed in 10% formalin buffer. Fixed specimens were processed into frozen blocks, sectioned according to standard procedures, and stained with hematoxylin-eosin (H&E). Microscopic quantification Sections were photographed under a microscope equipped with a digital camera (Nikon, Tokyo, Japan). The diameter and area of ​​adipocytes were measured using Image J software.

[0037] In order to determine the cause of the weight gain in mice, the present invention separated epididymal adipose tissue (eWAT), inguinal adipose tissue (iWAT) and BAT of mice. The results showed that the weight of eWAT, iWAT and BAT in the Ad-OGDHL group increased significantly compared with the GFP control group. Figure 3 However, after overexpression of OGDHL, the weight of spleen and kidney decreased significantly. However, the weight of liver did not change between GFP group and OGDHL group. The results of H&E staining of eWAT, iWAT and BAT pathological sections showed that after Ad-OGDHL administration, lipid droplets were larger than those in the control group.

[0038] Example 3 Mouse obesity test Comparative analysis of mouse weight: The present invention uses the method of tail vein injection to establish an animal model. Ad-OGDHL or Ad-GFP virus particles were injected respectively, and the weight changes were observed. The results showed that the weight of the Ad-OGDHL group increased significantly compared with the control group. Western blotting confirmed that the expression level of Ad-OGDHL in BAT increased by 1.5 times.

[0039] Adipose tissue morphology analysis: Adipose tissue was excised and fixed in 10% formalin buffer. Fixed specimens were processed into frozen blocks, sectioned according to standard procedures, and stained with hematoxylin-eosin (H&E). Microscopic quantification Sections were photographed under a microscope equipped with a digital camera (Nikon, Tokyo, Japan). The diameter and area of ​​adipocytes were measured using Image J software, as shown in Figure 2. Figure 4 shown.

[0040] Overexpression of OGDHL promotes lipid accumulation in mice The present invention isolated epididymal adipose tissue (eWAT), inguinal adipose tissue (iWAT) and BAT of mice, and the results showed that the weight of eWAT, iWAT and BAT in the Ad-OGDHL group increased significantly compared with the GFP control group. However, after overexpression of OGDHL, the weight of the spleen and kidney decreased significantly. The weight of the liver did not change between the GFP group and the OGDHL group. The results of H&E staining of pathological sections of eWAT, iWAT and BAT showed that after administration of Ad-OGDHL, the lipid droplets were larger than those in the control group.

[0041] Effects of OGDHL overexpression on plasma adipokine production in HFD-induced mice The present invention uses Western blotting to detect the expression of adiponectin, leptin, FGF21, and FABP4 proteins in plasma. It is worth noting that compared with the GFP group, the expression of plasma FGF21 protein was significantly reduced and the expression of plasma adiponectin protein was significantly enhanced after Ad-OGDHL administration. However, compared with the control group, the expression of plasma leptin and FABP4 proteins in Ad-OGDHL did not change.

[0042] Experimental process of OGDHL in mouse obesity In order to explore the effect of OGDHL on HFD-induced BAT lipid accumulation, the present invention uses the tail vein injection method to establish an animal model. Male, 8-week-old mice, weighing 20-22g. The animals were randomly divided into two groups: adenovirus-green fluorescent protein (Ad-GFP) or Ad-OGDHL, with 1×1011 Ad-OGDHL or Ad-GFP virus particles as control. These particles were injected into the tail vein of mice. Ad-GFP or Ad-OGDHL was injected once every three weeks, for a total of six injections. After injection of adenovirus, high-fat diet (HFD) was fed. The weight of mice was as follows Figure 2 As shown, protein expression of OGDHL in BAT was detected by Western blotting (β-Actin was loaded as a control). Quantitative western blot analysis (β-actin was loaded as a control). Data are expressed as mean ± SEM; N = 6 per group. *p<0.05, **p<0.01, ***p<0.001. Ad-OGDHL and Ad-GFP. Ad-GFP; BAT, brown adipose tissue; high-fat diet (HFD); OGDHL, member of the solute carrier family 25.

[0043] Overexpression of OGDHL promotes HFD-induced lipid accumulation In order to determine the cause of the weight gain in mice, the present invention isolated epididymal adipose tissue (eWAT), inguinal adipose tissue (iWAT) and BAT of mice. The results showed that the weight of eWAT, iWAT and BAT in the Ad-OGDHL group increased significantly compared with the GFP control group. Morphological comparison and weight statistics of iWAT, eWAT and BAT after the mice were sacrificed. The weight of spleen, kidney and liver was calculated after the mice were sacrificed. Data are expressed as mean ± SEM; N = 6 per group. * p < 0.05; Ad-OGDHL and Ad-GFP. iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue.

[0044] Effects of OGDHL overexpression on plasma adipokine production in HFD-induced mice To explore the molecular mechanism of OGDHL overexpression on adipocyte-related hormone secretion, such as Figure 5 As shown, the present invention uses Western blotting to detect the expression of adiponectin, leptin, FGF21, and FABP4 proteins in plasma. It is worth noting that compared with the GFP group, the expression of plasma FGF21 protein was significantly reduced and the expression of plasma adiponectin protein was significantly enhanced after Ad-OGDHL administration. However, compared with the control group, the expression of plasma leptin and FABP4 proteins in Ad-OGDHL did not change.

[0045] Sequence Listing Sequence Listing <110> Xi'an Medical College <120> OGDHL-promoted mouse obesity model and modeling method and application <141> 2024-10-21 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2193 <212> DNA <213> Artificial sequence <400> 1 atgagccagc tgaggctgct gccattccgg ctggggccac gggccaccaa gctcctggcc60 acacgggcta tcccggtgtt cagtgggtgc aggaggtctt ctggaccacc aacgaccatc120 ccgaggagca gaagcggagt cagctccagt tatgtggaag agatgtactt tgcctggttg180 gaaaacccac agagcgttca caagtcctgg gacagctttt ttcagagagc aagtaaggag240 gcctctgtgg gtcctgctca gccgcagctc ccagctgttc tccaagaaag caggacatca300 gtgtcaagct gcaccaagac cagcaagctg gtggaagacc acttggcagt ccagtccctg360 atccgggcct accagatccg aggccaccat gtggcccagc tggaccccct gggcatcctg420 gatgcagacc tagattcctt tgtgccctct gacttgatca caaccattga taaattggcc480 ttctatgacc tgcaggaggc tgatctggat aaggagttcc ggctgcccac cacgactttc540 atcggggcc cagagaacac actctctctt cgagagatca tacgtcgctt ggagagcacc600 tactgccagc atattggcct ggagttcatg tttattaatg acgtggaaca gtgccagtgg660 atcagacaga aatttgaaac tcctggcgtg atgcagttt ccgtcgagga gaagaggacc720 ctgctggcta gactggtgcg ctccatgagg tttgaagact tcctggccag gaagtggtct780 tcggagaagc ggtttggcct ggaaggctgc gaggtcatga ttcctgctct caagaccatt840 atcgacaagt ctagtgagat ggggattgag aacgtcatcc tggggatgcc acacaggggc900 aggctgaatg tgctggctaa cgtgatccgc aggaccctgg aacagatctt ctgtcagttt960 gatcccaagc tggaagcagc agatgagggc tctggtgatg tcaaatacca tctgggcatg1020 taccacgaga ggatcaaccg tgtcaccaac aggaacatca cattgtcact ggtagccaac1080 ccttcccatc tagaagctgt ggaccccgtg gtacaggga agacgaaggc agagcagttc1140 taccgtgggg atgcccaggg taggaaggtc atgtctatcc tggtccatgg ggatgccgcc1200 ttcgctggcc aggggtcgt ctatgagacc ttccatctca gtgacctgcc ttcttatacc1260 accaatggca cagtgcacgt tgtggtcaac aaccagatcg gcttcaccac agatccccga1 atggctcgct cctcccctta ccccactgat gtggcgcggg tggtcaatgc acccatcttc1380 catgtgaacg cagacgaccc agaggctgtg atctatgtgt gcagtgtggc agctgagtgg1440 aggaacacct tcaacaaaga tgttgttgta gacctggtct gttaccgccg gcgtggtcac1500 aatgagatgg acgagcccat gttcacgcag ccactcatgt acaagcagat ccacaagcag1 gtacccgtgc tgaagaagta tgcagacaag ctcatcgccg agggcaccgt cactctgcag1 gagtttgagg aggaaattgc caaatatgac cgtatctgtg aggaggccta cggcaggtcc1 aaggataaga agatcctgca fatheragcac tggctggact ccccctggcc tggcttcttc1 aatgtggatg gggagcccaa gagcatgaca tgccctacga caggcatccc tgaggagatg1800 ctgacccaca ttggcagtgt ggccagctct gtgcccctgg aggactttaa gatccacaca1 ggcctctctc gcatcctgcg tggccgtgca gacatgacaa agaagcgcac agtggactgg1920 gccctggcag agtacatggc ctttggttca ttgctgaagg agggcatcca cgtgcgactc1980 agcggccagg atgtggagag gggcacattc agccaccggc accatgttct tcatgaccag2040 gaagttgacc ggaggacgtg tgtcccgatg aatcatctgt ggcctgacca ggccccgtac2100 actgtgtgca atagctccct ctcagagtat ggagttctag ggtccagagc actcctcagc2160 aaggccagag aggttcctgc agatgagcaa tga2193。

Claims

1. Promoting mouse obesity model by OGDHL, characterized in that The mouse obesity model is obtained by inducing systemic high expression of OGDHL in mice using adenovirus overexpression technology.

2. The method for modeling a mouse obesity model by promoting OGDHL according to claim 1, characterized in that: Follow these steps: Step 1, construction of adenovirus OGDHL vector and cell infection; Step 2: The adenovirus OGDHL vector obtained in step 1 was injected into mice through the tail vein. After injection, the mice were fed with HFD for 6 weeks to obtain the mouse obesity model.

3. The method for modeling a mouse obesity model by promoting OGDHL according to claim 2, characterized in that: The specific construction steps of the adenovirus OGDHL vector in step 1 are as follows: Step 1.1, using Hind Ⅲ and Sal Ⅰ to double-digest mouse OGDHL cDNA plasmid, electroeluting to recover OGDHL cDNA fragment, the nucleotide sequence of the cDNA fragment is shown in SEQ ID NO: 1, using Hind Ⅲ and Sal Ⅰ to double-digest pCMV-OGDHL plasmid, electroeluting to recover pAdTrack-CMV Hind Ⅲ and Sal Ⅰ double-digested linear fragment, then ligating the OGDHL cDNA fragment with the pAdTrack-CMV Hind Ⅲ and Sal Ⅰ double-digested linear fragment using T4 DNA ligase, and transforming the ligated product into E. coli In DH5α competent cells, the pAdTrack-OGDHL recombinant plasmid was extracted by alkaline lysis after culture; Step 1.2: The pAdTrack-OGDHL recombinant plasmid obtained in step 1.1 was digested with PemⅠ, and the linear fragment was recovered by electroelution. The pAdTrack-OGDHL PemⅠ linear fragment was electroporated into the pAdEasy-1 containing E. coli BJ5183 electrocompetent cells, the transformation products were cultured and the pAdEasy-OGDHL recombinant plasmid was extracted by alkaline lysis method; Step 1.3, using PacⅠ to digest the pAdEasy-OGDHL obtained in step 1.2 to obtain a Pac I linearized recombinant plasmid pAdEasy-OGDHL fragment, using PacⅠ enzyme to linearize the recombinant plasmid pAdEasy-OGDHL fragment to transfect HEK293 cells, culturing the cells for 7 to 10 days after transfection, observing cell growth and green fluorescent protein expression every day, and when the green fluorescent protein expression in the cells is "comet"-shaped and cytopathic effect occurs, collecting the HEK293 cells and vortexing to lyse the cells, centrifuging after lysis, collecting the supernatant, which is the Ad-OGDHL virus solution, and storing the Ad-OGDHL virus solution at -80°C for later use; Step 1.4, on the day of infection, the Ad-OGDHL virus solution obtained in step 1.3 was added to 1.5 mL of DEME complete culture medium and cultured for 90 minutes, and then 3.5 mL of DMEM complete culture medium was added for culture. When the cells showed obvious cytopathic effect and were accompanied by shedding, the virus was collected and the infection with Ad-OGDHL virus solution was repeated 4 times. The total amount of the obtained virus was collected to obtain the adenovirus expression vector Ad-OGDHL of the present invention.

4. The method for modeling a mouse obesity model by promoting OGDHL according to claim 3, characterized in that: In step 1.3, HEK293 cells were cultured in a culture dish one day before transfection to ensure that the cell density of the cells was 30%-50% during transfection.

5. The method for modeling a mouse obesity model by promoting OGDHL according to claim 3, characterized in that: One day before infection in step 1.4, HEK293 cells were cultured in a culture dish to ensure that the cell density was 75%-85% during infection.

6. Use of the mouse obesity model promoted by OGDHL according to claim 1 in promoting mouse obesity.

7. Use of the mouse obesity model promoted by OGDHL according to claim 1 in promoting HFD-induced lipid accumulation.

8. Use of the mouse obesity model promoted by OGDHL according to claim 1 in inducing the production of plasma adipokines in mice.

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