Application of antifreeze protein in weight losing and blood sugar reducing

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of antifreeze protein in weight losing and blood sugar reducing. The NPAFP transgenic mouse model is successfully constructed by utilizing a genetic engineering technology, and the potential application value of the NPAFP protein in weight loss and blood sugar reduction is verified. Furthermore, based on the model, it is found that the NPAFP protein has remarkable effects in the aspects of reducing fat accumulation and improving the blood glucose level by adjusting brown fat metabolism and the liver lipid droplet content. Based on compound development or protein engineering modification of the NPAFP protein, novel drugs and strategies can be provided for treatment of obesity and diabetes mellitus. Meanwhile, the molecular mechanism of the NPAFP protein is further explored by combining a multi-omics technology, and clinical transformation and application of the NPAFP protein are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of an antifreeze protein in weight loss and blood sugar reduction. Background Art

[0002] As people's living standards improve, their dietary structure has also begun to change. Irregular eating habits have led to an increasing incidence of metabolic syndrome (MS) every year, and there is a trend of younger people. After the development of metabolic syndrome, it can cause a variety of harms to the human body, such as obesity, insulin resistance, hyperinsulinemia, diabetes, coronary heart disease, hypertension, stroke, and even certain cancers, including pancreatic cancer, hepatobiliary cancer, and colon cancer. Among them, weight loss and blood sugar reduction are important topics in today's metabolic disease research. Obesity is a controllable risk for many diseases, especially visceral adipose tissue can regulate proinflammatory cytokines (such as interleukin-6), leading to inflammation, oxidative stress and insulin resistance, thereby increasing the risk of chronic diseases (Thomas DM, Brdau C, Bosy-Wesiphal A, et al. Relationships between body roundness with body fat and viseeral adipose tissue e-menging from a new geometrical model [J]. Obesity (Silver Spring), 2013, 21 (11): 2264-2271.). The main characteristics of T2DM are hyperglycemia caused by insulin resistance and pancreatic β-cell dysfunction, as well as persistent low-grade inflammation in peripheral tissues. Obesity, unhealthy eating habits, lack of exercise, and a variety of pathophysiological disorders caused by environmental and genetic factors can cause impaired glucose homeostasis in T2DM (DeFronzo RA, Ferrannini E, Groop L, et al. Type 2diabetes mellitus. Nat Rev Dis Primers. 2015 Jul 23;1:15019.). Studies have shown that the prevalence of T2DM increases proportionally with weight gain, with BMI increasing from a normal 25 kg / m 2 Increase to 30 kg / m 2, the risk of death associated with T2DM more than doubles (Magkos F, Hjorth MF, Astrup A. Diet and exercise in the prevention and treatment of type 2 diabetes mellitus. Nat Rev Endocrinol. 2020 Oct;16(10):545-555.). In addition, excessive carbohydrate intake can lead to insulin resistance, which may advance the onset of T2DM in people who are genetically susceptible to it. In patients with T2DM and obesity, a weight loss of about 15 kg can relieve the condition of 80% of T2DM patients (Jao CL, Hung CC, Tung YS, et al. The development of bioactive peptides from dietary proteins as a dipeptidyl peptidase IV inhibitor for the management of type 2 diabetes. Biomedicine (Taipei). 2015 Sep;5(3):14.).

[0003] Current treatments include lifestyle intervention, drug therapy, and metabolic surgery. The 2017 AACE / ACE consensus statement newly proposed that weight loss should be a long-term goal. For patients with prediabetes and type 2 diabetes who are already overweight or obese, behavioral intervention and weight loss drugs should be used when necessary, emphasizing the importance of weight loss in disease management. Weight loss can reduce insulin resistance, increase blood sugar-lowering effects, and also improve lipid metabolism and blood pressure. The 2017 AACE / ACE consensus statement first recommends lifestyle intervention, and weight loss drugs can be added when necessary. Weight loss drugs can be used for all people with a BMI ≥ 27 kg / m 2 Patients with complications or BMI ≥ 30 kg / m 2Patients with diabetes mellitus (Garber AJ, Handelsman Y, Grunberger G, et al. CONSENSUS STATEMENT BY THE AMERICAN ASSOCIATION OF CLINICAL ENDOCRINOLOGISTS AND AMERICAN COLLEGE OF ENDOCRINOLOGY ON THE COMPREHENSIVE TYPE 2 DIABETES MANAGEMENT ALGORITHM -2020 EXECUTIVE SUMMARY. Endocr Pract. 2020 Jan;26(1):107-139.). Studies have shown that the mechanism of action of weight loss and glucose-lowering compounds involves multiple biological pathways, which mainly focus on the regulation of fat metabolism, regulation of glucose metabolism, and the balance of intestinal flora. Naturally derived compounds, such as polyphenols and flavonoids, have been widely studied due to their low toxicity and multi-target effects. For example, resveratrol and green tea polyphenols can promote lipolysis and enhance insulin sensitivity by activating the AMPK signaling pathway; quercetin and astragaloside IV can significantly improve insulin resistance and lipid metabolism by regulating the expression of metabolism-related genes. In addition, synthetic compounds, such as the GLP-1 receptor agonist semaglutide, and metabolites derived from intestinal flora (such as short-chain fatty acids), also show unique roles in lipolysis, glucose metabolism, and intestinal flora regulation. These studies reveal the complexity of metabolic regulation and suggest that multi-target strategies may be the key to future drug development. However, existing treatments have problems such as limited efficacy, obvious side effects, or limited scope of application. Therefore, developing more efficient and safe weight-loss and glucose-lowering compounds and exploring their mechanisms of action have become hot research directions.

[0004] Antifreeze proteins (AFPs) are a general term for ice-binding proteins of different structures that can improve the antifreeze ability of organisms. They were first discovered in Antarctic fish and defined as antifreeze agents. So far, AFPs have been found in different organisms, the main categories of which include: bony fish, insects, plants, fungi, nematodes, amphibians, bacteria and diatoms. There are huge differences in the structures of antifreeze proteins from different species, and no obvious homology has been found from the perspective of genetic analysis, only consistency in antifreeze function. The adsorption process of antifreeze proteins on the surface of ice crystals is specific. Different types of antifreeze proteins may bind to crystal faces with different crystal orientations on the surface of ice crystals. After binding to the surface of ice crystals, antifreeze proteins can lower the temperature of the ice crystal growth point. In this way, antifreeze proteins can limit the growth of ice crystals and inhibit the recrystallization of ice crystals (Graham LA, Hobbs RS, Flet alher GL, et al. Helical Antifreeze Proteins Have Independently Evolved in Fishes on Four Occasions[J]. Plos One, 2013, 8(12):81285.). The protective function of AFPs is due to their characteristics, including ice surface affinity, thermal hysteresis, ice recrystallization inhibition, and temporary binding of organisms to ice. Different characteristics produce different antifreeze effects. Although multiple antifreeze properties of AFPs can usually be detected, their antifreeze effect is mainly based on one property. Generally speaking, all these properties or effects originate from the ability of proteins to bind to ice (Xiang H, Yang X, Ke L, et al. The properties, biotechnologies, and applications of antifreeze proteins. Int J Biol Macromol. 2020 Jun 15;153:661-675.). In recent years, as the properties and mechanisms of AFP have become increasingly clear, its applications have become more and more extensive, including cryogenic medicine, agriculture, food industry, environmental regulation, and anti-icing materials.

[0005] Antifreeze proteins can be roughly divided into four categories according to their species sources: fish antifreeze proteins, insect antifreeze proteins, plant antifreeze proteins and microbial antifreeze proteins. Among them, antifreeze proteins isolated from fish are divided into six types according to the differences in the primary structure and configuration of the protein: antifreeze proteins I, II, III, IV (AFP I, AFP II, AFP III, AFP IV), antifreeze glycoproteins (AFGPs) and hyperactive antifreeze proteins (Hyperactive-AFP) (Guo Tinghe. Irreversible random adsorption inhibition model of thermal hysteresis activity of antifreeze proteins [D]. Inner Mongolia: Inner Mongolia University, 2022.). Among them, antifreeze protein III (AFP III) is one of the most widely used antifreeze protectants that has not been found to be toxic. There have been reports on the addition of AFP III to freeze ovaries, sperm and oocytes, but most of them tend to be developmental studies. There are huge differences in the structures of antifreeze proteins from different species, and no obvious homology has been found from the perspective of genetic analysis. Only the antifreeze function is consistent.

[0006] In the previous study (“Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict”, Deng et al. PNAS, 2010.), it was first discovered and proved that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein AFPIII through neofunctionalization in the process of escaping adaptive conflict, and named it LdAFPII. This protein evolved from SAS-B and has multiple repeated functional domains (such as LdAFPIII-1 with one repeated functional domain, LdAFPIII-4 with four repeated functional domains, LdAFPIII-12 with twelve repeated functional domains, etc.), each AFPIII domain contains 62 amino acids. The sequence of LdAFPII protein was published in the previous study (“Cloning and evolutionary analysis of the gene of the multimeric type III antifreeze protein of Antarctic eel Pout (Lycodichthys dearborni)”, Yu Jing et al., Acta Genetica Sinica, 2005, 32 (8): 789-794.). Summary of the invention

[0007] The present invention successfully constructed an NPAFP transgenic mouse model using genetic engineering technology, and found that the NPAFP protein has a significant effect in reducing fat accumulation and improving blood sugar levels by regulating brown fat metabolism and liver fat droplet content. Based on this, the present invention was completed.

[0008] In a first aspect, the present invention provides a drug composition for reducing weight and lowering blood sugar, wherein the active ingredient of the drug composition is NPAFP protein.

[0009] Further, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0010] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0011] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0012] Furthermore, the obesity is divided into primary obesity and secondary obesity.

[0013] Furthermore, the diabetes mellitus can be divided into insulin-dependent type 1 diabetes mellitus (T1DM) and non-insulin-dependent type 2 diabetes mellitus (T2DM).

[0014] In a second aspect, the present invention provides the use of NPAFP protein in preparing products for weight loss and blood sugar reduction.

[0015] Furthermore, the products include but are not limited to health products, slimming foods or functional drinks.

[0016] Further, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0017] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0018] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0019] In a third aspect, the present invention provides the use of NPAFP protein in the preparation of a drug for treating obesity and / or diabetes.

[0020] Further, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0021] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0022] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0023] Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug.

[0024] Furthermore, the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

[0025] Furthermore, the preparation may be one or more of a conventional preparation, a sustained-release preparation and / or a controlled-release preparation.

[0026] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0027] Furthermore, the drug can be administered via injection, cavity administration or respiratory tract administration.

[0028] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0029] Furthermore, the obesity is divided into primary obesity and secondary obesity.

[0030] Furthermore, the diabetes mellitus can be divided into insulin-dependent type 1 diabetes mellitus (T1DM) and non-insulin-dependent type 2 diabetes mellitus (T2DM).

[0031] In a fourth aspect, the present invention provides the use of NPAFP protein in constructing an animal model of obesity and / or diabetes.

[0032] Further, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0033] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0034] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0035] Furthermore, the animals in the animal model include rodents, large mammals and non-human primates.

[0036] Furthermore, the rodents include but are not limited to mice, rats and rabbits.

[0037] Furthermore, the large mammals include but are not limited to pigs and dogs.

[0038] Furthermore, the non-human primates include but are not limited to rhesus monkeys and cynomolgus monkeys.

[0039] Furthermore, the obesity is divided into primary obesity and secondary obesity.

[0040] Furthermore, the diabetes mellitus can be divided into insulin-dependent type 1 diabetes mellitus (T1DM) and non-insulin-dependent type 2 diabetes mellitus (T2DM).

[0041] In a fifth aspect, the present invention provides a method for constructing an animal model of obesity and / or diabetes, wherein the animal model is constructed by inserting the NPAFP gene into the mouse H11 gene site using gene editing technology to construct NPAFP transgenic mice expressing different numbers of AFP III functional domains.

[0042] Further, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0043] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0044] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0045] Beneficial Effects 1. The present invention successfully constructed an NPAFP transgenic mouse model using genetic engineering technology, and verified the potential application value of NPAFP protein in weight loss and blood sugar reduction.

[0046] 2. The present invention found that NPAFP protein has a significant effect in reducing fat accumulation and improving blood sugar levels by regulating brown fat metabolism and liver fat droplet content.

[0047] 3. The compound development or protein engineering based on the NPAFP protein of the present invention can provide new drugs and strategies for the treatment of obesity and diabetes. At the same time, further exploring the molecular mechanism of NPAFP protein in combination with multi-omics technology will help promote its clinical transformation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The following is a method for constructing NPAFP mice.

[0049] Figure 2NPAFP protein regulates brown fat metabolism.

[0050] Figure 3 NPAFP reduces the density of brown fat droplets.

[0051] Figure 4 NPAFP protein reduces hepatic lipid droplet accumulation.

[0052] Figure 5 NPAFP protein lowers blood glucose levels in mice. DETAILED DESCRIPTION

[0053] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0055] The NPAFP (NeuroProtect AFP) of the present invention is antifreeze protein III. The NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0056] Each AFPIII domain contains 62 amino acids. The present invention uses alphafold3 to predict and optimize the protein structure, changing the amino acids at positions 36 and 37 from KL to RI, and the amino acid at position 50 from D to E to increase the flexibility of the protein structure. In order to explore its application in neurological diseases, it is named NeuroProtection AFPIII (such as NPAFP1, NPAFP4, NPAFP12, etc.).

[0057] The nucleotide sequence of the NPAFP1 protein of the present invention is shown in SEQ ID NO.1: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA The nucleotide sequence of the NPAFP2 protein described in the present invention is as shown in SEQ ID NO.2: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA The nucleotide sequence of the NPAFP3 protein described in the present invention is as shown in SEQ ID NO.3: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAA The nucleotide sequence of the NPAFP4 protein described in the present invention is as shown in SEQ ID NO.4: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA The nucleotide sequence of the NPAFP12 protein described in the present invention is shown in SEQ ID NO.5: Example 1 Animal Model Construction In order to explore the regulatory effect of NPAFP protein on fat and blood sugar in vivo, NPAFP transgenic mice containing different AFPIII domains were constructed ( Figure 1 ). A plasmid (Donor vector) expressing NPAFP protein was constructed using the CAG promoter. First, the CRISPR / Cas9 system and Donor vector samples were microinjected into the fertilized eggs of mice with a C57BL / 6JGpt background. The NPAFP expression plasmid was inserted into the H11 site of the C57BL / 6 mouse by homologous recombination technology; the fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice, and they were allowed to become pregnant and give birth. The genomic DNA of the F0 generation pups born by the recipient mice was extracted for PCR and sequencing identification on day 5-7 to confirm the genotype. After the positive F0 generation mice reached sexual maturity, they were mated with wild-type background mice, and the genomic DNA of the F1 generation mice born was extracted for PCR and sequencing identification on day 5-7 to confirm the genotype. The F1 generation mice were further mated and bred with WT mice, and the offspring were genotyped, and the 3-month-old sibling heterozygotes and WT mice were perfused and fixed for further section staining and blood glucose measurement.

[0058] During the construction process, specific primers targeting the NPAFP gene were designed at the 5' and 3' ends of the insertion site, as well as in the upstream and downstream regions of the insertion site (see Figure 1 B) to identify the mouse genotype by PCR electrophoresis (see Figure 1 CD). At present, transgenic mice that stably inherit and express NPAFP1, NPAFP4 and NPAFP12 proteins have been successfully obtained. These transgenic mice will be used to verify the role of NPAFP in diseases related to weight loss and blood sugar reduction. The 3-month-old transgenic heterozygous mice and WT mice of the same siblings were perfused and fixed for further section staining and blood glucose measurement. Example 2 Histomorphological Detection 2.1 HE staining: Detection of liver and brown adipose tissue morphology, observation of lipid droplet distribution and organelle density Mice were anesthetized with isoflurane and then perfused with PBS and 4% neutral buffered formaldehyde solution, respectively. Fresh liver and brown adipose tissue were obtained and fixed with 4% neutral buffered formaldehyde solution. The fixed tissue was dehydrated in gradient concentrations (70%, 80%, 95%, 100%). The tissue was made transparent with xylene. The transparent tissue was immersed in liquid paraffin, embedded and paraffin blocks were prepared. The tissue was sliced, spread on a slide, dried and fixed. Dewaxed with xylene, then gradually rehydrated with gradient ethanol (100%, 95%, 80%, 70%), and then washed with distilled water. The slices were stained with hematoxylin stain, rinsed with tap water, then differentiated with 1% hydrochloric acid alcohol, and then rinsed with tap water to return to blue. The slices were stained with eosin stain and rinsed with distilled water. Dehydrated with gradient ethanol (70%, 80%, 95%, 100%), then transparent with xylene, and finally sealed with neutral gum. After the coverslips were dried, the histological structure and staining effect were observed under a microscope.

[0059] like Figure 2 As shown in the figure, larger lipid droplets appeared in the brown adipose tissue cells of the control group WT mice, and the density of organelles was low. However, the number of lipid droplets in the brown adipose tissue cells of NPAFP1 mice decreased, and the density of organelles increased relatively. The number of lipid droplets in the brown adipose tissue cells of NPAFP4 and NPAFP12 mice was very small, and the density of organelles increased significantly. The above results show that NPAFP protein can significantly improve the metabolic characteristics of brown fat, and its effect is positively correlated with the number of AFPIII domains.

[0060] 2.2 Oil Red O staining: Assessment of the number and size of lipid droplets in liver and brown adipose tissue Mice were anesthetized with isoflurane and perfused with PBS and 4% neutral buffered formaldehyde solution, respectively. Fresh liver and adipose tissue were obtained and fixed with 4% paraformaldehyde. The fixed tissue was dehydrated with a gradient of 15% and 30% sucrose solution, embedded in OCT cryoembedding medium, and the tissue was sliced ​​and placed on a slide. The slices were dried at room temperature and then washed with distilled water. The slices were immersed in 0.5% Oil Red O working solution for staining. Differentiation was performed with 75% ethanol until the background was clear, and then rinsed with distilled water. The nuclei were counterstained with hematoxylin staining solution, then rinsed with tap water and back to blue. The slides were mounted with glycerol gelatin. After the slides were dried, the red staining results of lipids and the blue background of the nuclei were observed under a microscope.

[0061] like Figure 3As shown in the figure, the brown adipose tissue of WT mice contains more lipid droplets, and the lipid droplets are larger. However, the lipid droplet content of brown fat in mice expressing the NPAFP gene is significantly reduced, and the density of lipid droplets of NPAFP4 and NPAFP12 is lower than that of NPAFP1. This shows that NPAFP can reduce the content of lipid droplets in brown adipocytes, and its effect is positively correlated with the number of AFPIII domains.

[0062] like Figure 4 As shown in the figure, the number and size of lipid droplets in the liver tissue of WT mice were larger; in contrast, the content of lipid droplets in the liver of NPAFP1, NPAFP4 and NPAFP12 mice was significantly reduced, and the effect of NPAF12 group was more significant. This indicates that NPAFP protein has an inhibitory effect on liver fat accumulation.

[0063] Example 3 Blood Glucose Level Measurement Collect blood from the tail vein of mice and use the glucose oxidase method to detect blood glucose content. Prepare tools such as blood glucose meter, matching test strips, blood collection needles, alcohol cotton balls, etc. Before the test, ensure that the mice are fasting (fasting blood glucose test), but can drink water freely to avoid eating affecting blood glucose levels. Collect blood from the ear vein, insert the blood glucose test strip into the blood glucose meter, and after the instrument prompts, drip the collected blood onto the sampling end of the test strip to ensure that the blood volume is sufficient. The blood glucose meter will display the blood glucose value within a few seconds. After the blood collection is completed, press the blood collection site to stop the bleeding, and observe whether the mouse has any abnormal reaction. Record the blood glucose results accurately, indicating the mouse number and test time.

[0064] like Figure 5 As shown, the blood glucose level of NPAFP1 mice was not significantly different from that of the WT group, while the blood glucose levels of NPAFP4 and NPAFP12 mice were significantly lower than those of the WT group and NPAFP1 group, and the blood glucose level of NPAFP12 was lower than that of NPAFP4. This indicates that NPAFP protein has a dose-dependent effect in regulating blood glucose, and its effect is closely related to the number of AFPIII functional domains.

Claims

1. Application of NPAFP protein in the preparation of a product for weight loss and blood sugar reduction, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12; the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

2. The use as claimed in claim 1, wherein the product includes but is not limited to health products, weight loss foods or functional drinks.

3. Use of NPAFP protein in the preparation of a drug for treating obesity and / or diabetes, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12; the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

4. The use as claimed in claim 3, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

5. Use of NPAFP protein in constructing an animal model of obesity and / or diabetes, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12; the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

6. The use as claimed in claim 5, wherein the animals in the animal model include rodents, large mammals and non-human primates.

7. A method for constructing an animal model of obesity and / or diabetes, wherein the animal model is constructed by inserting the NPAFP gene into the mouse H11 gene site using gene editing technology to construct NPAFP transgenic mice expressing different numbers of AFP III functional domains.

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