CN114437194B - Humanized hypoglycemic polypeptide and application thereof - Google Patents

Abstract

The application belongs to the technical field of biology, and relates to a humanized blood glucose reducing polypeptide and application thereof. The amino acid sequence of the humanized hypoglycemic polypeptide SDCBP2-108-267aa is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1. The hypoglycemic polypeptide has the effect of promoting the absorption of glucose by cells. Can improve hyperglycemia metabolism, and can be used for preparing medicine for treating and/or preventing diabetes.

Description

Humanized hypoglycemic polypeptide and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a humanized blood glucose reducing polypeptide and application thereof.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

With the prevalence of global obesity, sedentary physical and high calorie dietary flows, diabetes has become a global health problem threatening human health following cardiovascular diseases/tumors, with a significant rise in prevalence, by 2018, about 4.15 million diabetics worldwide, predicted to be more than 6.25 million in 2040, had become profound physical and psychological distress to patients and families, and had a great burden on the health care system. Over 90% of diabetics are type II diabetics (T2D), which are mainly caused by obesity-induced target organ insulin resistance or relative insulin deficiency caused by pancreatic beta cell dysfunction, ultimately resulting in hyperglycemia. The current clinical medicines for treating diabetes mainly comprise the following medicines: insulin has obvious hypoglycemic effect, but causes hypoglycemia and weight gain; metformin is mainly used for reducing gluconeogenesis and hepatic glucose production, however, long-term use can cause gastrointestinal tract and lactic acidosis; sulfonylurea drugs (and insulin secretagogues) can increase insulin secretion by islet cells, and can cause hypoglycemia and increase cardiovascular disease risk after long-term use; sodium-glucose cotransporter (SGLT 2) inhibitors, which block the reabsorption of glucose by the proximal tubule of the kidney, can cause ketoacidosis, genital mycosis and fracture after long-term use; an incretin analog, which promotes insulin secretion, delays gastric emptying, suppresses appetite, and causes nausea, vomiting, and pancreatitis after long-term use; peroxisome proliferator-activated receptor gamma (ppary) antagonists (thiazolidinediones), which enhance the lipid storage capacity of adipose tissue, reduce abnormal lipid storage of liver and muscle tissue, improve insulin sensitivity of liver and muscle, cause weight gain, bladder cancer and fracture after long-term use, and increase cardiovascular disease risk; alpha-glucosidase inhibitor, interfere with intestinal glucose absorption, reduce glucose production, increase glucose utilization, and cause diarrhea, abdominal pain, nausea, and emesis after long-term use. In short, these drugs have low efficacy, poor tolerance and obvious side effects, and thus cannot fully improve the endocrine-metabolism abnormality problem of type II diabetes, and therefore, intensive research on pathogenesis of type II diabetes is needed to develop safe and effective drugs for the root cause of the disease.

Secreted proteins, hormones and cytokines are a class of factors secreted by islet beta cells and insulin responsive tissues such as muscle, liver, adipose tissue, play an important role in the food intake, insulin sensitivity and energy metabolism of the body, and changes in the levels of these secreted factors in serum are critical to maintaining glucose metabolism and energy homeostasis in the body. Insulin is a hormone secreted by islet beta cells, and can promote uptake of glucose by skeletal muscle and adipose tissue and reduce glucose production in the liver, and the decrease in insulin secretion causes hyperglycemia. Leptin is secreted from adipose tissue and acts as an antidiabetic agent by inhibiting glycogen synthesis, inhibiting glycogen response and inhibiting hypothalamic-pituitary-adrenal axis. Incretins glucose-dependent insulin release polypeptide (GIP) and glycogen-like polypeptide-1 (GLP-1) are released upon ingestion of food and absorption of glucose, greatly promoting insulin secretion. In addition, GLP-1 plays an important role in inhibiting glycogen secretion and promoting insulin synthesis. High concentrations of retinol binding protein-4 (RBP 4) in serum cause antagonism of insulin by reducing the level of GLUT4 in skeletal muscle. The hormone Irisen acts on white adipose tissue cells to promote UCP1 expression and brown fat transition, and is increased in serum of obese patients. The adipokine Apelin acts as a G-protein coupled receptor, playing an important role in maintaining cardiovascular function, humoral homeostasis, angiogenesis, food intake and cell proliferation, and secretion is increased in type II diabetes patients. FGF21 is used as endocrine hormone to regulate liver ketogenesis, gluconeogenesis and lipolysis, can effectively resist obesity caused by diet and enhance glucose tolerance, the level of FGF21 in serum is positively correlated with obesity and insulin resistance, and the metabolic disorder of diabetic mice can be effectively improved by injecting FGF 21. Therefore, research on the relationship between secreted proteins and blood glucose and blood lipid has an extremely important role in the treatment of diabetes. The secreted protein is secreted by self tissue organs, has little toxic and side effects, and is extremely safe for organisms, so that research on the secreted protein provides theoretical basis for treatment of diabetes polypeptide.

Syndecan binding protein (SDCBP), also known as syntenin or melanoma differentiation associated gene 9 (MDA-9), was originally thought to be a connexin linking Syndecan-mediated signaling pathways and cytoskeleton. It modulates the functions of shuttling of transmembrane receptors, tumor cell degeneration, and neuronal-synaptic transmission through interactions of PDZ domains with multiple proteins. SDCBP has been found to inhibit P53 protein levels by binding to transcription initiation factor 5A (eIF 5A), thereby inhibiting apoptosis. SDCBP is involved in tumor progression and is associated with cell migration, invasion, and pseudopodia formation. SDCBP is involved in the regulation of cytoskeletal system by participating in FAK kinase, p38 mitogen-activated protein kinase, c-jun NH2 terminal kinase and nuclear factor KB signaling pathway. It is reported in the literature that PKC alpha activation causes SDCBP expression, further promoting FN-mediated assembly of integrin-beta 1 signaling pathway complex, leading to FAK activation as well as downstream signaling pathway activation. SDCBP can also be directly combined with c-Src, which is favorable for forming an activated FAK/c-Src signal complex and is very important for regulating and controlling a migration complex. Although SDCBP plays an important role in the regulation of cytoskeleton, the specific mechanism is not clear, and whether or not it is involved in cytoskeletal-mediated glucose uptake is still blank.

Disclosure of Invention

Aiming at the problems in the prior art, the application aims to provide a humanized hypoglycemic polypeptide and application thereof.

In order to solve the technical problems, the technical scheme of the application is as follows:

in a first aspect, the humanized blood glucose reducing polypeptide SDCBP2-108-267aa has an amino acid sequence which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1.

The hypoglycemic polypeptide has the effect of promoting the absorption of glucose by cells.

In a second aspect, there is provided a nucleotide encoding the hypoglycemic polypeptide comprising any one of the group:

(a) A nucleotide encoding a polypeptide having the amino acid sequence;

(b) A nucleotide complementary to the nucleotide of (a).

In a third aspect, the application of the hypoglycemic polypeptide in preparing medicines for treating and/or preventing diabetes is provided.

Further, the hypoglycemic polypeptide is a basic skeleton or a methylated or acylated derivative of the amino acid sequence.

In a fourth aspect, a medicament for the treatment and/or prophylaxis of diabetes, the medicament comprising as an active ingredient a humanized hypoglycemic polypeptide as defined above.

According to the application, the medicament further comprises at least one pharmaceutically inactive ingredient.

The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.

The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In yet another embodiment of the present application, the carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

In yet another embodiment of the application, the medicament of the application may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present application may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In yet another embodiment of the present application, the subject to be administered can be human or non-human mammal, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

In a fifth aspect, a method of promoting glucose uptake by a cell in vitro, the method comprising administering in vitro purified SDCBP2-108-267aa protein.

Further, the amino acid sequence of the hypoglycemic polypeptide is SEQ ID NO.1.

Further, the concentration of SDCBP2-108-267aa protein is 0.02-0.2mg/ml; preferably 0.05mg/ml.

Further, the cells include HeLa cells.

One or more of the technical schemes of the application has the following beneficial effects:

the application discloses an SDCBP2 amino acid fragment which is an effective polypeptide for reducing blood sugar, and researches of the application show that the SDCBP2 is a secreted protein, and the secreted SDCBP2 can be combined to a cell membrane by relying on 108-267 amino acid sequences to promote glucose absorption. The method mainly comprises the following three aspects: intracellular SDCBP2 is a secreted protein, SDCBP2 promotes GLUT4 up-membranes, SDCBP2-108-267 amino acids promote GLUT4 up-membranes, and SDCBP2-108-267 amino acids promote intracellular glucose uptake.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.

FIG. 1 is a diagram showing the PCR reaction conditions in example 2;

FIG. 2 is a graph showing secretion of 293T cells transiently overexpressing SDCBP2 of example 1 under glucose or serum starvation.

FIG. 3 is an in vitro purified SDCBP2-108-267aa protein of example 2 facilitating the membrane coating of GLUT 4. FIG. a shows the protein purification of SDCBP2 and deletion mutants; panel b is the SDCBP2 full-length protein facilitating the upper membrane of GLUT 4; panel c is SDCBP2-108-267aa promotes the membrane feeding of GLUT 4.

FIG. 4 is an in vitro purified SDCBP2-108-267 protein of example 3, which promotes intracellular glucose uptake.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. The application will be further illustrated by the following examples

Example 1

SDCBP2 protein is a secreted protein.

1. Experimental materials

1.1 cell and culture consumables

Human kidney embryo cells 293T, human cervical carcinoma cells HeLa, cell culture dishes, culture plates, cell culture medium: DMEM+10% foetal calf serum

1.2 reagents

Flag-M2-beads(sigma)、Protein A/G beads(Abmart)

Formulated reagents

Adjusting the pH value to 7.4, fixing the volume to 1L, and sterilizing by high-pressure steam for 40min for later use.

IP lysis buffer

RIPA buffer

RIPA 100ml

Adding water to volume to 100ml, filtering, packaging, and adding protease inhibitor before use.

Basic reagents for calcium phosphate transfection are prepared:

2×HEBES 1L

NaCl 16.4g

HEPES 11.9g

Na ₂ HPO ₄ 0.21g

the pH value is regulated to 7.05, fine adjustment is carried out to the most proper pH value, the volume is fixed to 1L, and the filtration is carried out by a filter membrane with the thickness of 0.22 mu m.

2.5M CaCl ₂ 100mL

CaCl ₂ 3.874g

H ₂ O 50mL

Filtering with 0.22 μm filter membrane, and packaging.

2. Experimental method

2.1 The specific experimental procedure of Western blot is as follows:

(1) Preparing modified polyacrylamide gel: cleaning the rubber plate, and arranging the rubber plate on a rubber preparation frame according to a correct sequence; leak detection; preparing separating gel, ddH ₂ O sealing; after the separating glue is solidified, preparing the concentrated glue, then inserting a comb, and after the concentrated glue is completely solidified, the concentrated glue can be used.

(2) Electrophoresis: placing the prepared glue and an electrophoresis tank; adding a running buffer into an electrophoresis tank; loading a sample; and (3) adjusting the voltage of the electrophoresis apparatus to 80V to start electrophoresis, and when loading completely enters the separation gel, adjusting the voltage to 120V until the loading reaches the bottom of the separation gel, and ending electrophoresis.

(3) Transferring: soaking PVDF membrane in methanol for 30s, and then placing the PVDF membrane into a transfer buffer for standby; adding transfer buffer into a transfer film tank, and cooling by an ice-water mixture in advance; placing the sponge, the filter paper, the glue and the PVDF film according to the correct sequence, and then placing the sponge, the filter paper, the glue and the PVDF film in a clamping groove of a film transferring groove; the electrophoresis apparatus was started and 80V was used for transfer, and the time required was generally 90min.

(4) And (3) blocking: the PVDF membrane with the transferred membrane is put into 5% skimmed milk prepared by PBST, and incubated for 1-1.5 hours at room temperature by a shaking table.

(5) Adding an antibody: specific primary antibodies were diluted to appropriate concentrations with 5% skim milk and incubated on a room temperature shaker for 2 hours or overnight at 4 ℃. Then washed 3-4 times with PBST at room temperature for 10min each.

(6) Adding a secondary antibody: the corresponding secondary antibody (HRP conjugated) was diluted to the desired concentration with 5% skim milk, incubated for 2-2.5 hours at room temperature, and then washed with PBST for 4-5 times at room temperature for 10 minutes each time.

(7) Developing and fixing: in a darkroom, sucking the liquid on the PVDF film, and dripping the mixed luminous reagent A, B on the film; placing PVDF film in a light-proof box, and covering with X-ray film; developing and fixing the X-ray film in turn; and washing the X-ray film, and drying in an oven to obtain an analysis result.

2.2 transfection of calcium phosphate particles

(1) Preparing solution A (16 ul CaCI+target plasmid+sterile water 300 ul), preparing solution B (300 ul 2X HEPES), uniformly mixing the two reagents, uniformly blowing bubbles into the solution B by using an electric pipette, and simultaneously sucking the solution A into the solution B by using the pipette, wherein the speed is uniform.

(2) After blowing, A, B solution was gently mixed, 600ul of the mixture was gently and evenly added dropwise into a 6cm dish to be transfected, and gently mixed by shaking.

(3) The transfected cells are placed into an incubator for continuous culture for about 1 hour, and the size of the formed calcium particles can be observed under a microscope.

(4) About 4-6 hours, the transfected cells are changed to fresh medium, the specific time being determined by the calcium particle size.

(5) After 12 hours, the expression of GFP fluorescent protein can be observed under a fluorescence microscope, and the transfection efficiency can be judged about 16 hours according to the expression of fluorescence, and the target protein expression is about 24 hours.

(6) According to the experimental requirement, the cells can be harvested after the target protein is expressed for subsequent experiments.

3. Results and analysis

As shown in fig. 2, flag-SDCBP2 was transfected into 293T cells, blood glucose treatment and serum starvation treatment were performed on the cells at different concentrations after 24 hours, and after 12 hours, medium and cells were collected separately and lysed with RIPA to obtain cell Lysate (Lysate); the culture medium is enriched with Flag-M2-beads for extracellular Flag-tagged proteins, and the enrichment condition in the culture medium is detected by Western blot experiments, so that obvious enrichment of SDCBP2 is found, and the SDCBP2 is proved to be secreted protein.

Example 2

SDCBP2-108-267aa protein promotes the upper membrane of GLUT 4.

1. Experimental materials

1.1 cells

Human cervical cancer cell HeLa

Culture medium: DMEM+10% foetal calf serum

1.2 plasmid

His-GFP-SDCBP2-108-267aa deletion mutant is constructed by taking His-GFP-SDCBP2 as a template

1.3 reagents

4% paraformaldehyde

2. Experimental method

The 2.1SDCBP2 delet was constructed as follows:

(1) Primer design

Through browsing NCBI website, downloading the sequence of SDCBP2 gene, using related software to find out the enzyme cutting site contained in the gene sequence, researching the map of the used vector, selecting proper restriction endonuclease, dividing SDCBP2 into five sections as shown in figure 3, and designing the required upstream and downstream primers with the aid of primer-5 and other software.

(2) PCR amplification of target Gene

The PCR system is as follows:

mixing the prepared PCR reaction liquid, instantly separating, and placing into a PCR instrument for reaction. The reaction conditions are shown in FIG. 1.

(3) Recovery of amplified target fragment by tapping and enzyme digestion

After the PCR is finished, a proper amount of products are taken for agarose gel electrophoresis detection, whether main product bands with expected molecular weights exist on the gel is observed, and target bands are subjected to rubber tapping recovery.

a. Agarose gel (with appropriate EB added) was prepared at appropriate concentration according to the size of the recovered DNA fragment.

b. To the PCR product, 6 XDNA loading was added to dilute to 1X, and spotting was started.

c. Immediately after sample addition, electrophoresis was performed by applying a current at 120V for 20 minutes, and then the electrophoresis was stopped.

d. Taking out gel, observing the position of the strip under an ultraviolet lamp, photographing and preserving by using a gel imaging system, cutting off a target strip, and recovering the target gene by using a root agar gel recovery kit.

e. The pcDNA3.0 Flag vector is selected, and the vector and the PCR product are respectively and simultaneously digested with the same enzyme to generate the same digestion site. The digestion conditions are 3-4 hours at 37 ℃. The cut products of the vector and PCR products were separated by agarose gel electrophoresis, and then the desired product was recovered by using a gel recovery kit (kit was purchased from Tiangen Biochemical technologies Co., ltd.). The specific steps are shown in the instruction book of the kit.

(4) Connection

Experimental principle: under certain conditions, T4 DNA ligase catalyzes the formation of a phosphodiester linkage between adjacent 5 '-terminal phosphate and 3' -terminal hydroxyl groups of two double-stranded DNA fragments, thereby ligating the two fragments. 1. Mu.l of vector, 1. Mu. l T4 of ligase, 1. Mu.l of buffer and 7. Mu.l of target gene fragment are added into the system, and after being uniformly mixed, the mixture is placed into a water bath kettle at 16 ℃ for 8-12 hours.

(5) Transformation

The transformation is carried out by utilizing the heat shock method principle, 50 microliter of just-melted competence is added into the connection product, the mixture is placed on ice for 30 minutes, then the mixture is placed into a water bath with the temperature of 42 ℃ for heat shock for 60 seconds, then the mixture is placed on ice for 5 minutes, then 700 microliter of non-resistant LB is added, the mixture is resuscitated by a shaking table with the temperature of 37 ℃ for 45 minutes, after low-speed centrifugation, the mixture is coated on a flat plate with corresponding resistance, and the mixture is placed into an incubator with the temperature of 37 ℃ for 12 hours in an inverted manner.

(6) Shaking and extracting plasmid

In an ultra-clean workbench, clones obtained by transformation are picked up, inoculated into LB culture medium with corresponding resistance, cultured for 12-16 hours at 37 ℃ under shaking at 200rpm/min, then bacteria are preserved, and plasmids are extracted from the residual bacterial liquid by using a root plasmid small extraction kit, and detailed steps can be referred to the specification.

(7) Restriction enzyme identification, sequencing analysis and verification of expression

Taking 1ug plasmid, selecting corresponding enzyme and Buffer for enzyme digestion identification. The enzyme was digested at 37℃for 3 hours, and then detected by agarose gel electrophoresis. And (3) picking a clone from the plasmid with correct enzyme digestion verification, sending the clone to a sequencing company for sequencing, and analyzing the feedback result to detect whether the construction is successful. The successfully constructed plasmid was transiently transferred into 293T cells and expression was verified by immunoblotting experiments.

2.2 His protein purification

(1) Constructing plasmids required by protein purification, converting the plasmids into a Transetta strain after sequencing correctly, picking monoclonal, performing small mutagenesis, setting different induction temperatures and different ODs, and exploring the optimal conditions of protein induction.

(2) According to the inducing conditions of fumbling when small amount of protein is induced, large induction is performed by enlarging culture. The activated bacterial liquid is added into LB, the mixture is shaken at 220rpm at 37 ℃ to reach an OD value for small fumbling, IPTG is added, then the temperature of the shaking table is adjusted to reach fumbling temperature, and the bacterial liquid is collected after a proper time.

(3) Centrifuging the bacterial liquid, adding lysis buffer, lysozyme, PMSF, DTT and the like, standing on ice for a period of time, and carrying out ultrasonic treatment by an ultrasonic instrument.

(4) Centrifuging the ultrasonic-finished lysate at a high speed, collecting the supernatant in a new 100ml centrifuge tube, adding nickel-coupled beads, placing in a 4-DEG C refrigerator, and shaking for 2-4 hours by using a mute mixer.

(5) The solution was passed through the column and then washed 3 times with a wash buffer.

(6) Proteins were eluted by using an Elutation buffer, dialyzed against PBS at 4℃and the concentration was measured to determine the quality of the purified proteins.

2.3 immunofluorescent staining:

(1) And (3) treating a plate: placing the cover glass into a 24-well plate, treating each well with 200ul of polylysine, and air-drying by ultraviolet irradiation for about 45min;

(2) And (3) accessing cells: inoculating a proper amount of cells to be detected, carrying out transfection when the cells grow to 50%, and carrying out the transfection for 24-48 hours according to the experiment requirement;

(3) Cell fixation: washing off the culture medium, rinsing the cells once with PBS at normal temperature, fixing the cells with 4% paraformaldehyde, and slowly shaking on a shaker for 10 minutes;

(4) Cell perforation: the fixative was aspirated and washed twice with PBS and incubated with 0.3% Triton X-100 in PBS for 10min.

(5) Cell blocking: the PBS of Triton X-100 was blotted off, washed 3 times with PBS for 5 minutes each, and then blocked with 2% BSA for 30 minutes at room temperature;

(6) Adding an antibody: primary antibody was diluted proportionally with 2% bsa, incubated for 2 hours at room temperature or overnight at 4 ℃;

(7) Adding a secondary antibody: washing off the primary antibody, washing with PBS for 3 times, each time for 5 minutes, diluting the fluorescent secondary antibody with 2% BSA in proportion, and incubating for 2 hours at room temperature, wherein the steps are protected from light;

(8) Nuclear dyeing: washing off the secondary antibody, washing 3 times by PBS, and then acting DAPI for 2 minutes;

(9) Sealing piece: DAPI was blotted, blocked, observed under a fluorescence microscope and photographed.

3. Results and analysis

As shown in FIG. 3, wherein FIG. a. Prokaryotic expression plasmid His-GFP-SDCBP2 and deletion mutants (1-107 aa, 1-87 aa,1-267aa,108-267aa,188-292 aa) were constructed and purified to give proteins. Panel b. Flag-GLUT4 was transfected into HeLa cells, after 24 hours, his-SDCBP2 protein was incubated with HeLa cells for 12 hours, cells were collected, cells were fixed with 4% paraformaldehyde, flag-GLUT4 was stained red with Flag antibody, and nuclei were stained blue with DAPI. FIG. c. Flag-GLUT4 was transfected into HeLa cells, after 24 hours, his-SDCBP2 protein was incubated with HeLa cells for 12 hours, cells were collected, cells were fixed with 4% paraformaldehyde, flag-GLUT4 was stained red with Flag antibody, SDCBP2 was stained green with His antibody and used to indicate localization of SDCBP2 protein in cells. The above results indicate that SDCBP2 protein promotes GLUT4 membrane-up, and that amino acid sequence 108-267 is an essential fragment for promoting GLUT4 membrane-up.

Example 3

The in vitro purified SDCBP2-108-267aa protein can promote the absorption of glucose by cells.

1. Experimental materials

1.1 cells and Medium

Human hepatoma cell HepG2, culture medium: DMEM+10% FBS

1.2 reagents

Grape carbohydrase mark detection reagent box (purchased from Nanjing built biological company)

2. Experimental method

Detection was performed according to the kit instructions.

3. Experimental results

HepG2 cells were cultured using six well plates, four wells were used, respectively, with only medium added to the first well, no cells were cultured, cells were cultured from the second well, and the number of cells in each well was the same, only medium was added to the second well, no protein was added to the third well, medium and purified His-SDCBP2-1-107aa protein were added to the fourth well, medium and purified His-SDCBP2-108-267aa protein were added to the fourth well, the concentration of protein contained in the medium of each treatment group was 0.05mg/ml, and the glucose content in the cell culture medium was measured after 24 hours of culture. As shown in FIG. 4, the right bar graph corresponding to each horizontal coordinate point in FIG. 4 is SDCBP2-108-267aa, the left bar graph represents SDCBP2-1-107aa, and the SDCBP2-108-267aa protein can be obtained to promote the absorption of glucose by cells.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shanghai first people Hospital

<120> a humanized hypoglycemic polypeptide and application thereof

<130> 202027499

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 159

<212> PRT

<213> humanized blood glucose reducing polypeptide

<400> 1

Ile His Leu Cys Lys Asp Glu Arg Gly Lys Thr Gly Leu Arg Leu Arg

1 5 10 15

Lys Val Asp Gln Gly Leu Phe Val Gln Leu Val Gln Ala Asn Thr Pro

20 25 30

Ala Ser Leu Val Gly Leu Arg Phe Gly Asp Gln Leu Leu Gln Ile Asp

35 40 45

Gly Arg Asp Cys Ala Gly Trp Ser Ser His Lys Ala His Gln Val Val

50 55 60

Lys Lys Ala Ser Gly Asp Lys Ile Val Val Val Val Arg Asp Arg Pro

65 70 75 80

Phe Gln Arg Thr Val Thr Met His Lys Asp Ser Met Gly His Val Gly

85 90 95

Phe Val Ile Lys Lys Gly Lys Ile Val Ser Leu Val Lys Gly Ser Ser

100 105 110

Ala Ala Arg Asn Gly Leu Leu Thr Asn His Tyr Val Cys Glu Val Asp

115 120 125

Gly Gln Asn Val Ile Gly Leu Lys Asp Lys Lys Ile Met Glu Ile Leu

130 135 140

Ala Thr Ala Gly Asn Val Val Thr Leu Thr Ile Ile Pro Ser Val

145 150 155

Claims (3)

1. A method for promoting glucose uptake by human HepG2 cells in vitro, characterized by: the method comprises administering in vitro purified SDCBP2-108-267aa protein;

the amino acid sequence of the SDCBP2-108-267aa protein is SEQ ID NO.1.

2. The method of promoting glucose uptake in human HepG2 cells in vitro according to claim 1, wherein: the concentration of SDCBP2-108-267aa protein is 0.02-0.2mg/ml.

3. The method of promoting glucose uptake in human HepG2 cells in vitro according to claim 2, wherein: the concentration of SDCBP2-108-267aa protein was 0.05mg/ml.