CN114539380A

CN114539380A - Humanized hypoglycemic polypeptide, preparation method and application thereof

Info

Publication number: CN114539380A
Application number: CN202011325365.3A
Authority: CN
Inventors: 王传贵; 孙莲慧; 张胜萍; 范广建
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2022-05-27

Abstract

The invention discloses a humanized hypoglycemic polypeptide, a preparation method and application thereof, wherein the amino acid sequence of the humanized hypoglycemic polypeptide is GLAWSKTGPVAKELSGLPSGPSAGSCPPPPPPCPPPPPVSTISCSYESASRSSLFAQINQGESITHALKHVSDDMKTHKNPALKAQSGPVRSGPKPFSAPKPQTSPSPKRATKKEPAVLELEGKKWR. The invention shows that the re-entry of the secretory protein CAP1 into cells depends on the 204-330 amino acid sequence; CAP1-204-330 amino acids promote membrane formation on GLUT1/GLUT 4; can also promote the absorption of glucose in cells. The invention provides a novel method for treating and/or preventing hyperglycemia, which is beneficial to developing medicaments and health-care products for treating and/or preventing diabetes.

Description

Humanized hypoglycemic polypeptide, preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to humanized hypoglycemic polypeptide, and a preparation method and application thereof.

Background

With the prevalence of global obesity, physical sedentary and high-calorie diet trends, diabetes has become a global health problem threatening human health after cardiovascular diseases/tumors, the prevalence rate thereof has increased significantly, by 2018, about 4.15 million diabetic patients worldwide, more than 6.25 million are predicted in 2040 years, causing profound physical and mental pain to patients and families, and causing a huge burden on health care systems. More than 90% of diabetics are type II diabetes (T2D) which is mainly caused by relative insulin deficiency due to obesity-induced insulin resistance of target organs or pancreatic gland beta cell dysfunction, eventually leading to hyperglycemia. At present, the clinical medicines for treating diabetes mainly comprise the following types: insulin has obvious effect of reducing blood sugar, but can cause low blood sugar and weight gain; metformin, mainly used for reducing gluconeogenesis and hepatic glucose production, however, gastrointestinal tract and lactic acidosis are caused by long-term use; sulfonylurea drugs (and insulin secretagogues) can increase insulin secretion of islet cells, cause hypoglycemia after long-term use, and increase cardiovascular disease risk; sodium glucose cotransporter (SGLT2) inhibitors block glucose reabsorption from the proximal kidney tubule, which can cause ketoacidosis, genital mycosis, and bone fracture in long-term use; incretin analogs, promote insulin secretion, delay gastric emptying, suppress appetite, and cause nausea, vomiting, and pancreatitis in long-term use; peroxisome proliferator-activated receptor gamma (PPAR gamma) antagonists (thiazolidinediones), enhance the storage capacity of adipose tissues on lipid, reduce the abnormal storage of lipid by liver and muscle tissues, improve the sensitivity of the liver and muscle on insulin, cause weight gain, bladder cancer and fracture after long-term use, and increase the risk of cardiovascular diseases; alpha-glucosidase inhibitors interfere with intestinal glucose absorption, reduce glucose production, increase glucose utilization, and cause diarrhea, abdominal pain, nausea, and vomiting after long-term use. In conclusion, due to the characteristics of low efficacy, poor tolerance and obvious side effect, the endocrine metabolism disorder problem of the type II diabetes cannot be comprehensively improved, so that the pathogenesis of the type II diabetes needs to be deeply researched, and a safe and effective medicine aiming at the root cause of the disease is developed.

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, and play important roles in 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 pancreatic islet beta cells, and can promote glucose uptake in skeletal muscle and adipose tissue and reduce glucose production in the liver, with reduced insulin secretion resulting in hyperglycemia. Leptin is secreted from adipose tissue and acts as an antidiabetic agent by inhibiting glycogen synthesis, inhibiting the glycogen response and inhibiting the hypothalamic-pituitary-adrenal axis. The incretin glucose-dependent insulin-releasing polypeptide (GIP) and glycogen-like polypeptide-1 (GLP-1) are released when food is taken and glucose is absorbed, and insulin secretion is greatly promoted. In addition, GLP-1 also has important effects in inhibiting glycogen secretion and promoting insulin synthesis. Retinol binding protein-4 (RBP4) in high serum concentrations causes insulin antagonism by reducing GLUT4 levels in skeletal muscle. The hormone Irisen acts on white adipose tissue cells to promote the expression of UCP1 and the conversion of brown adiposity, and is increased in the serum of obese patients. The fat factor Apelin is used as a G protein coupled receptor, plays an important role in maintaining cardiovascular function, fluid homeostasis, angiogenesis, food intake and cell proliferation, and has increased secretion in type II diabetes patients. FGF21 is used as endocrine hormone for regulating liver ketone generation, gluconeogenesis and lipolysis, and can effectively resist obesity caused by diet and enhance glucose tolerance, FGF21 level in serum is in positive correlation with obesity and insulin resistance, and FGF21 injection can effectively improve metabolic disorder of diabetic mice. Therefore, the research on the relationship between secreted protein and blood sugar and blood fat has an extremely important role in the treatment of diabetes. The secretory protein is secreted by self tissues and organs, almost has no toxic or side effect, and is extremely safe to organisms, so the research on the secretory protein provides a theoretical basis for the treatment of the polypeptide for the diabetes.

The adenylate cyclase-related protein family is a protein closely related to cytoskeletal movement and comprises two members, namely CAP1 and CAP2, wherein CAP1 is widely distributed and can be detected in many tissues, and CAP2 is relatively narrowly distributed and is obviously expressed only in brain, cardiac skeletal muscle and skin. CAP protein has negative regulation and control effect on actin polymerization, maintains actin in the state of monomer actin, and inhibits transformation between monomer actin and poly actin, thereby inhibiting movement of cytoskeleton. CAP1 plays an important role in cell signaling. The CAP1 protein is involved in the insulin pathway, and during insulin-stimulated glucose transport and absorption, the CAP1 protein plays a role in regulating glucose absorption. The Glucose-dependent Glucose transporter proteins are taken in by cells, GLUT1/4 is distributed in intracellular storage vesicles in a basic state, GLUT1/4 can be displaced from the vesicles in cytoplasm to cell membranes when the cells are stimulated by insulin, and the Glucose transporter proteins can play a role in transporting Glucose after being transferred to the cell membranes. The process is mainly completed by two ways, CAP1 protein is involved in one of the pathways, insulin firstly interacts with the insulin receptor on the cell membrane, then beta subunit of the insulin receptor is self-phosphorylated, the beta subunit of the insulin receptor is positioned at the inner side of the cell membrane, the phosphorylated beta subunit can activate the downstream CBL protein, however, the CBL protein can not directly interact with the insulin receptor IR, the CBL needs to firstly combine with SH3 functional domain of CAP1, then the CAP1 recruits the CBL protein to the vicinity of the insulin receptor, and further activates the phosphorylation of the CBL, then the CBL-CAP1 complex is dissociated from the insulin receptor IR and transported to lipid-rich raft, the phosphorylated CBL recruits CrkII-C3G complex, so that the complex is also transferred to the lipid raft, and the C3G protein can specifically activate TC10 in the lipid raft region, thereby promoting the movement of cytoskeleton, and transferring the glucose transport protein glut from the vesicle of the cell matrix to the cell membrane to start the glucose transport. In this pathway, eventually the TC10 protein follows the transfer of C3G protein to "lipid rafts", activating the small GTP-binding protein TC10 in the lipid raft area, and then promoting cytoskeletal movement to bring the upper membrane of the glucose transporter GLUT into place is a critical step, whereas the "lipid raft" microenvironment, centered on CBL, CAP1, C3G and TC10, is necessary for the activation of TC10 protein. The CAP1 protein is an important regulator of the process, in which the CAP1 plays a role in recruiting CBL protein to the vicinity of insulin receptor IR for phosphorylation, depending on the interaction between the SH3 domain of CAP1 and CBL, and the abundance of CAP1 protein and related modifications of CAP1 protein affect the phosphorylation of CBL protein under the action of insulin receptor, thereby affecting downstream signal pathways. CAP1 therefore plays a role in the Insulin pathway and is an important regulator of glucose uptake by cells.

However, there is no extensive research and discussion in the literature on how CAP1 regulates glucose absorption, and there is still a technical barrier to whether CAP1 can be applied in the field of glucose lowering.

Disclosure of Invention

The invention mainly aims to provide a humanized hypoglycemic polypeptide with good safety and better hypoglycemic effect, and the amino acid sequence of the polypeptide is shown as the sequence table SEQ ID NO: 1, specifically, the compound shown in the specification,

GLAWSKTGPVAKELSGLPSGPSAGSCPPPPPPCPPPPPVSTISCSYESASRSS LFAQINQGESITHALKHVSDDMKTHKNPALKAQSGPVRSGPKPFSAPKPQTSP SPKRATKKEPAVLELEGKKWR。

preferably, the hypoglycemic polypeptide is of human origin and is the amino acid at position 204-330 of the secretory protein CAP1, which is labeled as CAP1-204aa-330 aa.

Preferably, the humanized hypoglycemic polypeptide is any one of the following:

(a) the amino acid sequence is SEQ ID NO: 1;

(b) as shown in SEQ ID NO: 1, optionally methylated or acylated derivatives of the amino acid sequence shown in 1.

As a second aspect of the present invention, there is also provided a cell-penetrating peptide fragment comprising the sequence of SEQ ID NO: 1.

Preferably, the cell-penetrating peptide segment further comprises a peptide represented by SEQ ID NO: 1 as basic skeleton, and performing extension, shortening, amino acid substitution and amino acid modification to obtain peptide segment as membrane penetrating carrier.

As a third aspect of the invention, the invention also provides a preparation method of the hypoglycemic polypeptide, which is artificially synthesized by a genetic engineering method.

Preferably, the preparation method of the hypoglycemic polypeptide comprises the following steps:

(1) constructing a plasmid His-CAP1-204-303aa required by protein purification, transforming the plasmid into a Transetta strain after the sequencing is correct, selecting a single clone, performing small mutagenesis, and exploring the optimal conditions of protein induction;

(2) according to the induction condition of groping when small amount of induction is carried out on the protein, the amplification culture is carried out for large induction; adding the activated bacterial liquid into an LB culture medium, shaking at 220rpm at 37 ℃ until the OD value is 0.6, adding IPTG, adjusting the temperature of a shaking table to 25 ℃, and collecting the bacterial liquid after 8 hours;

(3) centrifuging the bacterial liquid, adding lysine buffer, lysozyme, PPMSF and DTT, standing on ice for 15 minutes, performing ultrasonic treatment by using an ultrasonic instrument, wherein the ultrasonic treatment is performed for 3 seconds and 7 seconds, and repeating the ultrasonic treatment for 90 times;

(4) centrifuging the lysate subjected to ultrasonic treatment at a high speed of 10000rpm for 30 minutes, collecting the supernatant in a new 100ml centrifuge tube, adding beads of coupled nickel, placing the centrifuge tube in a 4-DEG refrigerator, and shaking the centrifuge tube for 2 to 4 hours by using a mute mixer;

(5) passing the solution through a column, and then washing for 3 times by using a wash buffer;

(6) the protein was eluted with an Elution buffer, dialyzed against PBS at 4 ℃ in a refrigerator, and the concentration was measured to determine the quality of the purified protein.

As a fourth aspect of the present invention, there is also provided the use of the hypoglycemic polypeptide, which can reduce the intracellular glucose content;

furthermore, the invention also provides application of the hypoglycemic polypeptide in preparing medicines for treating and/or preventing diabetes. The hypoglycemic polypeptide increases the glucose intake in cells, reduces the glucose outside the cells and achieves the purpose of reducing the blood sugar.

As a fifth aspect of the present invention, there is provided a hypoglycemic product comprising a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1, wherein the derivative of the hypoglycemic polypeptide refers to the amino acid sequence shown in the sequence table SEQ ID NO: 1 or a derivative obtained by methylation or acylation of an amino acid sequence shown in a sequence table SEQ ID NO: 1 is a basic skeleton, and is a derivative obtained by extension, shortening, amino acid substitution and amino acid modification and used as a hypoglycemic polypeptide.

Preferably, the hypoglycemic product comprises the hypoglycemic polypeptide and derivatives obtained by modification of the hypoglycemic polypeptide, and pharmaceutically acceptable carriers.

Compared with the prior art, the invention has the beneficial effects that: the invention discloses that the CAP 1-204-330-amino acid fragment is an effective polypeptide for reducing blood sugar, and particularly, the research of the invention shows that the intracellular CAP1 is a secretory protein, and the reentering of the CAP1 protein into cells depends on the 204-330-amino acid sequence; CAP1-204-330 amino acids promote membrane formation on GLUT1/GLUT 4; the CAP1-204-330 amino acid sequence promotes intracellular glucose uptake. The invention provides a novel method for treating and/or preventing hyperglycemia, which is beneficial to developing medicaments and health-care products for treating and/or preventing diabetes.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1. detection of secretion and transmembrane status of CAP1 in example 1; FIG. a is a graph of the secretion of CAP1 in serum starvation of 293T cells transiently overexpressing CAP 1; panel b shows the amino acid sequences upon which CAP1 and mutant proteins purified in vitro are dependent for entry into cells.

FIG. 2. in vitro purified CAP1-204-330aa protein from example 2 promoted the upper membrane of GLUT1 and GLUT 4.

FIG. 3. in example 3, in vitro purified CAP1-204 and 330aa proteins promoted intracellular glucose uptake; it is shown that the CAP1-204 and 330aa proteins promote glucose uptake in HeLa cells and HepG2 cells.

FIG. 4 reaction conditions for PCR amplification of the gene of interest in example 1.

FIG. 5, the quality of the purified His-CAP1-204 and 330aa proteins was examined in example 2.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples of the specification. Reagents and equipment used in the present invention are conventional in the art unless otherwise specified. Of course, the use of the instruments and materials in the embodiments is not limited to the enumeration of the present examples, but is based on the idea of being able to solve the technical problems of the present invention and achieve the corresponding technical effects. In addition, molecular biological methods which are not described in detail in the examples are all conventional methods in the field, and specific operations can be referred to molecular biological guidelines or product specifications.

Example 1: confirmation that the CAP1 protein is a secreted protein assay:

1. experimental Material

1.1 cell and culture consumables

Human kidney embryo cell 293T, human cervical carcinoma cell HeLa, cell culture dish, culture plate, cell culture medium:

DMEM + 10% fetal bovine serum

1.2 reagents

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

Formulated reagent

Adjusting pH to 7.4, diluting to 1L, sterilizing with high pressure steam for 40min, and storing.

IP lysis buffer

RIPA buffer

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

Lysine buffer (pH adjusted to 8.0 with 5N NaOH)

Wash buffer (pH adjusted to 8.0 with 5N NaOH)

Elution buffer (pH adjusted to 8.0 with 5N NaOH)

10 × Running buffer (Running buffer)

10 × Transfer buffer (Transfer buffer)

Tris 165g

Glycine 720g

ddH 2O-5L

Calcium phosphate transfection basic reagent preparation:

adjusting pH to 7.05, fine-adjusting to the most suitable pH, diluting to 1L, and filtering with 0.22 μm filter membrane.

2.5M CaCl₂ 100mL

CaCl₂ 3.874g

H₂O 50mL

Filtering with 0.22 μm filter membrane, packaging, and storing.

2. Experimental methods

2.1Western blot the specific experimental procedures were as follows:

(1) preparing modified polyacrylamide gel: cleaning the rubber plate, and arranging the rubber plate on a rubber making frame according to a correct sequence; leak detection is carried out; preparation of separation gel, ddH₂O sealing; and (4) preparing concentrated glue after the separation glue is solidified, inserting a comb, and using after the concentrated glue is completely solidified.

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

(3) Film transfer: soaking the PVDF membrane in methanol for 30s, and then putting the PVDF membrane into a transfer buffer for later use; adding the transfer buffer into a film transfer tank, and cooling by using an ice-water mixture in advance; placing the sponge, the filter paper, the glue and the PVDF membrane in a correct sequence, and then placing the sponge, the filter paper, the glue and the PVDF membrane in a clamping groove of a membrane rotating groove; the electrophoresis apparatus is started, and the membrane is rotated by using 80V voltage, and the time required is generally 90 min.

(4) Blocking: the PVDF membrane after membrane conversion is put into 5% skimmed milk prepared by PBST and incubated for 1-1.5 hours in a shaking table at room temperature.

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

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

(7) Developing and fixing: in a dark room, sucking off the liquid on the PVDF membrane, and dripping the mixed luminescent reagent A, B on the membrane; placing the PVDF membrane in a light-proof box, and covering an X-ray film; sequentially developing and fixing the X-ray film; and (5) washing the X-ray film, and drying in an oven to obtain an analysis result.

2.2 calcium phosphate particle transfection

(1) Preparing solution A (300ul of 16ul of CaCI + target plasmid + sterile water), preparing solution B (300ul of 2 XHEPES), mixing the two reagents, uniformly bubbling the solution B by using an electric pipetting gun, and simultaneously sucking the solution A by using the pipetting gun and dripping the solution A into the solution B at a uniform speed.

(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 (293T cells were previously plated), and the mixture was gently shaken and evenly mixed.

(3) The transfected 293T cells are put into an incubator for continuous culture, and the size of formed calcium particles can be observed under a microscope for about 1 hour.

(4) About 4-6 hours, the transfected cells were replaced with fresh medium for a specific time determined according to the size of the calcium particles.

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

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

2.3 construction of CAP1 deletion body, the specific operation is as follows:

(1) primer design

By browsing NCBI website, downloading CAP1 gene sequence, using related software to find out enzyme cutting sites contained in the gene sequence, researching map of used vector, selecting proper restriction enzyme, dividing CAP1 into five segments as shown in figure 2, and designing required upstream and downstream primers by aid of software such as primer-5.

(2) PCR amplification of target genes

The PCR system was as follows:

and mixing the prepared PCR reaction solution uniformly, instantly separating, and putting the mixture into a PCR instrument for reaction. The reaction conditions are shown in FIG. 4: firstly, 94 ℃ for 3 min; then 94 ℃ for 1min, 55 ℃ for 1min, 72 ℃ for 1min, this step is 35 cycles; finally, 10min at 72 ℃.

(3) Tapping recovery and enzyme digestion of amplified target fragment

And after the PCR is finished, taking a proper amount of products to perform agarose gel electrophoresis detection, observing whether the gel has a main product band with the predicted molecular weight, and performing gel cutting recovery on a target band.

a. Agarose gel (with appropriate amount of 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 and diluted to 1X, and spotting was started.

c. Immediately after sample application, the electrophoresis was carried out by applying a current at a voltage of 120V, and the electrophoresis was stopped after 20 minutes.

d. And taking out the gel, observing the position of the strip under an ultraviolet lamp, then photographing and storing by using a gel imaging system, cutting off the target strip, and recovering the target gene by using a Tiangen agar recovery kit.

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

(4) Connection of

The experimental principle is as follows: under certain conditions, T4 DNA ligase catalyzes the formation of a phosphodiester bond between the adjacent 5 'phosphate and 3' hydroxyl groups of two double-stranded DNA fragments, thereby joining the two fragments. Adding 1. mu.l of vector, 1. mu. l T4 ligase, 1. mu.l of buffer and 7. mu.l of target gene fragment into the system, uniformly mixing, and placing the mixture into a water bath kettle at the temperature of 16 ℃ for 8-12 hours.

(5) Transformation of

And (3) converting by utilizing a heat shock method principle, adding 50 microliters of just melted competence into the ligation product, standing on ice for 30 minutes, then putting into a 42 ℃ water bath kettle for heat shock for 60 seconds, then putting on ice for 5 minutes, then adding 700 microliters of nonresistant LB, recovering on a 37 ℃ shaking table for 45 minutes, centrifuging at a low speed, coating a plate on a corresponding resistant plate, and inversely putting the plate in a 37 ℃ incubator for culturing for 12 hours.

(6) Shaking and extracting plasmid

And (3) in an ultra-clean workbench, selecting the clone obtained by transformation, inoculating the clone into an LB culture medium with corresponding resistance, carrying out shaking culture at 37 ℃ and 200rpm/min for 12-16 hours, then preserving the bacteria, and extracting the plasmids from the residual bacteria liquid by using a small Tiangen plasmid extraction kit, wherein the detailed steps can be referred to the instruction.

(7) Enzyme digestion identification, sequencing analysis and expression verification

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

2.4His protein purification

(1) Constructing the plasmid His-CAP1-204-330aa required for protein purification, firstly carrying out PCR to obtain the CAP1-204-330aa fragment in the same way as the above, and then connecting the sequence of the fragment to a pet28a-His vector to construct the His-CAP1-204-330 aa. After the sequencing is correct, the DNA is transformed into a Transetta strain, a single clone is selected, small inducers are made, different induction temperatures and different OD are set, and the optimal conditions for protein induction are explored.

(2) According to the induction conditions which are groped when a small amount of protein is induced, the large induction is carried out by the amplification culture. Adding the activated bacterial liquid into LB culture medium, shaking at 220rpm at 37 deg.C until OD value is 0.6, adding IPTG, adjusting temperature of shaking table to 25 deg.C, and collecting bacterial liquid after 8 hr.

(3) And (3) centrifuging the bacterial liquid, adding lysine buffer, lysozyme, PMSF and DTT, standing on ice for 15 minutes, performing ultrasonic treatment by using an ultrasonic instrument, wherein the ultrasonic treatment procedure comprises ultrasonic treatment for 3 seconds and ultrasonic treatment for 7 seconds, and repeating the ultrasonic treatment for 90 times.

(4) Centrifuging the lysate subjected to ultrasonic treatment at a high speed of 10000rpm for 30 minutes, collecting the supernatant in a new 100ml centrifuge tube, adding beads of coupled nickel, placing the centrifuge tube in a 4-DEG refrigerator, and shaking the centrifuge tube for 2 to 4 hours by using a mute mixer.

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

(6) The protein was eluted by Elution buffer, dialyzed by PBS at 4 ℃ in a refrigerator, and the concentration was measured to determine the quality of the purified His-CAP1-204-330aa protein, as shown in FIG. 5.

3. Results and analysis

Transfecting Flag-CAP1 into 293T cells, performing serum starvation treatment on the cells after 24 hours, collecting culture medium and the cells respectively after 4 hours, and cracking the cells by RIPA to obtain cell Lysate (Lysate); the culture medium is enriched with extracellular Flag-tagged proteins by Flag-M2-beads, Western blot experiment detects the enrichment condition in the culture medium, as shown in figure 1, CAP1 is found to be obviously enriched, and CAP1 is proved to be a secreted protein.

Constructing prokaryotic expression plasmid His-GFP-CAP1 and deletion mutant (1-203aa, 204-330aa, 331-475aa, delta204-330aa), purifying to obtain proteins, incubating each protein and HeLa cells for 12 hours, discarding cell culture medium, washing with PBS for three times, then cracking the cells with RIPA, and detecting proteins absorbed in the cells by WB, as shown in figure 1, b, the result shows that only CAP1 full-length protein and 204-330aa protein are detected in cell lysate.

Example 2CAP1-204-330aa protein promotion of upper membrane validation assays of GLUT1 and GLUT4

1. Experimental materials

1.1 cells

Human cervical carcinoma cell HeLa

Culture medium: DMEM + 10% fetal bovine serum

1.2 plasmids

The His-GFP-CAP1-204 and 330aa deletion mutant is constructed by taking His-GFP-CAP1 as a template

Mutant deletion

1.3 reagents

4% Paraformaldehyde

2. Experimental methods

The immunofluorescence technique is involved in this example, and the specific operation is as follows:

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

(2) cell inoculation: inoculating a proper amount of cells to be detected, performing transfection when the cells grow to 50%, and performing 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 shaking table for 10 minutes;

(4) cell perforation: the fixative was aspirated, then washed twice with PBS and incubated with 0.3% TritonX-100 in PBS for 10 min.

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

(6) adding a primary antibody: diluting the primary antibody by using 2% BSA according to a proportion, and incubating for 2 hours at room temperature or incubating overnight at 4 ℃;

(7) adding a secondary antibody: washing the primary antibody, washing with PBS for 3 times, 5 minutes each time, diluting the fluorescent secondary antibody with 2% BSA according to a proportion, incubating for 2 hours at room temperature, and keeping out of the sun after the step;

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

(9) sealing: the DAPI was aspirated, mounted, observed under a fluorescent microscope and photographed.

3. Results of the experiment

The deletion mutant (1-203aa, 204-475aa,1-330aa, 331-475aa, delta204-330aa) of Flag-CAP1 is constructed by using the construction method of the mutation deletion body, then the deletion mutant is transfected into 293T cells, after 24 hours, the cells are subjected to serum starvation for 4 hours, culture mediums and the cells are respectively collected, RIPA is used for cracking the cells, Flag-M2-beads are used for enriching the extracellular Flag-tagged protein, WB detects that the CAP1 is full-length, 204-475aa and 1-330aa can be obviously secreted out of the cells, and 1-203aa,331-475aa and delta204-330aa can not be secreted out, which indicates that the secretion of CAP1 depends on 204-330 aa. The results are shown in FIG. 2, indicating that the 204-330 amino acid sequence mediates the secretion of the CAP1 protein.

Example 3 in vitro purified CAP1-204-330aa protein facilitates the absorption of glucose by cells in a confirmation assay:

1. experimental Material

1.1 cells and culture Medium

Human cervical cancer cell HeLa, human liver cancer cell HepG2, medium: DMEM + 10% FBS

1.2 reagents

Glucose enzyme labeling detection kit (procurement from Nanjing to build biology company)

2. Experimental methods

The detection is carried out according to the kit specification, and the specific detection process is as follows:

1. Poly-D lysine plates were cultured in growth medium at 50,000-80,000 cells/well/100. mu.L/96 well or 12,500-20,000 cells/well/25. mu.L/384-well black wall/clear bottom cells for 4-6 hours.

2. Remove the cell plate from the incubator, aspirate the medium from the wells, and use 10Cells were removed in 0. mu.l/well (96-well plate) or 25. mu.l/well (384-well plate) serum-free medium. Cells were incubated at 37 ℃ with 5% CO₂Incubate in incubator for 6 hours to overnight.

3. The cell plate was removed from the incubator, the medium was aspirated from the wells, and the cells were gently washed twice with 100. mu.L/well of 1 XKRPH buffer.

4. Add 90. mu.L/well glucose uptake buffer (fraction B) and 5% CO at 37 ℃₂Cells were incubated in the incubator for 1 hour.

5. With or without insulin or test compound for 20 minutes. 10 μ L/well of 10 × insulin solution was added to a final concentration of 1 μ M or 10 × compound test solution. And also to untreated wells 10. mu.L of insulin carrier buffer or complex carrier buffer was added as a control and 5% CO at 37 ℃ C₂Incubate for 20 minutes in the incubator.

6. To each well was added 10. mu.L/well of 2-DG solution (component A) and 5% CO at 37 deg.C₂Incubate in incubator for 20-40 min. For negative controls, some wells were left untreated with insulin, inhibitor and 2-DG.

7. After treatment, the solution in each well was removed and the cells were gently washed 3 times with KRPH buffer at 100. mu.L/well to remove additional 2-DG from the solution. Remove KRPH buffer from the wells.

8. To each well 25. mu.L/well of acidic lysis buffer (component C) was added and incubated at 37 ℃ for 20 min to lyse the cells. And a 2DG uptake assay mixture can be prepared simultaneously.

9. To each well was added 25. mu.L/well of neutralization buffer (component D), mixed well, and left at room temperature for 5-10 minutes to neutralize the cell lysate.

10. To each well of 2DG6P standard or cell lysate, 50 μ L of 2DGUptake Assay working solution was added.

11. The reaction was incubated at room temperature for 30 minutes to 2 hours, protected from light.

12. The increase in absorbance ratio at 570/610nm was monitored using an absorbance plate reader.

3. Results of the experiment

An experimental group for detecting glucose uptake comprising the following procedures: the cells were cultured in a six-well plate, four wells were used, and only medium was added to the first well, no cells were cultured, and the cells were cultured from the second well, and the number of cells in each well was the same, only medium and no protein were added to the second well, medium and purified His-GFP protein were added to the third well, medium and purified His-CAP1-204 and 330aa protein were added to the fourth well, and the concentration of protein contained in the medium of each treatment group was 0.05mg/ml, and the measurement of the glucose content in the cell medium was started after 24 hours of culture.

The glucose content (glu) is measured by a microplate reader colorimetry, and the calculation formula is as follows:

the above grouping experiment for detecting glucose uptake was performed on two cell lines of Hela and HepG2, and the experimental results are shown in FIG. 3, and it can be seen that the CAP1-204 and 330aa proteins promote the uptake of glucose by the cells.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Hospital for the first person in Shanghai City

<120> humanized hypoglycemic polypeptide, preparation method and application thereof

<130> 20201119

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 127

<212> PRT

<213> Artificial Synthesis

<400> 1

Gly Leu Ala Trp Ser Lys Thr Gly Pro Val Ala Lys Glu Leu Ser Gly

1 5 10 15

Leu Pro Ser Gly Pro Ser Ala Gly Ser Cys Pro Pro Pro Pro Pro Pro

20 25 30

Cys Pro Pro Pro Pro Pro Val Ser Thr Ile Ser Cys Ser Tyr Glu Ser

35 40 45

Ala Ser Arg Ser Ser Leu Phe Ala Gln Ile Asn Gln Gly Glu Ser Ile

50 55 60

Thr His Ala Leu Lys His Val Ser Asp Asp Met Lys Thr His Lys Asn

65 70 75 80

Pro Ala Leu Lys Ala Gln Ser Gly Pro Val Arg Ser Gly Pro Lys Pro

85 90 95

Phe Ser Ala Pro Lys Pro Gln Thr Ser Pro Ser Pro Lys Arg Ala Thr

100 105 110

Lys Lys Glu Pro Ala Val Leu Glu Leu Glu Gly Lys Lys Trp Arg

115 120 125

Claims

1. The humanized hypoglycemic polypeptide is characterized in that the amino acid sequence of the humanized hypoglycemic polypeptide is shown as the sequence table SEQ ID NO: 1 is shown.

2. The humanized hypoglycemic polypeptide of claim 1, wherein the humanized hypoglycemic polypeptide is any one of the following:

(a) the amino acid sequence is SEQ ID NO: 1;

3. A membrane-penetrating peptide fragment is characterized by comprising the amino acid sequence shown in a sequence table SEQ ID NO: 1.

4. The cell-penetrating peptide fragment of claim 3, wherein the cell-penetrating peptide fragment comprises the amino acid sequence shown in SEQ ID NO: 1 is a basic skeleton, and the peptide segment obtained by extension, shortening, amino acid substitution and amino acid modification is used as a membrane-penetrating vector.

5. The method for preparing hypoglycemic polypeptide of claim 1, wherein the hypoglycemic polypeptide is artificially synthesized by genetic engineering.

6. The method for preparing the hypoglycemic polypeptide according to claim 5, comprising the following steps:

(1) constructing a plasmid His-CAP1-204-303aa required by protein purification, transforming the plasmid into a Transetta strain after the sequencing is correct, selecting a single clone, performing small induction, and groping the optimal condition of protein induction;

(2) performing amplification culture for large induction according to the optimal protein induction condition searched in the step (1); adding the activated bacterial liquid into an LB culture medium, shaking at 220rpm at 37 ℃ until the OD value is 0.6, adding IPTG, adjusting the temperature of a shaking table to 25 ℃, and collecting the bacterial liquid after 8 hours;

(3) centrifuging the bacterial liquid, adding lysine buffer, lysozyme, PMSF and DTT, standing on ice for 15 minutes, performing ultrasonic treatment by using an ultrasonic instrument, wherein the ultrasonic treatment is performed for 3 seconds and 7 seconds, and repeating the ultrasonic treatment for 90 times;

(4) centrifuging the lysate subjected to ultrasonic treatment at a high speed, collecting the supernatant in a new 100ml centrifuge tube, adding beads of coupled nickel, placing the centrifuge tube in a 4-DEG refrigerator, and shaking the centrifuge tube for 2 to 4 hours by using a mute mixer;

7. The use of a hypoglycemic polypeptide according to any of claims 1 or 2, wherein the intracellular glucose level is reduced.

8. Use of the polypeptide for lowering blood glucose according to any one of claims 1 or 2 in the preparation of a medicament for treating and/or preventing diabetes.

9. A hypoglycemic product, which comprises the hypoglycemic polypeptide or the derivative of the hypoglycemic polypeptide of claim 1 or 2, wherein the derivative of the hypoglycemic polypeptide is represented by the amino acid sequence shown in the sequence table SEQ ID NO: 1 or a derivative obtained by methylation or acylation of an amino acid sequence shown in a sequence table SEQ ID NO: 1 is a basic skeleton, and is a derivative obtained by extension, shortening, amino acid substitution and amino acid modification.

10. The hypoglycemic product of claim 9, which comprises the hypoglycemic polypeptide of claim 1 or 2, its modified derivative, and a pharmaceutically acceptable carrier.