CN106749524A - A kind of anti-fat heptapeptide NPVWKRK - Google Patents

Abstract

The invention discloses an anti-obesity heptapeptide NPVWKRK. The amino acid sequence of the synthetic polypeptide is as follows: Asn-Pro-Val-Trp-Lys-Arg-Lys, abbreviated as NPVWKRK, with a molecular weight of 927.12 and a purity of 98.95%. The heptapeptide NPVWKRK of the present invention is synthesized by a solid-phase synthesis method using a polypeptide synthesizer. Through the detection of mouse preadipocyte 3T3-L1 proliferation inhibitory activity in vitro, the polypeptide of the present invention exhibits a certain inhibitory effect on 3T3-L1 within the concentration range of 0.125-2 mg/mL. At 2 mg/mL, the in vitro proliferation inhibition rate of 3T3‑L1 was 45.38%. The invention provides a synthetic polypeptide with potential anti-obesity activity in vitro, which can be applied in the field of biopharmaceuticals.

Description

An anti-obesity heptapeptide NPVWKRK

technical field

The invention belongs to the field of biopharmaceuticals, in particular to a synthetic polypeptide and its application.

Background technique

Bioactive peptides are special protein fragments that have a positive effect on the function or state of the body and ultimately affect the health of the body. Compared with proteins, the advantages of small-molecule peptide fragments are mainly reflected in: easier to be absorbed and utilized by the human body; high activity, which can exert its unique physiological effects at a small concentration; small molecular weight, easy to modify and transform, can By artificial chemical synthesis etc. Compared with single amino acids, small molecule peptides not only have special physiological activities, but also have incomparable advantages of amino acids in absorption channels and absorption speed. Studies have confirmed that there are special oligopeptide absorption channels in the small intestine of the human body, and the protein ingested by the human body is hydrolyzed by various digestive enzymes, and is mainly absorbed in the form of low peptides. Many studies have shown that bioactive peptides from various sources have various functions such as anti-oxidation, anti-tumor, antibacterial, antihypertensive, and hypoglycemic, and have become a hot spot in the development of biomedicine and health care products. Obesity increases the risk of atherosclerosis, coronary heart disease, hypertension, diabetes, gout, fatty liver and other diseases. Therefore, the development and research of safe and harmless bioactive peptides with weight-loss and fat-lowering effects has become particularly important. Currently, peptides with weight-loss and lipid-lowering effects include liraglutide, perch active peptide, silkworm chrysalis peptide, etc.

Contents of the invention

The present invention selects mouse preadipocyte 3T3-L1 as the research object, and uses the MTT method to measure the in vitro inhibitory activity of the synthetic peptide. The purpose of the present invention is to provide a synthetic polypeptide with anti-obesity activity in vitro, which can be applied in the field of biopharmaceuticals.

The anti-obesity heptapeptide synthesized by the present invention is abbreviated as NPVWKRK, the molecular weight is 927.12, and the sequence is: Asn-Pro-Val-Trp-Lys-Arg-Lys. in,

Asn means the corresponding residue of the amino acid whose English name is Asparagine and whose Chinese name is Asparagine;

Pro means the corresponding residue of the amino acid whose English name is Proline and whose Chinese name is proline;

Val represents the corresponding residue of the amino acid whose English name is Valine and whose Chinese name is valine;

Trp represents the corresponding residue of the amino acid whose English name is Tryptophan and whose Chinese name is tryptophan;

Lys represents the corresponding residue of the amino acid whose English name is Lysine and whose Chinese name is lysine;

Arg represents the corresponding residue of the amino acid whose English name is Arginine and whose Chinese name is arginine;

Lys represents the corresponding residue of the amino acid whose English name is Lysine and Chinese name is lysine.

The amino acid sequence described in the present invention adopts the standard Fmoc scheme, through resin screening, and a reasonable polypeptide synthesis method. Link the C-terminal carboxyl group of the target polypeptide to an insoluble polymer resin in the form of a covalent bond, and then use the amino group of this amino acid as the starting point to form a peptide bond with the carboxyl group of another molecule of amino acid. By repeating this process continuously, the target polypeptide product can be obtained. After the synthesis reaction is completed, the protecting group is removed, and the peptide chain is separated from the resin to obtain the target product. Peptide synthesis is a process of repeated addition of amino acids, and the solid-phase synthesis sequence is synthesized from the C-terminus to the N-terminus.

In the present invention, the synthetic polypeptide with a final concentration of 0.125-2 mg/mL is mixed with 3T3-L1, and after incubation for 48 hours, the inhibitory rate on adipocytes reaches 14.59%-45.38% by MTT method, which can be used in the field of preparation of biomedicine in the application.

When the heptapeptide NPVWKRK concentration is 2 mg/mL, the in vitro proliferation inhibition rate of 3T3-L1 is 45.38%.

Compared with the prior art, the present invention has the following advantages and technical effects:

The present invention synthesizes the peptide for the first time, and uses the MTT method to detect the in vitro adipocyte proliferation inhibitory activity of the synthetic polypeptide, and the synthetic polypeptide has a certain adipocyte inhibitory ability.

Description of drawings

Figure 1a is the HPLC chart of the synthetic polypeptide Asn-Pro-Val-Trp-Lys-Arg-Lys.

Figure 1b is the MS chart of the synthetic polypeptide Asn-Pro-Val-Trp-Lys-Arg-Lys.

Fig. 2 is a bar graph showing the inhibitory activity of synthetic polypeptide Asn-Pro-Val-Trp-Lys-Arg-Lys on adipocyte 3T3-L1.

detailed description

The present invention will be further described below in conjunction with specific examples, but the implementation and protection scope of the present invention are not limited thereto.

Peptide Solid Phase Synthesis

Select polymer resin (China Peptide Biochemical Co., Ltd.), according to the characteristics of the amino acid sequence Asn-Pro-Val-Trp-Lys-Arg-Lys, first connect the carboxyl group of Lys to a resin in the form of a covalent bond, and then the Lys The amino group shrinks with the carboxyl group of Arg. After treatment, add Lys, the amino group of Arg reacts with the carboxyl group of Lys, add amino acids from right to left in turn, add the last Asn amino acid, and then cut off the resin to obtain the target polypeptide. Purify by high performance liquid chromatography, the chromatographic column model is Phenomenex C ₁₈ , size 4.6*150mm, mobile phase A: water containing 0.1% trifluoroacetic acid (TFA); mobile phase B: solution containing 0.09% TFA (80% Acetonitrile + 20% water); Phase B increased from 14.0% to 24.0% within 20 min, the flow rate was 1.0mL/min, and the detection wavelength was 220nm. Quick-frozen in liquid nitrogen and freeze-dried to obtain the final product, which requires a purity of over 98.95% and a structure identified by MS (as shown in Figure 1).

In vitro inhibitory activity of synthetic peptides

The growth inhibitory effect of peptide fractions on 3T3-L1 was analyzed by MTT colorimetry. The specific operation steps are as follows:

1) Take the cells in the logarithmic growth phase, digest them with 0.25% (volume) trypsin-EDTA digestion solution, add the corresponding complete medium to stop the digestion and resuspend the cells, and adjust the concentration of the cell suspension to 5×10 ⁴ cells/mL, add to 96-well plate, 100 µL per well, culture in 37 ℃ constant temperature CO2 incubator;

2) After 24 hours of culture, the cells adhered to the wall, sucked out the waste culture medium, and added a final volume of 200 µL of fresh complete medium containing different concentrations of the samples to be tested, and used the complete medium as a negative control, and cultured at a constant temperature of 37 °C in CO ₂ Cultivate in the box;

3) After 48 hours, suck out the drug solution, wash the plate twice with PBS, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37 °C;

4) After 4 h, discard the culture solution containing MTT, add 150 µl DMSO, shake on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490 nm and calculate the inhibition rate:

Adipocyte growth inhibition rate (%)=((control group OD-blank group OD)-(administration group OD-blank group OD))/((control group OD-blank group OD))×100.

Application Example 1

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a constant temperature CO ₂ incubator at 37 °C. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 125 μg/mL polypeptide sample with a final volume of 100 μL, and use the complete medium as a negative control, incubate in a constant temperature _CO2 incubator at 37 °C, suck out the drug solution after 48 h, and wash with PBS Plate twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO2 incubator at 37°C; after 4 h, discard the culture solution containing MTT, add 150 µl DMSO Oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 125 µg/mL polypeptide on adipocyte 3T3-L1 is 14.59%.

Application Example 2

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a 37°C constant temperature CO ₂ incubator. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 250 μg/mL polypeptide sample with a final volume of 100 μL, and use the complete medium as a negative control, culture in a constant temperature _CO2 incubator at 37 °C, suck out the drug solution after 48 h, and wash with PBS Plate twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37°C; after 4 h, discard the culture solution containing MTT and add 150 µl DMSO After oscillating on a micro-oscillator for 15 min, measure the optical density value at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 250 µg/mL polypeptide on adipocyte 3T3-L1 is 24.93%.

Application Example 3

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a 37°C constant temperature CO ₂ incubator. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 500 μg/mL polypeptide sample with a final volume of 100 μL, and use the complete medium as a negative control, culture in a constant temperature _CO2 incubator at 37 °C, suck out the drug solution after 48 h, and wash with PBS Plate twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh base complete medium; continue culturing in a constant temperature CO2 incubator at 37 °C; after 4 h, discard the culture solution containing MTT and add 150 µl DMSO After oscillating on a micro-oscillator for 15 min, measure the optical density value at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 500 µg/mL polypeptide on adipocyte 3T3-L1 is 31.73%.

Application Example 4

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a 37°C constant temperature CO ₂ incubator. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 1000 μg/mL peptide sample with a final volume of 100 µL, and use the complete medium as a negative control, incubate in a constant temperature CO ₂ incubator at 37 °C, suck out the drug solution after 48 hours, and wash the plate with PBS Twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37 °C; after 4 h, discard the culture solution containing MTT, add 150 µl DMSO Oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 1000 µg/mL polypeptide on adipocyte 3T3-L1 is 33.57%.

Application Example 5

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a constant temperature CO ₂ incubator at 37 °C. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 2000 μg/mL peptide sample with a final volume of 100 µL, and use the complete medium as a negative control, incubate in a constant temperature CO ₂ incubator at 37 °C, suck out the drug solution after 48 hours, and wash the plate with PBS Twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37 °C; after 4 h, discard the culture solution containing MTT, add 150 µl DMSO Oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 2000 µg/mL polypeptide on adipocyte 3T3-L1 is 45.38%.

SEQUENCE LISTING

<110> South China University of Technology

<120> An anti-obesity heptapeptide NPVWKRK

<130>

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Artificial sequence

<400> 1

Asn Pro Val Trp Lys Arg Lys

1 5

Claims (3)

1. an anti-obesity heptapeptide NPVWKRK, characterized in that the amino acid sequence of the heptapeptide NPVWKRK is Asn-Pro-Val-Trp-Lys-Arg-Lys. 2. A kind of anti-obesity heptapeptide NPVWKRK as claimed in claim 1, it is characterized in that when described heptapeptide NPVWKRK concentration is 0.125-2 mg/mL, the in vitro proliferation inhibitory rate to preadipocyte 3T3-L1 is 14.59% %-45.38%. 3. A kind of anti-obesity heptapeptide NPVWKRK according to claim 2, it is characterized in that when the concentration of the heptapeptide NPVWKRK is 2 mg/mL, the in vitro proliferation inhibition rate of 3T3-L1 is 45.38%.