阅读说明：本技术 蚕蛹蛋白源四肽sgqr及其用途 (Silkworm pupa protein source tetrapeptide SGQR and application thereof ) 是由 孙素玲 王伟 张玉 王君虹 朱作艺 李雪 于 2019-09-23 设计创作，主要内容包括：本发明公开了一种蚕蛹蛋白源四肽SGQR,其氨基酸序列为Ser-Gly-Gln-Arg。本发明还同时公开了上述蚕蛹蛋白源四肽SGQR在制备降血脂药物中的应用。SGQR可以显著地降低HMGCR基因和蛋白量,因此证明SGQR能降低胆固醇的合成量。(The invention discloses a silkworm pupa protein source tetrapeptide SGQR, the amino acid sequence of which is Ser-Gly-Gln-Arg. The invention also discloses an application of the silkworm pupa protein source tetrapeptide SGQR in preparation of a blood fat reducing medicine. SGQR can obviously reduce HMGCR gene and protein amount, thereby proving that SGQR can reduce the synthesis amount of cholesterol.)

1. The silkworm pupa protein source tetrapeptide SGQR is characterized in that: the amino acid sequence of the silkworm pupa protein source tetrapeptide SGQR is Ser-Gly-Gln-Arg.

2. the silkworm chrysalis protein-derived tetrapeptide SGQR according to claim 1, which is characterized in that: is a micromolecular peptide for reducing blood fat.

3. The use of the silkworm pupa protein-derived tetrapeptide SGQR according to claim 1 or 2 for preparing a blood lipid lowering medicament.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a tetrapeptide Ser-Gly-Gln-Arg (SGQR) capable of inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and reducing blood fat (cholesterol).

Background

At present, 1.8 hundred million people are broken through by hyperlipemia patients in China. With the gradual progress of China into aging society and the improvement of people's diet living standard, the number of patients with hyperlipidemia will continue to increase. Lipid metabolism disorders, especially cholesterol metabolism disorders, are well-known major cardiovascular disease-causing factors.

Silkworm chrysalis (bombyx mori) is a novel healthy food raw material, and is generally raised in China and other east Asia countries, and annual silkworm cocoons produced in China account for 70% of the total cocoon weight in the world. Silkworm is rich in protein and essential amino acids. Researches find that the silkworm pupa protein has the effects of improving obesity and regulating blood fat. Furthermore, the protein extracted from silkworm pupae is less expensive than protein derived from other protein sources. Therefore, the development and utilization of silkworm pupa protein resources have important significance and broad prospects.

Silkworm pupa protein can be hydrolyzed into polypeptide by protease in stomach and intestinal tract. Meanwhile, studies have reported that silkworm pupa protein hydrolysate can reduce the blood lipid (cholesterol) level in the plasma of mice and rats. However, at present, it is not clear which peptide monomer in silkworm pupa protein hydrolysate plays a role in regulating blood lipid (cholesterol) and the action mechanism thereof.

Most of the conventional blood lipid (cholesterol) lowering tetrapeptides are derived from amaranth (VGVL), soybean (LPYP), and the like; the inhibition rate of the protein on HMGCR is about 45 percent and 48 percent respectively.

Disclosure of Invention

the invention aims to solve the technical problem of providing the silkworm pupa protein source tetrapeptide SGQR and application thereof.

In order to solve the technical problems, the invention provides the silkworm pupa protein source tetrapeptide SGQR, wherein the amino acid sequence of the silkworm pupa protein source tetrapeptide SGQR is as follows: Ser-Gly-Gln-Arg.

The silkworm pupa protein source tetrapeptide SGQR is a small molecular peptide for reducing blood fat (cholesterol).

The invention also provides the application of the silkworm pupa protein source tetrapeptide SGQR in preparing the hypolipidemic medicament, namely, the application in improving hyperlipidemia (cholesterol).

The silkworm pupa protein source tetrapeptide SGQR is derived from silkworm pupa protein, after enzymolysis, separation and purification, a peak with the best HMGCR activity inhibition is used as a target peak for identifying the primary structure of the silkworm pupa protein hypolipidemic (cholesterol) peptide, and meanwhile, 20 HMGCR inhibitory peptides with published structure and activity data are used as a training set, and an MOE molecular software is utilized to generate a pharmacophore model SGQR of the HMGCR inhibitory peptides.

The tetrapeptides of the invention are obtained by:

(1) Degrading silkworm pupa protein by using neutral protease:

The hydrolysis degree and the HMGCR inhibition rate are used as dual-function indexes to research the enzymolysis conditions of the silkworm pupa protein. Through experiments such as response surface optimization conditions and the like, the optimal enzymolysis conditions for preparing the silkworm pupa protein hypolipidemic (cholesterol) peptide are as follows: the dosage of neutral protease is 5.1% (w/w), the enzymolysis time is 5h, the enzymolysis temperature is 52 ℃, the enzymolysis pH is 7.0, and the bottom/water ratio (w/v) is 3.9%. Under the condition, the inhibition rate of the silkworm pupa protein lipopeptide on HMGCR is 45.24%.

(2) Separating and purifying the silkworm pupa protein enzymatic hydrolysate:

Separating the peptide segment with the molecular weight less than 3kDa by using a column of sephadex G-15 to obtain 4 peaks which are named as a No. 1 peak, a No. 2 peak, a No. 3 peak and a No. 4 peak in sequence. The inhibition rates of the peak 1, the peak 2, the peak 3 and the peak 4 on HMGCR are respectively 51.05%, 31.38%, 49.47% and 52.86%.

(3) Identification of the hypolipidemic peptide:

And (3) taking the peak No. 4 obtained by the separation in the step (2) as a target peak for identifying the primary structure of the silkworm pupa protein hypolipidemic (cholesterol) peptide. Meanwhile, 20 HMGCR inhibitory peptides with published structure and activity data are used as a training set to construct a pharmacophore model of the HMGCR inhibitory peptides. And (3) constructing an active peptide structure by adopting a build-sequence module of the MOE, and performing structure optimization by utilizing an energy minimize module to obtain a lowest energy structure. The Pharmacophore search module in the MOE software is used for calculating and generating a Pharmacophore model with the predictable ability based on the activity of the compounds in the training set by using the MMFF94x force field. The pharmacophore model SGQR of HMGCR inhibitory peptide was generated using MOE molecular software.

(4) SGQR chemical synthesis and detection of HMGCR inhibition effect

And (4) chemically synthesizing the SGQR identified in the step (3), and detecting the inhibition effect of the SGQR on the HMGCR by using an RP-HPLC method. The inhibition rate of HMGCR by SGQR is found to be 77.70%.

The invention has the advantages and positive effects that:

(1) The tetrapeptide of the invention has the definite effect of reducing the expression level of HMGCR gene and protein:

SGQR can obviously reduce the HMGCR gene and protein amount, and shows that SGQR can reduce the synthesis amount of cholesterol.

(2) The tetrapeptide of the invention has the definite effects of activating LDLR gene and protein expression level:

SGQR increases LDLR gene and protein expression levels, suggesting that SGQR can effectively clear LDL in blood and ultimately lead to a decrease in LDL concentration in blood.

(3) The tetrapeptide of the invention has the definite effect of inhibiting SQS gene and protein expression level:

SGQR reduces the expression level of SQS genes and proteins, and shows that SGQR can effectively inhibit the biosynthesis of cholesterol.

According to the amino acid sequence, the amino acid sequence can be synthesized by Jile Biochemical (Shanghai) Co., Ltd, so that the blood fat (cholesterol) reducing peptide (tetrapeptide SGQR for short) can be obtained.

The application and dosage of the hypolipidemic (cholesterol) peptide (or tetrapeptide SGQR for short) are as follows:

The tetrapeptide of the invention is orally taken, and the dosage is about 0.5g (adult dosage) once taken orally, and 2-3 times daily.

Drawings

the following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a chromatogram and mass spectrum of SGQR;

A: SGQR total ion current chromatogram; b: MS of SGQR²A spectrogram;

SGQR was analyzed by LC-MS/MS.

FIG. 2 is a graph comparing the reduction of gene and protein expression levels of HMGCR by SGQR;

A: effect of SGQR on HMGCR gene expression level; b: effect of SGQR on HMGCR protein levels.

FIG. 3 is a graph showing comparison between gene and protein expression levels of LDLR reduced by SGQR;

A: the effect of SGQR on LDLR gene expression level; b: effect of SGQR on LDLR protein levels.

FIG. 4 is a graph comparing gene and protein expression levels of SQS reduced by SGQR;

A: influence of SGQR on SQS gene expression level; b: effect of SGQR on SQS protein levels.

FIG. 5 is a graph of the interaction of SGQR with HMGCR, LDLR and SQS;

Molecular docking technology is adopted to research interaction of SGQR and HMGCR, LDLR and SQS

a: interaction of SGQR with HMGCR; b: interaction of SGQR with LDLR; c: interaction of SGQR with SQS;

The left panel is the map of the active pocket, and the right panel is the map of the molecular docking results of SGQR and the active pocket.

In the above figure, SGQR is Ser-Gly-Gln-Arg.

Detailed Description

in order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.