LEPTIN ACTIVE PEPTIDE HAVING CD LOOP AND E HELIX REGION MUTATIONS, CODING GENE THEREOF, AND APPLICATION THEREOF

Abstract

A leptin active peptide having CD-loop and helix E region mutations, a coding gene thereof, and an application thereof are provided in the present invention. An amino acid sequence of the leptin active peptide having CD-loop and helix E region mutations is shown in SEQ ID NO.1.

Claims

1. A leptin active peptide having CD-loop and helix E region mutations, comprising an amino acid sequence as shown in SEQ ID NO.1.

2. A coding gene for coding the leptin active peptide having CD-loop and helix E region mutations according to claim 1.

3. An application of the leptin active peptide having CD-loop and helix E region mutations according to claim 1 in preparation of adipocyte-degrading dugs, weight loss drugs or hypoglycemic drugs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a design of a leptin active peptide having CD-loop and helix F region mutations; A: a three-dimensional structural diagram simulated and constructed by taking a human-derived leptin sequence as a template; B: amino acid sequence alignment and function division of No. 3 exon of the human-derived leptin (upper) and the leptin active peptide having CD-loop and helix E region mutations (lower); *: mutation site;

[0011] FIG. 2 shows activity analysis of a leptin active peptide having CD-loop and helix E region mutations; Lip-lowering activity analysis of 3T3-L1 preadipocytes (MTT detection); PEP-E: the leptin active peptide having CD-loop and helix E region mutations, as well as 10.sup.6, 10.sup.9 and 10.sup.11 after the PEP-E respectively refer to concentrations, such as 10.sup.6M, 10.sup.9M and 10.sup.11M, of the leptin active peptide having CD-loop and helix E region mutations; H-LEP: human-derived leptin protein (positive control), and 10.sup.9 refers to 10.sup.9M; Ros: rosiglitazone (positive control 2); Control: negative control. Preadipocytes: induced preadipocytes stage; D0-D2: 0-2nd day of induced differentiation (an early stage of differentiation); D9: the 9th day of induced differentiation (a mature adipocyte stage). Statistical analysis: a, positive control (H-LEP/Ros)vs. negative control (Control), adipocyte lysis activity of the positive control is obviously better than that of the negative control, P<0.05; b, PEP-E vs. H-LEP, adipocyte lysis activity of the PEP-E is obviously better than that of the H-LEP, P<0.05; c, PEP-E vs. Ros, and adipocyte lysis activity of the PEP-E is obviously better than that of the Ros. P<0.05; and*, P<0.01.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0012] Embodiments below are further descriptions of the present invention, rather than limitations to the present invention.

Embodiment 1

[0013] 1. Material and Method

[0014] 1.1. Design and Synthesis of Leptin Active Peptide having CD-Loop and Helix E Region Mutations

[0015] A leptin active peptide having CD-loop and helix E region mutations is designed in a functional region thereof according to hydrophilic/hydrophobic property change conditions of mutation sites by taking, an amino acid sequence of human-derived leptin as a template, and the amino acid sequence is SCHLHWAPGLETLDSGVQEASGY (specifically shown as SEQ ID NO.1). The leptin active peptide having CD-loop and helix E region mutations is synthesized by Shanghai Sangon Biotech Co., Ltd. Through HPLC detection, a concentration of the chemosynthetic leptin active peptide having CD-loop and helix E region mutations is greater than 23%, and a requirement for subsequent activity analysis is met. The accuracy of the synthesized leptin active peptide having CD-loop and helix E region mutations is detected by mass spectrometry, and the amino acid sequence of the leptin active peptide is confirmed as SCHLHWAPGLETLDSGVQEASGY (specifically shown as SEQ ID NO.1).

[0016] 1.2 In-Vitro Lipid-Lowering Activity Analysis of the Leptin Active Peptide

[0017] Lipid degradation activity of the synthesized leptin active peptide having CD-loop and helix E region mutations is analyzed on a cellular level by taking a 3T3-L1 preadipocyte line as a model. Specific operations are as follows:

[0018] 1.2.1 Experimental Design

[0019] (1) Grouping:

[0020] a: preadipocytes;

[0021] b: an early differentiation stage D0-D2 (0-2nd day of induced differentiation); and

[0022] c: a mature adipocyte stage D9 (the 9th day of induced differentiation);

[0023] (2) Leptin peptide and control

[0024] a. leptin active peptide having CD-loop and helix E region mutations (PEP-E): diluted to 10.sup.3M with purified water and preserved at 20 C. for later use. After an experiment is started, working concentrations have 3 concentration gradients, that is, 10.sup.6M, 10.sup.9M and 10.sup.11M;

[0025] b. positive control 1: H-LEP recombinant human-derived leptin protein (10221-HNAE, Beijing Sino Biological Inc.), diluted to 10.sup.6M with purified water and preserved at 20 C. for later use. After an experiment is started, the working concentration is 10.sup.9M.

[0026] c. positive control 2: Ros rosiglitazone, R2408, Sigma), diluted to a stock solution with a concentration of 2518 M with DMSO and preserved at 20 C. for later use. After an experiment is started, the stock solution is diluted to a working solution of 0.5 M with cell culture fluid.

[0027] d. negative control (Control): equivalent quantity of PBS

[0028] (3) cell differentiation induced liquid

[0029] a. cell differentiation induced liquid I: a complete medium containing 0.5 M IBMX(3-lsobutyl-1-methylxanthine, 15879, Sigma), 1 M dexamethasone (dexamethasone, D4902, Sigma) and 167 nM insulin (insulin, 15500, Sigma.);

[0030] b. cell differentiation induced liquid II: a complete medium containing 167 nM insulin (insulin, 15500, Sigma).

[0031] 1.2.2 3T3-L Preadipocyte Culture

[0032] (1) a complete culture solution: DMEM culture medium of 10% FBS (fetal calf serum)DMEM 45 ml+FBS 5 ml+double-antibody 0.5-0.8 ml (DMEM, Hyclone SH30243.01B500 ml high glucose, containing L-glutamine, sodium pyruvate, Fetal Bovine Serum, Invitrogen 10091-148, Penicillin-Streptomycin, Invitrogen 15140-122 100).

[0033] (2) passage: sucking old liquid, washing twice with calcium-magnesium-free PBS, adding 0.5 ml/T25 0.25% pancreatin, digesting for about 2 min (when most of cells float), and stopping digestion with complete culture solution; directly inoculating in a novel 25 cm.sup.2 culture bottle (T25) according to a ratio of 1:3 without centrifuging; and changing the liquid after 3-4 hours, and performing passage in time while growing by about 70%.

[0034] 1.2.3 Induced Differentiation of 3T3-L1 Preadipocytes into Mature Adipocytes

[0035] (1) inoculating 3T3-L1 preadipocytes in corning cell bind 96-well plates (totally inoculated with 3 plates, respectively plate a, plate b and plate c), wherein the inoculation density is 4500 cells/well; and marking, and standing and culturing at 37 C. in a 5% CO.sub.2 incubator. Each sample of each plate is operated repeatedly for 3 times, and the whole experiment is repeated for 3 times;

[0036] (2) continuously culturing for 48 hours after the cells are merged to 100% (enabling the cells to exit from a growth cycle, and starting induced differentiation.); and

[0037] (3) after the cells exit from the growth cycle,

[0038] a. preadipocytes:

[0039] i, taking out the plate a, respectively adding different amounts of leptin active peptide having CD-loop and helix E region mutations (PEP-E) to enable the working concentrations to be respectively 3 concentration gradients, that is, 10.sup.6M, 10.sup.9M and 10.sup.11M, setting 2 positive controls, a negative control (PBS) and a blank control (3 repeated holes are set for each sample), and continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator.

[0040] ii, when culturing to 45 hours (that is, 3 hours before the end of drug treatment), taking out the plate a, and measuring MTT (preadipocytes). Specific operations: adding 20 L of 5 mg/mL of MTT solution into each well, continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator for 3 hours, sucking and discarding the culture solution in the wells, adding 150 L of DMSO into each well, horizontally shaking for 10 min, detecting a light absorption value at 492 nm by a microplate reader, and drawing a growth curve.

[0041] b, the early differentiation stage (D0-D2):

[0042] i, taking out the plate b; adding the differentiation induced liquid I (D0); respectively adding the leptin active peptide having CD-loop and helix E region mutations (PEP-E,); setting 2 positive controls, a negative control (PBS) and a blank control (3 repeated holes are set for each sample), and continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator.

[0043] ii, when culturing to 45 hours (that is, 3 hours before the end of drug treatment), taking out the plate b, and measuring MTT (at the early differentiation stage, D0-D2). Specific operations: adding 20 L of 5 mg/mL of MTT solution into each well; continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator for 3 hours, sucking and discarding the culture solution in the wells; adding 150 L of DMSO into each well, horizontally shaking for 10 min, detecting a light absorption value at 492 nm by a microplate reader, and drawing a growth curve;

[0044] c. the mature adipocyte stage (D9):

[0045] i, taking out the plate c, adding the differentiation induced liquid I (D0) into each well; and continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator for 48 hours;

[0046] ii, after treating with the differentiation induced liquid I (D2) for 48 hours, taking out the plate c, adding the differentiation induced liquid II into each well, and continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator;

[0047] iii, after treating with the differentiation induced liquid II (D4) for 48 hours, taking out the plate c and replacing with a normal complete medium, and continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator;

[0048] vi, culturing for 48 hours (D6) after replacing with the normal complete medium, taking out the plate c to replace the liquid, and c standing and culturing at 37 C. in the 5% CO.sub.2 incubator for 24 hours;

[0049] v, culturing to D7 (the 7.sup.th day,); taking out the plate c to replace the liquid (a complete medium.); respectively adding the leptin active peptide having CD-loop and helix E region mutations (PEP-E), setting 2 positive controls, a negative control (PBS) and a blank control (3 repeated holes are set for each sample), and continuously standing and culturing at 37 C. in the 5% CO2 incubator for 48 hours; and

[0050] iv, when treating with drugs for 45 hours (that is, 3 hours before the end of drug treatment), taking out the plate c, and measuring MTT (mature adipocyte stage, D9), Specific operations: adding 20 L of 5 mg/mL of MTT solution into each well, continuously standing and culturing at 37 C. in the 5% CO.sub.2 incubator for 3 hours, sucking and discarding the culture solution in the wells, adding 150 L of DMSO into each well, horizontally shaking for 10 min, detecting a light absorption value at 492 nm by a microplate reader, and drawing a growth curve.

[0051] 1.2.4 Statistical Analysis

[0052] The MTT detection data is subjected to one-way analysis of variance (ANOVA) by using SPSS17.0 software.

[0053] 2. Results

[0054] 2.1 Design and Synthesis of Leptin Active Peptide having CD-Loop and Helix E Region Mutations

[0055] The leptin active peptide having CD-loop and helix E region mutations is designed and has 3 amino acid mutation sites compared with human-derived leptin. A peptide sequence is as follows: SCHLHWAPGLETLDSGVQEASGY (molecular weight: 2457.66, see FIG. 1 for details). Through HPLC detection, a concentration of the chemosynthetic leptin active peptide having CD-loop and helix E region mutations is greater than 95.23%, and a requirement for subsequent activity analysis is met.

[0056] 2.2 MTT Lipid-Lowering Activity Analysis

[0057] Cell activity analysis results show that the adipocyte-degrading activity of the synthesized leptin active peptide having CD-loop and helix E region mutations is obviously better than that of positive control-human-derived leptin protein (H-LEP) and a commercially available drug rosiglitazone (Ros,) at 3 cell differentiation stages (that is, the preadipocytes stage, the early differentiation stage and the mature adipocyte stage), and the lipid-lowering activity of the leptin active peptide having CD-loop and helix E region mutations has an optimal effect at the D0-D2 stage when the concentration is 10.sup.6M. (FIG. 2).

TABLE-US-00001 Sequencetable <110> SUNYAT-SENUNIVERSITY <120> LEPTINACTIVEPEPTIDEHAVINGCDLOOPANDEHELIXREGION MUTATIONS,CODINGGENETHEREOF,ANDAPPLICATION THEREOF <130> D-ZSDX-00201-NUS <160> 1 <170> PatentInversion15 <210> 1 <211> 23 <212> PRT <213> ArtificialSequence <220> <223> Anaminoacidsequenceoftheleptinactivepeptidehaving CD-loopandhelixEregionmutations <400> 1 SerCysHisLeuHisTrpAlaProGlyLeuGluThrLeuAspSerGly 151015 ValGlnGluAlaSerGlyTyr