APPLICATION OF ARTIFICIALLY EXPRESSED HSP27 PROTEIN IN DETECTING RESIDUE OF A ß2-ADRENERGIC RECEPTOR AGONIST BASED DRUG

Abstract

Provided is an application of an artificially expressed HSP27 protein in detecting 2-adrenergic receptor stimulant drug residue.

Claims

1. Use of an artificially expressed HSP27 protein in detecting residue of a 2-adrenergic receptor agonist based drug.

2. The use according to claim 1, wherein, the artificially expressed HSP27 protein can specifically bind to a 2-adrenergic receptor agonist based drug.

3. The use according to claim 1, comprising any artificial expression mode for the artificially expressed HSP27 protein with the protein as a core, including but not limited to protein expression by a prokaryotic expression system, a eukaryotic expression system and a yeast expression system.

4. The use according to claim 1, wherein the artificially expressed HSP27 protein is used for the detection of a 2-adrenergic receptor agonist based drug, including but not limited to an enzyme-linked immunosorbent assay.

5. The use according to any one of claims 1 to 4, wherein the artificially expressed HSP27 protein is used in the quantitative and qualitative detection of a 2-adrenergic receptor agonist based drug.

6. The use according to claim 2, wherein the artificially expressed HSP27 protein can specifically bind to a 2-adrenergic receptor agonist based drug, and can be prepared into a detection kit, a test strip or a test card for 2-adrenergic receptor agonist based drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the results of SDS-PAGE identification of the purified HSP27 protein. In the figure, M is a broad-spectrum 180KD protein Marker, and 1 is a purified HSP27 protein.

[0019] FIG. 2 shows the results of the ELISA reaction of the purified HSP27 protein and small molecules. In the figure, the abscissa represents different coating concentrations of HSP protein, and the ordinate represents OD value; the columns corresponding to coating concentrations of each HSP protein listed from left to right are CL-BIO, RAC-BIO, FH-BIO (BIO represents biotin).

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0020] Specific embodiments of the present invention will be further described in detail below with reference to the Examples.

Example 1: Small Molecule Docking Technology

[0021] 1. Preparation for Docking

[0022] The molecular structure of the small molecule 2-adrenergic receptor agonist required for docking was downloaded from ZINC, and its structure was optimized to obtain its energy-minimized state for subsequent docking.

[0023] The proteins were 2 adrenergic receptor protein 2RH1 and heat shock protein 27 (HSP27) 3ROR, both of them were downloaded from the PDB database, the corresponding ligands and non-essential parts were removed, and hydrogenation and energy minimization were performed.

[0024] According to the literature, the 2RH1 docking pocket was set as the region including Asp113, Asn312, Ser203, Phe193, Phe289, Phe290, Va1114, Val117, Trp109, Tyr308, Tyr316, Tyr199, Trp286 and Thr118 residues, Threshold was defined as 0.50, Bloat was defined as 10 A.

[0025] According to the specific conditions of 3ROR, the docking pocket was set as the region including Glu99, Asp65, Arg209, Phe100, Tyr50, Cys103, Asp203, Tyr202, Ser200, Asn199, Glu198, Trp62, Met66, Met67, Va198 and Ser45 residues and the like, Threshold was defined as 0.50, Bloat was defined as 10 A.

[0026] 2. Molecular Docking

[0027] The above-mentioned region for protein docking was used as a receptor, a small molecule was used as a ligand, the FlexX method was used for docking work, and the docking conditions were set based on the default condition in SYBYL. The Total_Score value in the docking results was used as the final criterion. The docking results of the same small molecule for two proteins were shown in Table 1.

TABLE-US-00001 TABLE 1 Results of docking between 2-adrenergic receptor agonist based drug and proteins Total_Score 2-adrenergic receptor 2- adrenergic agonist based drug CAS HSP27 receptor protein Dobutamine 34368-04-2 8.4035 7.5982 (Dobutamine hydrochloride) (49745-95-1) Fenoterol 13392-18-2 7.8134 7.3290 Ractopamine 97825-25-7 7.0472 6.8865 Salbutamol 18559-94-9 6.3827 5.7219 Pirbuterol 38677-81-5 6.3238 5.5702 Terbutaline 23031-25-6 6.2437 5.7557 Mabatero 56341-08-3 6.0874 5.0646 Metaproterenol 586-06-1 5.8220 5.8766 Clenbuterol 129138-58-5 5.8202 4.5850 Brombuterol 41937-02-4 5.7717 4.5829 Isoproterenol 7683-59-2 5.4242 5.8632 Cimaterol 54239-37-1 5.4104 5.2559 Adrenaline 51-43-4 5.2629 5.4472 Clorprenaline 3811-25-4 4.7317 5.1257 Phenylethanolamine 7568-93-6 4.2296 4.6572

[0028] 3. Analysis of Docking Results

[0029] The analysis of docking results showed that the docking results between 2-adrenergic receptor agonist based drug and HSP27 were mostly higher than those between 2-adrenergic receptor agonist based drug and 2-adrenergic receptor, indicating that the 2-adrenergic receptor agonist based drug has better binding ability to HSP27.

Example 2: HSP27 Protein Expression Experiment

[0030] According to the HSP27 gene sequence published in GenBank, the corresponding primers were designed to amplify the sequence of the CDS region, the resultant sequence was enzyme-digested and ligated to a pLSLa vector, which was subjected to PCR identification and sequencing, then transformed into BL21 (DE3) host bacteria, and subjected to induced expression by adding IPTG. The target protein was purified by an agarose resin column, and the characteristics of the expressed and purified protein were identified by SDS-PAGE (see FIG. 1 for the results). The results in FIG. 1 show that, the artificially synthesized protein of the present invention has a high yield with good purification effect.

Example 3: Identification of HSP27 Protein

[0031] the artificially expressed HSP27 protein at an optimal concentration was selected as a coating antigen, and coated on the ELISA plate in an amount of 50 L/well. The plate was placed in a 37 C. constant temperature incubator for reaction for 2 h, washed with PBST for 4 times, and air-dried at room temperature. BSA with a mass fraction of 1% was added as a blocking solution in amount of 200 L/well, and reaction was carried out at a constant temperature of 37 C. for 1 h. The resultant plate was washed with PBST for 4 times, and air-dried at room temperature. Then, HSP27 polyclonal antiserum with gradient concentrations was added at 50 L/well, a negative control (NC) was set, and the plate was held at 37 C. for 30 min, and washed for 4 times. 50 L of goat anti-mouse secondary antibody (1:1000) was added to each well, and the plate was held at 37 C. for 30 min, and then washed for 4 times. The color development reaction was carried out with TMB color developing solution in an amount of 100 L/well, and 10 minutes later, the reaction was terminated with 50 L of 2 mol/L H.sub.2SO.sub.4. The absorbance of each well was read at 450 nm with a microplate reader.

TABLE-US-00002 TABLE 2 ELISA identification results of artificially expressed HSP27 protein Protein concentration Serum dilution ratio of HSP27 5000 10000 20000 40000 80000 160000 320000 640000 1280000 2560000 1 g/mL 4.00 3.92 4.00 3.77 3.50 2.60 1.63 0.97 0.67 0.51

Example 4: Binding Experiment Between HSP27 Protein and Small Molecules

[0032] 1. The bacteria solution of BL21 (DE3) host bacteria of artificially expressed HSP27 was subjected to ultrasonication, and then coated in gradient on a ELISA plate with an initial concentration of 100 g/mL (based on the protein) in an amount of 50 L/well;

[0033] 2. 100 ng/mL of small molecules conjugated biotin was added into the resultant ELISA plate in an amount of 50 L/well, mixed well on a shaker, and incubated at 37 C. for 30 min in the dark; the ELISA plate was washed with an ELISA plate washing machine for 4 times, or the liquid in the wells of the ELISA plate was manually removed by shaking out, each well was filled with diluted buffer solution, and then shaken out the liquid to dry again, and washing as repeated in this way for 4 times;

[0034] 3. HRP-labeled streptavidin conjugate was added into the ELISA plate, and the reaction was carried out at 37 C. for 30 min; washing was performed in the same way as the above step for 4 times.

[0035] 4. TMB solution was added into corresponding microwells according to the required amount of 100 L per well, and the plate was shaken on the ELISA plate shaker for 30 s, and subjected to color development at room temperature for 10 min;

[0036] 5. 50 L of 2 mol/L sulfuric acid was added to each well to terminate the reaction, and the plate was shaken on the ELISA plate shaker for 30 s, and then the absorbance at 450 nm of each well was read with a microplate reader, and the results were judged (see FIG. 2 for the results).

[0037] The results in FIG. 2 indicate that adrenergic receptor agonists such as small molecule FH (fenoterol), CL (Clenbuterol) and RAC (ractopamine) and the like can bind to HSP27; HSP27 can play a role of binding to adrenergic receptor agonist in vitro, which lays the foundation for the detection of adrenergic receptor agonist by HSP.