Screening method of anticancer medicine

Abstract

The invention discloses a screening method of an anticancer medicine. The screening method of the anticancer medicine comprises the following steps: synthesizing a probe for an oligonucleotide chain coupling antibody, culturing cells, treating the cells with a medicine, detecting the interaction of cells of Hsp90 and Cdc37 by adopting an in-situ proximity ligation assay technology, and analyzing data. The interaction of cells of Hsp90 and Cdc37 can be positioned and quantified accurately, so that the high-throughput screening method of the anticancer medicine has broad prospect in the field of anticancer medicine preparation.

Description

technical field

[0001] The invention relates to the technical field of drug screening, in particular to a method for screening anticancer drugs capable of destroying the interaction between Hsp90 and Cdc37 by adopting an in situ adjacent junction technique. Background technique

[0002] Proximity ligation assay (PLA) is a highly specific and sensitive protein analysis technique. The ability to detect protein expression levels, protein-protein interactions, and protein post-transcriptional modifications (such as phosphorylation) has been applied to a range of biological research systems. The method recognizes two interacting proteins in a complex by two antibodies, each specific for the target protein. This method modifies single-stranded DNA oligonucleotides on species-specific antibodies to form proximity probes to recognize specific antibodies against the target protein. If the antigens bound by these two specific antibodies interact, the DNA linked by two pairs of corre...

Examples

Embodiment 1

[0020] Example 1 Probes for Synthetic Oligonucleotide Chain Conjugated Antibodies

[0021] (1) Add 1 μL 4mM DBCO (Dibenzylcyclooctyne, dibenzylcyclooctyne) to 2 μg / μL 10 μL anti-mouse IgG antibody and react at room temperature for 30 minutes to add NHS groups to the N-terminal; (2) Add 1 μL 1M Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid buffer) was incubated at room temperature for 5 minutes to terminate the reaction; (3) the antibody modified with NHS group and the synthesized single-chain Arm1 modified with azide group at the 5' end (SEQ ID NO:1) 1.5 μL was incubated overnight at 4°C to couple the anti-mouse IgG antibody to Arm1 to form probe 1.

[0022] Similarly, Arm2 (SEQ ID NO: 2) was added to the anti-rabbit IgG antibody to form probe 2.

Embodiment 2

[0023] Example 2 Cell culture and drug treatment

[0024] (1) Incubate the Panc1 pancreatic cancer cell line in a retort flask at 37°C, 5% CO 2 For culture, the medium is DMEM + 10% serum + 1% penicillin-streptomycin mixed solution; (2) After the cells are full, digest the cells with 0.25% trypsin and count the cells; (3) A circular glass piece with a diameter of 10 mm was placed in a 24-well cell culture plate and rinsed with DMEM; (4) Add 500 μL of 2×10 to each well of the cell culture plate 5 cells / ml, 37°C, 5% CO 2Cultivate overnight to make the cells adhere to the wall; (5) On the second day, add the drugs to be screened at a final concentration of 10 μM to each well, such as tripterine, tetrandrine, andrographolide, sophocarpine, and oxymatrine , slender diosgenine, aconitine, neoaconitine, hypoaconitine, asiaticoside, hanhuanglingsu, silymarin, evodiamine, magnolan, imperatorin, basartone, And set up a negative control. Treat the cells at 37°C for 1 hour; (6) Take...

Embodiment 3

[0025] Example 3 Detection of Hsp90-Cdc37 Interaction at the Cellular Level Using In Situ Proximity Ligation

[0026] (1) Add 50 μl blocking solution (1×PBS (phosphate buffer), add 5 mM EDTA (ethylenediaminetetraacetic acid), 20% sheep serum, 2.5 μg / mL salmon sperm ( Salmon Sperm Deoxyribonucleic acid), 25mM cysteine, 0.1% Tween20) at 37°C for blocking for 2h; (2) Add rabbit antibody specific to Hsp90 and mouse antibody specific to Cdc37, overnight at 4°C; (3) Wash with TBST Three times, add 1:50 times diluted probe 1 and probe 2, and incubate at 37°C for 1h; (4) Wash three times with TBST, add long single-stranded ligated DNA (SEQ ID NO:3) and short single-stranded ligated DNA (SEQ ID NO:3) ID NO: 4), ligation reaction buffer and T4 ligase, react at 37°C for 30 minutes; (5) wash three times with TBST, add DNA polymerase and reaction buffer, react at 37°C for 90 minutes; (6) wash three times with TBST, add fluorescence Detect single-stranded DNA (SEQ ID NO:5) and nuclear dy...

technical field

[0001] The invention relates to the technical field of drug screening, in particular to a method for screening anticancer drugs capable of destroying the interaction between Hsp90 and Cdc37 by adopting an in situ adjacent junction technique. Background technique

[0002] Proximity ligation assay (PLA) is a highly specific and sensitive protein analysis technique. The ability to detect protein expression levels, protein-protein interactions, and protein post-transcriptional modifications (such as phosphorylation) has been applied to a range of biological research systems. The method recognizes two interacting proteins in a complex by two antibodies, each specific for the target protein. This method modifies single-stranded DNA oligonucleotides on species-specific antibodies to form proximity probes to recognize specific antibodies against the target protein. If the antigens bound by these two specific antibodies interact, the DNA linked by two pairs of corre...

Examples

Embodiment 1

[0020] Example 1 Probes for Synthetic Oligonucleotide Chain Conjugated Antibodies

[0021] (1) Add 1 μL 4mM DBCO (Dibenzylcyclooctyne, dibenzylcyclooctyne) to 2 μg / μL 10 μL anti-mouse IgG antibody and react at room temperature for 30 minutes to add NHS groups to the N-terminal; (2) Add 1 μL 1M Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid buffer) was incubated at room temperature for 5 minutes to terminate the reaction; (3) the antibody modified with NHS group and the synthesized single-chain Arm1 modified with azide group at the 5' end (SEQ ID NO:1) 1.5 μL was incubated overnight at 4°C to couple the anti-mouse IgG antibody to Arm1 to form probe 1.

[0022] Similarly, Arm2 (SEQ ID NO: 2) was added to the anti-rabbit IgG antibody to form probe 2.

Embodiment 2

[0023] Example 2 Cell culture and drug treatment

[0024] (1) Incubate the Panc1 pancreatic cancer cell line in a retort flask at 37°C, 5% CO 2 For culture, the medium is DMEM + 10% serum + 1% penicillin-streptomycin mixed solution; (2) After the cells are full, digest the cells with 0.25% trypsin and count the cells; (3) A circular glass piece with a diameter of 10 mm was placed in a 24-well cell culture plate and rinsed with DMEM; (4) Add 500 μL of 2×10 to each well of the cell culture plate 5 cells / ml, 37°C, 5% CO 2Cultivate overnight to make the cells adhere to the wall; (5) On the second day, add the drugs to be screened at a final concentration of 10 μM to each well, such as tripterine, tetrandrine, andrographolide, sophocarpine, and oxymatrine , slender diosgenine, aconitine, neoaconitine, hypoaconitine, asiaticoside, hanhuanglingsu, silymarin, evodiamine, magnolan, imperatorin, basartone, And set up a negative control. Treat the cells at 37°C for 1 hour; (6) Take...

Embodiment 3

[0025] Example 3 Detection of Hsp90-Cdc37 Interaction at the Cellular Level Using In Situ Proximity Ligation

[0026] (1) Add 50 μl blocking solution (1×PBS (phosphate buffer), add 5 mM EDTA (ethylenediaminetetraacetic acid), 20% sheep serum, 2.5 μg / mL salmon sperm ( Salmon Sperm Deoxyribonucleic acid), 25mM cysteine, 0.1% Tween20) at 37°C for blocking for 2h; (2) Add rabbit antibody specific to Hsp90 and mouse antibody specific to Cdc37, overnight at 4°C; (3) Wash with TBST Three times, add 1:50 times diluted probe 1 and probe 2, and incubate at 37°C for 1h; (4) Wash three times with TBST, add long single-stranded ligated DNA (SEQ ID NO:3) and short single-stranded ligated DNA (SEQ ID NO:3) ID NO: 4), ligation reaction buffer and T4 ligase, react at 37°C for 30 minutes; (5) wash three times with TBST, add DNA polymerase and reaction buffer, react at 37°C for 90 minutes; (6) wash three times with TBST, add fluorescence Detect single-stranded DNA (SEQ ID NO:5) and nuclear dy...

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