CN112680476A

CN112680476A - KLHL22-DEPDC5 protein interaction screening system

Info

Publication number: CN112680476A
Application number: CN202011534849.9A
Authority: CN
Inventors: 高凯瑜; 夏培雪; 刘颖
Original assignee: Nanjing Jingruikang Molecular Medicine Technology Co ltd
Current assignee: Nanjing Jingruikang Molecular Medicine Technology Co ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-04-20

Abstract

The invention discloses a KLHL22 protein and DEPDC5 protein interaction screening system. The system comprises pHTC-KLHL22 plasmid and pNLF1-N-DEP plasmid; the pHTC-KLHL22 plasmid is a recombinant plasmid obtained by inserting a gene coding KLHL22 protein into a multiple cloning site of the pHTC plasmid; the pNLF1-N-DEP plasmid is a recombinant plasmid obtained by inserting a DNA molecule coding the DEP structure domain of DEPDC5 protein into the multiple cloning site of the pNLF1-N plasmid.

Description

KLHL22-DEPDC5 protein interaction screening system

Technical Field

The invention belongs to the field of drug screening, and particularly relates to a KLHL22-DEPDC5 protein interaction screening system.

Background

Currently, in the field of drug screening, there are many detection systems for protein-protein interaction, which are mainly classified into protein-based drug screening systems and cell-based drug screening systems, wherein the protein-level drug screening systems include an ALPHA system, an HTRF system, and the like, and are characterized by being in a lysis state or a protein purification state at the time of final detection. In addition, the cell-based drug screening system detects protein interaction in a cell survival state, and mainly comprises FRET, BRET and other technologies.

KLHL22 is an important E3 ubiquitination ligase, and substrates of the ligase comprise PLK1, DEPDC5, PD-1 and the like, and have important regulation and control functions in the aspects of cell growth, proliferation and cancer formation. In previous reports, KLHL22 activated mTOR signaling pathway via ubiquinated DEPDC5 to promote the proliferation of triple negative breast cancer. Based on this, the inventors hoped to be able to develop a system indicating DEPDC5-KLHL22 interaction, and further develop drugs related to triple negative breast cancer.

Disclosure of Invention

The invention aims to provide a KLHL22 protein and DEPDC5 protein interaction screening system.

The KLHL22 protein and DEPDC5 protein interaction screening system provided by the invention comprises a pHTC-KLHL22 plasmid and a pNLF1-N-DEP plasmid;

the pHTC-KLHL22 plasmid is a recombinant plasmid obtained by inserting a gene coding KLHL22 protein into a multiple cloning site of the pHTC plasmid;

the pNLF1-N-DEP plasmid is a recombinant plasmid obtained by inserting a DNA molecule coding the DEP structure domain of DEPDC5 protein into the multiple cloning site of the pNLF1-N plasmid.

Further, the pHTC-KLHL22 plasmid is a recombinant plasmid obtained by inserting a gene coding KLHL22 protein between a SacII enzyme digestion recognition site and an XbaI enzyme digestion recognition site of the pHTC plasmid;

furthermore, the pNLF1-N-DEP plasmid is a recombinant plasmid obtained by inserting a DNA molecule encoding the DEP structural domain of the DEPDC5 protein between the SacII enzyme digestion recognition site and the XbaI enzyme digestion recognition site of the pNLF1-N plasmid.

Wherein the protein amino acid sequence of the KLHL22 is shown as a sequence 1 in a sequence table; the amino acid sequence of the DEPDC5 protein DEP structural domain is shown as a sequence 2 in a sequence table;

the nucleotide sequence of the gene for coding the KLHL22 protein is shown as a sequence 3 in a sequence table; the nucleotide sequence of the DNA molecule for coding the DEP structural domain is shown as a sequence 4 in a sequence table.

Further, the mass ratio of the pHTC-KLHL22 plasmid to the pNLF1-N-DEP plasmid can be 1: (0.001-1), preferably 1: (0.001-0.1), more preferably 1: (0.001-0.01).

Further, the screening system also comprises HEK-293T cells.

Further, the system also includes a ligand and a fluorescein substrate.

The ligand may in particular be

NanoBRET^TM618Ligand, the fluorescein substrate can be specifically

A luciferase substrate.

The invention also provides an application of the KLHL22 protein and DEPDC5 protein interaction screening system.

The KLHL22 protein and DEPDC5 protein interaction screening system provided by the invention is applied to the preparation of a drug screening kit.

The medicament can be a medicament related to the prevention and/or treatment of breast cancer (such as triple negative breast cancer).

The invention also provides an in vitro screening method of the medicine related to preventing and/or treating breast cancer.

The in vitro screening method of the medicine related to preventing and/or treating breast cancer provided by the invention comprises the following steps:

1) transfecting a cell line with pHTC-KLHL22 plasmid and pNLF1-N-DEP plasmid in the screening system and expressing;

2) and adding a ligand into the transfected cells, simultaneously adding a compound to be screened, adding a luciferase substrate after 8 hours, uniformly mixing, detecting bioluminescent signals of 450nm and 620nm by using an enzyme-labeling instrument, and taking the 620nm signal/450 nm signal x 1000 as final protein interaction strength.

3) Comparing the protein interaction strength in a system added with the compound to be screened with the protein interaction strength in a system without the compound to be screened, if the protein interaction strength is reduced and has a significant difference (P is less than 0.05), judging that the compound has an inhibition effect on the proliferation of breast cancer cells, namely the compound sample has the potential of being used as a medicine related to the prevention and/or treatment of breast cancer; otherwise, the compound has no potential as a medicine related to preventing and/or treating breast cancer.

Wherein, the cell line is HEK-293T cell.

The ligand may in particular be

NanoBRET^TM618Ligand, the fluorescein substrate can be specifically

A luciferase substrate.

The breast cancer may in particular be triple negative breast cancer.

In order to model the KLHL22-DEPDC5 interaction, the inventors have tried the ALPHA, Nanobit, et al system. KLHL22-DEPDC5 expressed in the prokaryotic cells was found to be unable to work on the ALPHA system. It is presumed that it is difficult for prokaryotically expressed KLHL22 and DEPDC5 to interact with each other. Similarly, the inventors have utilized the NanoBit system to construct the KLHL22-DEPDC5 system, which is also not workable. Finally, the NanoBRET screening system was chosen, in which we selected the interaction of KLHL22 with the DEP domain, where depc 5 interacts with KLHL 22.

In constructing KLHL22-DEP interaction system, the inventors constructed KLHL22 and DEP to pHTC, pHTN, pNLF1-N and pNLF1-C of Nanobret, thereby obtaining 8 plasmids. And the connection of different proteins at N and C ends is tested, and finally, the pHTC-KLHL22 and pNLF1-N-DEP have the best interaction effect (figure 2). In addition to the different connection modes of the proteins, the inventors also tested the transfection ratios of the plasmids and found that the conditions were more suitable for drug screening when the plasmid amount of pHTC-KLHL22 was 2ug and the plasmid amount of pNLF1-N-DEP was 0.02ug, with higher interaction. In addition, the inventors overexpressed the ha-DEP plasmid simultaneously with the overexpression of pHTC-KLHL22 and pNLF 1-N-DEP. The NanoBRET signal of KLHL22-DEP was found to be suppressed by the overexpression of ha-DEP, indicating that the system of the present invention truly reflects the interaction of KLHL 22-DEP.

Drawings

FIG. 1 is a schematic diagram of the interaction between the proteins detected by the NanoBRET method; the NanoLuc generates 460nm fluorescence, activates 618ligand and fluorescence above 600nm, and the intensity of 600nm/460nm is the interaction intensity of two proteins.

Fig. 2 shows the detection of different KLHL22 and DEP connections.

FIG. 3 is a graph showing the detection of the intensity of the interaction between KLHL-HT and NL-DEP at different transfection ratios; KLHL-HT: NL-DEP: a1 mug +1 mug, B2 mug +0.2 mug, C2 mug +0.02 mug, D2 mug +0.002 mug and negative 2 mug +0 mug; positive 2. mu. g P53-HT + 0.2. mu.g NL-MDM 2.

Fig. 4 shows that the signal intensity of the NanoBRET system is detected by overexpression of an empty vector or ha-DEP based on overexpression of a NanoBRET-related plasmid, and the signal intensity is significantly different from the signal intensity of the NanoBRET system, wherein p is 0.0062.

FIG. 5 shows the inhibition of the KLHL22-DEP protein interaction by Compound 1A 8.

FIG. 6 is a graph of the inhibitory effect of Compound 1A8 on MD A-MB-231.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

In the following examples: human KLHL22 (uniprot: Q53GT1), DEP (DEPDC5 DEP domain)

KLHL-HT for pHTC-KLHL 22; HT-KLHL represents pHTN-KLHL 22;

KLHL-NL stands for pNLF1-C-KLHL 22; NL-KLHL represents pNLF1-N-KLHL 22;

DEP-NL stands for pNLF 1-C-DEP; NL-DEP stands for pNLF 1-N-DEP;

DEP-HT represents pHTC-DEP; HT-DEP stands for pHTN-DEP.

The following examples employ the methods: NanoBRET (promega, https:// www.promega.com.cn/Products/Protein-Interactions/Live-Cell-Protein-Interactions/NanoBRET-PPI-Starter-Systems /)

Compound 1A8, SMILE-encoded, as used in the examples below:

OC 1/C (/ O) C (═ CC ═ 1[ C @ ]3([ H ]) NC ═ 2C ═ CC ═ 2C4 ═ CC (C) ═ NN34) C (C) C has a structural formula shown in formula I:

compound 1A8 was purchased from ChemDiv library, no CAS number, and the ChenDiv library number: 7210-1961.

Example 1 construction of KLHL22-DEPDC5 protein interaction screening System

The experimental method comprises the following steps:

firstly, a related plasmid (promega, N1811) for over-expression of KLHL22 and DEP is constructed by using a traditional plasmid construction method, and specifically, KLHL22 and DEP are constructed on pHTC, pHTN, pNLF1-N and pNLF1-C of Nanobret, so that 8 plasmids are obtained. After the plasmid was obtained, the plasmid was transfected with PEI reagent (1mg/mL, biosciences, 24765).

The protein amino acid sequence of KLHL22 is shown as a sequence 1 in a sequence table; the amino acid sequence of the DEPDC5 protein DEP structural domain is shown as a sequence 2 in a sequence table;

When the 8 recombinant plasmids are constructed, the adopted enzyme cutting sites are SacII and XbaI.

pCDNA3.1-HA: available from addgene, 128034.

pCDNA3.1-HA-DEP: the DNA sequence encoding the DEP domain (as shown in sequence 4 of the sequence listing) was ligated into the pCDNA3.1-HA plasmid using BamHI and XhoI using conventional methods.

First, HEK-293T cells were grown to a suitable growth state and plated at 30% cell density in 6-well plates. One day later, 0.8-1.2 x 10 pairs⁶The number of cells was plasmid transfected 1ug (plasmid related to KLHL22 overexpression) +1ug (plasmid related to DEP overexpression). One day after transfection, cells were digested and then usedThe cells were counted in a counter (Thermofeisher) and the cell density was adjusted to 2 x 10 using Opti-MEM (Gibco) containing 4% serum (Gibco)⁶Per mL, and adding

NanoBRET^TM618Ligand (1000-fold dilution, promega, N1662). After 24 hours, 25uL of luciferin substrate(s) was added to the cells

Luciferase substrate) (100-fold dilution, promega, N1662), mixed well and added to wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer Envision). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. The results are shown in FIG. 2. As can be seen from FIG. 2, pHTC-KLHL22 has the strongest bioluminescent signal compared to the pNLF1-N-DEP group (KLHL-HT + NL-DEP) compared to the other 7 groups of conditions.

In addition to the different modes of linkage of the proteins, the inventors further examined the transfection ratios of the plasmids. pHTC-KLHL22 was transfected with pNFL1-N-DEP in the same manner as A at 1. mu.g + 1. mu.g, B at 2. mu.g + 0.2. mu.g, C at 2. mu.g + 0.02. mu.g, D at 2. mu.g + 0.002. mu.g, negative at 2. mu.g +0. mu.g, or positive at 2. mu.g pHTC-TP53+ 0.2. mu.g pNLF1-N-MDM 2. First, HEK-293T cells were grown to a suitable growth state and plated at 30% cell density in 6-well plates. One day later, cells were grouped as above for plasmid transfection. One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶Per mL, and adding

Luciferase substrate) (100-fold dilution, promega, N1662), mixed well and added to 96-well platesIn the hole. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer Envision). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. And the intensity homogenization of the group A is adjusted to be 1, and other experimental groups are correspondingly adjusted. The results are shown in FIG. 3. In four groups A-D, 2. mu.g: 0.02 μ g group (group C) and 2 μ g: the group of 0.002. mu.g (group D) had higher strength of interaction than the positive control group (positive) given by the kit.

In FIG. 4, pHTC-KLHL22 was transfected with the pNFL1-N-DEP group at 1. mu.g + 0.01. mu.g +1ug of pCDNA3.1-HA or 1. mu.g + 0.01. mu.g +1ug of pCDNA3.1-HA-DEP. One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶one/mL and 618ligand (1000-fold dilution, promega, N1662) was added. After 24 hours, 25uL of fluorescein substrate (100 fold dilution, promega, N1662) was added to the cells, mixed well and added to the wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. The intensity of the control (control) was adjusted to 1 for uniformity and the other experimental groups were adjusted accordingly. The results in fig. 4 show that the over-expressed ha-DEP group (o.v. ha-DEP) is able to competitively inhibit the interaction of KLHL22 with DEP.

Example 2 inhibition of KLHL22-DEP protein interaction by Compound 1A8

HEK-293T cells were cultured to the appropriate growth state and plated at 30% cell density in 6-well plates. Transfection was performed at 2. mu.g + 0.02. mu.g using pHTC-KLHL22 and pNFL1-N-DEP plasmid (1. mu.10)⁶Individual cells). One day after transfection, cells were digested and counted with a counter (Thermofeisher) and cell density was adjusted to 2 x 10 with Opti-MEM (Gibco) containing 4% serum (Gibco)⁶Per mL, and adding

NanoBRET^TM618Ligand (1000-fold dilution, promega, N1662). After 16 hours, cells were treated with a 10. mu.M concentration of chemolysisCompound 1A8 compound (compound) or DMSO solvent (ctrl). After 8 hours, 25uL of luciferin substrate(s) was added to the cells

Luciferase substrate) (100-fold dilution, promega, N1662), mixed well and added to wells of a 96-well plate. And bioluminescent signals at 450nm and 620nm were detected using a microplate reader (perkin elmer Envision). And 620nm signal/450 nm signal 1000 as the final protein interaction intensity. And the intensity homogenization of the control (ctrl) was adjusted to 1, with corresponding adjustments for the other experimental groups. The results are shown in FIG. 5. As can be seen from FIG. 5, the KLHL22-DEP interaction intensity was significantly lower than that of the control (ctrl) in the cells added to the compound group (compound). Indicating that the compound has the function of inhibiting the KLHL22-DEP interaction.

Example 3 inhibitory Effect of Compound 1A8 on triple negative Breast cancer cell MD A-MB-231

Typical triple negative breast cancer cells MD A-MB-231 were seeded into 96-well plates at 5000 cells per well at 100. mu.L, and after the cells were attached, 1uL of 10mM compound (compound) or DMSO solvent (ctrl) was added per well at a final concentration of 100. mu.M. After 48 hours, the number of cells was counted, and the number of cells in the drug-added group (compound) was much lower than that in the control group (ctrl), indicating that the compound had the effect of inhibiting the growth of cancer cells.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> Nanjing Jingruikang molecular medicine science and technology Co., Ltd

<120> KLHL22-DEPDC5 protein interaction screening system

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 634

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Glu Glu Gln Glu Phe Thr Gln Leu Cys Lys Leu Pro Ala Gln

1 5 10 15

Pro Ser His Pro His Cys Val Asn Asn Thr Tyr Arg Ser Ala Gln His

20 25 30

Ser Gln Ala Leu Leu Arg Gly Leu Leu Ala Leu Arg Asp Ser Gly Ile

35 40 45

Leu Phe Asp Val Val Leu Val Val Glu Gly Arg His Ile Glu Ala His

50 55 60

Arg Ile Leu Leu Ala Ala Ser Cys Asp Tyr Phe Arg Gly Met Phe Ala

65 70 75 80

Gly Gly Leu Lys Glu Met Glu Gln Glu Glu Val Leu Ile His Gly Val

85 90 95

Ser Tyr Asn Ala Met Cys Gln Ile Leu His Phe Ile Tyr Thr Ser Glu

100 105 110

Leu Glu Leu Ser Leu Ser Asn Val Gln Glu Thr Leu Val Ala Ala Cys

115 120 125

Gln Leu Gln Ile Pro Glu Ile Ile His Phe Cys Cys Asp Phe Leu Met

130 135 140

Ser Trp Val Asp Glu Glu Asn Ile Leu Asp Val Tyr Arg Leu Ala Glu

145 150 155 160

Leu Phe Asp Leu Ser Arg Leu Thr Glu Gln Leu Asp Thr Tyr Ile Leu

165 170 175

Lys Asn Phe Val Ala Phe Ser Arg Thr Asp Lys Tyr Arg Gln Leu Pro

180 185 190

Leu Glu Lys Val Tyr Ser Leu Leu Ser Ser Asn Arg Leu Glu Val Ser

195 200 205

Cys Glu Thr Glu Val Tyr Glu Gly Ala Leu Leu Tyr His Tyr Ser Leu

210 215 220

Glu Gln Val Gln Ala Asp Gln Ile Ser Leu His Glu Pro Pro Lys Leu

225 230 235 240

Leu Glu Thr Val Arg Phe Pro Leu Met Glu Ala Glu Val Leu Gln Arg

245 250 255

Leu His Asp Lys Leu Asp Pro Ser Pro Leu Arg Asp Thr Val Ala Ser

260 265 270

Ala Leu Met Tyr His Arg Asn Glu Ser Leu Gln Pro Ser Leu Gln Ser

275 280 285

Pro Gln Thr Glu Leu Arg Ser Asp Phe Gln Cys Val Val Gly Phe Gly

290 295 300

Gly Ile His Ser Thr Pro Ser Thr Val Leu Ser Asp Gln Ala Lys Tyr

305 310 315 320

Leu Asn Pro Leu Leu Gly Glu Trp Lys His Phe Thr Ala Ser Leu Ala

325 330 335

Pro Arg Met Ser Asn Gln Gly Ile Ala Val Leu Asn Asn Phe Val Tyr

340 345 350

Leu Ile Gly Gly Asp Asn Asn Val Gln Gly Phe Arg Ala Glu Ser Arg

355 360 365

Cys Trp Arg Tyr Asp Pro Arg His Asn Arg Trp Phe Gln Ile Gln Ser

370 375 380

Leu Gln Gln Glu His Ala Asp Leu Ser Val Cys Val Val Gly Arg Tyr

385 390 395 400

Ile Tyr Ala Val Ala Gly Arg Asp Tyr His Asn Asp Leu Asn Ala Val

405 410 415

Glu Arg Tyr Asp Pro Ala Thr Asn Ser Trp Ala Tyr Val Ala Pro Leu

420 425 430

Lys Arg Glu Val Tyr Ala His Ala Gly Ala Thr Leu Glu Gly Lys Met

435 440 445

Tyr Ile Thr Cys Gly Arg Arg Gly Glu Asp Tyr Leu Lys Glu Thr His

450 455 460

Cys Tyr Asp Pro Gly Ser Asn Thr Trp His Thr Leu Ala Asp Gly Pro

465 470 475 480

Val Arg Arg Ala Trp His Gly Met Ala Thr Leu Leu Asn Lys Leu Tyr

485 490 495

Val Ile Gly Gly Ser Asn Asn Asp Ala Gly Tyr Arg Arg Asp Val His

500 505 510

Gln Val Ala Cys Tyr Ser Cys Thr Ser Gly Gln Trp Ser Ser Val Cys

515 520 525

Pro Leu Pro Ala Gly His Gly Glu Pro Gly Ile Ala Val Leu Asp Asn

530 535 540

Arg Ile Tyr Val Leu Gly Gly Arg Ser His Asn Arg Gly Ser Arg Thr

545 550 555 560

Gly Tyr Val His Ile Tyr Asp Val Glu Lys Asp Cys Trp Glu Glu Gly

565 570 575

Pro Gln Leu Asp Asn Ser Ile Ser Gly Leu Ala Ala Cys Val Leu Thr

580 585 590

Leu Pro Arg Ser Leu Leu Leu Glu Pro Pro Arg Gly Thr Pro Asp Arg

595 600 605

Ser Gln Ala Asp Pro Asp Phe Ala Ser Glu Val Met Ser Val Ser Asp

610 615 620

Trp Glu Glu Phe Asp Asn Ser Ser Glu Asp

625 630

<210> 2

<211> 75

<212> PRT

<213> Artificial sequence

<400> 2

Ser Thr Gly Val Gln Leu Leu Ser Glu Gln Lys Gly Leu Ser Pro Tyr

1 5 10 15

Cys Phe Ile Ser Ala Glu Val Val His Trp Leu Val Asn His Val Glu

20 25 30

Gly Ile Gln Thr Gln Ala Met Ala Ile Asp Ile Met Gln Lys Met Leu

35 40 45

Glu Glu Gln Leu Ile Thr His Ala Ser Gly Glu Ala Trp Arg Thr Phe

50 55 60

Ile Tyr Gly Phe Tyr Phe Tyr Lys Ile Val Thr

65 70 75

<210> 3

<211> 1905

<212> DNA

<213> Artificial sequence

<400> 3

atggcagagg agcaggagtt cacccagctc tgcaagttgc ctgcacagcc ctcacaccca 60

cactgcgtga acaacaccta ccgcagcgca cagcactccc aggctctgct ccgagggctg 120

ctggctctcc gggacagcgg aatcctcttc gatgttgtgc tggtggtgga gggcagacac 180

atcgaggccc atcgcatcct gctggctgcg tcctgcgatt acttcagagg aatgtttgct 240

gggggattga aggagatgga acaggaagag gtcctgatcc acggtgtgtc ctacaatgct 300

atgtgccaaa tcctacattt catatacacc tccgagctgg agctcagcct gagcaatgta 360

caagagacac tggtggctgc ctgccagctt cagatcccag aaattatcca tttctgctgt 420

gatttcctca tgtcctgggt ggacgaagag aacattctcg atgtctaccg gctggcagag 480

ctgtttgact tgagccgcct gactgagcaa ctggacacct atatcctcaa aaactttgtg 540

gccttctctc ggactgacaa gtaccgccag cttccattgg agaaggtcta ctccctcctc 600

agcagcaatc gcctggaggt ctcctgcgag accgaggtat atgagggggc ccttctctac 660

cattatagcc tggagcaggt gcaggctgac cagatctcgc tgcacgagcc cccaaagctc 720

cttgagacag tgcggtttcc gctgatggaa gctgaggtcc tgcagcggct gcatgacaag 780

ctggacccca gccctttgag ggacacagtg gccagcgccc tcatgtacca ccggaacgag 840

agcctacagc ccagcctgca gagcccgcaa acggagctgc ggtcggactt ccagtgcgtt 900

gtgggcttcg ggggcattca ctccacgccg tccactgtcc tcagcgacca ggccaagtat 960

ctaaacccct tactgggaga gtggaagcac ttcactgcct ccctggcccc ccgcatgtcc 1020

aaccagggca tcgcggtgct caacaacttc gtatacttga ttggagggga caacaatgtc 1080

caaggatttc gagcagagtc ccgatgctgg aggtatgacc cacggcacaa ccgctggttc 1140

cagatccagt ccctgcagca ggagcacgcc gacctgtccg tgtgtgttgt aggcaggtac 1200

atctacgctg tggcgggccg tgactaccac aatgacctga atgctgtgga gcgctacgac 1260

cctgccacca actcctgggc atacgtggcc ccactcaaga gggaggtgta tgcccacgca 1320

ggcgcgacgc tggaggggaa gatgtatatc acctgcggcc gcagagggga ggattacctg 1380

aaagagacac actgctacga tccaggcagc aacacttggc acacactggc tgatgggcct 1440

gtgcggcgcg cctggcacgg catggcaacc ctcctcaaca agctgtatgt gatcgggggc 1500

agcaacaacg atgccggata caggagggac gtgcaccagg tggcctgcta cagctgcacg 1560

tctggacagt ggtcatctgt ctgcccactc cctgctgggc acggtgagcc tggcattgct 1620

gtgctggaca acaggatcta tgtgttaggt ggccgctcac acaaccgcgg cagccgcaca 1680

ggctacgtgc acatttacga tgtggagaag gactgctggg aggaagggcc ccagctggac 1740

aactccatct caggcctggc ggcctgtgtg ctcaccctgc cccgctccct gctccttgag 1800

ccgccccgcg ggacccctga ccgcagccag gccgacccgg actttgcctc tgaggtgatg 1860

agtgtgtctg actgggagga gtttgacaac tccagtgagg actag 1905

<210> 4

<211> 225

<212> DNA

<213> Artificial sequence

<400> 4

tcgacaggag tccagctgct ctctgaacag aagggcctct caccgtactg cttcatcagc 60

gcggaggtgg tacactggtt ggtgaaccac gtggagggga tccagacaca ggcgatggcc 120

attgacatca tgcagaaaat gctggaagag cagctcatca cacatgcatc tggcgaagcc 180

tggcggacct tcatctacgg cttctatttc tacaagatag taacg 225

Claims

1. A KLHL22 protein and DEPDC5 protein interaction screening system comprises pHTC-KLHL22 plasmid and pNLF1-N-DEP plasmid;

2. The system of claim 1, wherein: the protein amino acid sequence of the KLHL22 is shown as a sequence 1 in a sequence table; the amino acid sequence of the DEPDC5 protein DEP structural domain is shown as a sequence 2 in a sequence table.

3. The system of claim 2, wherein: the nucleotide sequence of the gene for coding the KLHL22 protein is shown as a sequence 3 in a sequence table; the nucleotide sequence of the DNA molecule for coding the DEP structural domain is shown as a sequence 4 in a sequence table.

4. A system as claimed in any one of claims 1 to 3, wherein: the mass ratio of the pHTC-KLHL22 plasmid to the pNLF1-N-DEP plasmid is 1: (0.001-1), preferably 1: (0.001-0.1), more preferably 1: (0.001-0.01).

5. The system of claim 4, wherein: the mass ratio of the pHTC-KLHL22 plasmid to the pNLF1-N-DEP plasmid is 1:0.01 or 1: 0.001.

6. the system of any of claims 1 to 5, wherein: the system also includes HEK-293T cells.

7. The system of any of claims 1 to 6, wherein: the system also includes a ligand and a luciferin substrate.

8. The system of claim 6, wherein: the ligand is

NanoBRET^TM618Ligand, the luciferase substrate is

A luciferase substrate.

9. Use of the system of any one of claims 1-8 in the preparation of a drug screening kit.

10. Use according to claim 9, characterized in that: the medicament is a medicament related to preventing and/or treating triple negative breast cancer.