CN109371102B

CN109371102B - Anti-tumor drug screening model with RHBDD1 as target and method thereof

Info

Publication number: CN109371102B
Application number: CN201811234898.3A
Authority: CN
Inventors: 宋伟; 赵宏; 王晓娟; 阿曼尼萨·图尔荪托合提; 杜伯雨; 缪时英; 王琳芳
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2021-11-05
Anticipated expiration: 2038-10-23
Also published as: CN109371102A

Abstract

The invention discloses an anti-tumor drug screening model taking RHBDD1 as a target spot and a method thereof. Specifically, the invention discloses an anti-tumor drug screening model taking RHBDD1 as a target, which comprises RHBDD1 protein or its truncated protein, a fluorescence-labeled substrate short peptide, a liposome transfection reagent and a model buffer solution. The invention also discloses a method for establishing an anti-tumor drug screening model taking RHBDD1 as a target spot, which comprises the following steps: a) adding RHBDD1 protein or its truncated protein, fluorescently-labeled substrate short peptide, liposome transfection reagent and model buffer solution, and mixing; b) adding a drug to be tested; c) reacting for 2-4 hours in a dark place; d) the change in fluorescence value is measured. Therefore, the invention establishes a drug screening platform with RHBDD1 as a target spot, screens the anti-tumor drug based on the RBDD1 as the target spot and provides a basis for clinical cancer treatment.

Description

Anti-tumor drug screening model with RHBDD1 as target and method thereof

Technical Field

The invention relates to the field of tumor drug screening, in particular to an anti-tumor drug screening model and method with RHBDD1 as a target spot.

Background

The tumor greatly threatens the health of human beings, and the research of the anti-tumor drug is a field which is very challenging and has great significance in the present life science. With the deep research on tumor pathogenic mechanism, the change process of signal channels in cells is being gradually clarified, and the discovery of novel targeted drugs with high efficiency, low toxicity and strong specificity by taking the signal channels as drug screening targets becomes one of the effective ways of the current research on antitumor drugs. For different diseases andmany different screening protocols have been reported and have contributed to the study of tumors: for liver microsomes, a novel multilayer microsome device has been reported for drug screening^[1](ii) a High throughput screening of dipeptidase DPP 4-targeted inhibitors has entered clinical trials^[2](ii) a Screening of epileptic compounds by stimulation of electric current^[3](ii) a And a method of screening for a drug by phenotype and the like^[4]The screening of the drugs is carried out by different tumors and specific targets in the process of exploring the tumor drugs.

Colorectal cancer is one of the common malignant tumors in the world, the mortality rate is the third place of the malignant tumors, and the colorectal cancer tends to increase year by year^[5]. Rhomboid family proteins are a highly conserved class of transmembrane serine proteases in all species, with a conserved Rhomboid domain^[6]RHBDD1 is a novel member of Rhomboid family found in the previous spermatogenesis process in the group, is highly expressed in testis tissue and is closely related to apoptosis of spermatogonia^[7,8]. The study shows that the inactivation of RHBDD1 can inhibit the growth of colorectal cancer tumor cells, and further research shows that RHBDD1 interacts with proTGF alpha, induces ADAM independent shearing and promotes proTGF alpha secretion, and the secreted proTGF alpha further activates downstream EGFR/Raf/MEK/ERK signaling pathways and is proved in a colon cancer animal model induced by colitis; RHBDD1 can also influence the occurrence and development of colorectal cancer through wnt/beta-catenin (beta-catenin) signaling pathway, and the fact that RHBDD1 can be used as a molecular marker in the prognosis of colorectal cancer tumor and is a potential drug target^[9,10,11]. In addition, RHBDD1 can influence the cell cycle by regulating CDK2 through AKT pathway, and further influence the occurrence and development of breast cancer. All the studies show that the RHBDD1 can be used as a molecular marker in tumor prognosis and is a potential drug target. The RHBDD1 has important significance in exploring new molecular markers and drug targets of colorectal cancer, and further provides important clues for targeted therapy of cancer.

Therefore, a drug screening platform with RHBDD1 as a target is constructed, screening of antitumor drugs with RBDD1 as the target is carried out, and a basis is provided for clinical treatment of cancer.

Disclosure of Invention

In view of the above objects, in a first aspect, the invention provides an antitumor drug screening model with RHBDD1 as a target, which comprises RHBDD1 protein or its truncated protein, a fluorescently-labeled substrate short peptide, a lipofection reagent and a model buffer solution.

In a particular embodiment, the sequence of the truncated protein of RHBDD1 is shown in SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.

In a specific embodiment, the fluorescently labeled substrate short peptide is fluorescently labeled KEREN short peptide, and the sequence of the KEREN short peptide is shown as SEQ ID NO. 4.

In a specific embodiment, the lipofection agent is Invitrogen^TM Lipofectamine^TM3000。

In a specific embodiment, the model buffer is an aqueous solution containing 20mM Hepes and 150mM NaCl.

In a second aspect, the present invention provides a method for establishing an anti-tumor drug screening model with RHBDD1 as a target, comprising: a) adding RHBDD1 protein or its truncated protein, fluorescently-labeled substrate short peptide, liposome transfection reagent and model buffer solution, and mixing; b) adding a drug to be tested; c) reacting for 2-4 hours in a dark place; d) the change in fluorescence value is measured.

In a specific embodiment, in step a), 0.4-1.2. mu.g of RHBDD1 protein or its truncated protein, 0.5-3.5. mu.M of fluorescently labeled substrate short peptide and 1-3. mu.l of lipofectin are added to the model buffer to 20-80. mu.l and mixed well.

In a specific embodiment, in the step b), 2-300 μ M of the drug to be tested is added to every 20-80 μ l of the screened model.

In a preferred embodiment, in step a) 0.8. mu.g of the truncated protein of RHBDD1 with the sequence shown in SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3, 2. mu.M of the fluorescently labeled KEREN short peptide and 2. mu.l of Invitrogen are added^TM Lipofectamine^TM3000, make up model buffer to 50. mu.l and mix well.

In a third aspect, the invention provides an RHBDD1 truncated protein for establishing an anti-tumor drug screening model taking RHBDD1 as a target spot, and the sequence of the protein is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

In a fourth aspect, the present invention provides a nucleotide sequence encoding the truncation protein of RHBDD1 described above.

In a fifth aspect, the invention provides the application of the RHBDD1 truncated protein in preparing a model for screening an anti-tumor drug taking RHBDD1 as a target.

In a sixth aspect, the present invention provides a method for screening an anti-tumor drug by using the anti-tumor drug screening model with RHBDD1 as a target, which comprises: a) adding the drug to be tested into the constructed anti-tumor drug screening model with RHBDD1 as the target; b) reacting for 2-4 hours at 37 ℃ in a dark place; c) the change in fluorescence at 485/528nm was measured.

In particular embodiments, the addition of the test agent does not significantly increase the fluorescence compared to a control without the test agent, indicating that the test agent may have anti-tumor properties.

In a seventh aspect, the invention provides a model for screening RHBDD1 inhibitors, which comprises RHBDD1 protein or its truncated protein, fluorescently-labeled substrate short peptide, lipofectin and model buffer. And a method of screening for an inhibitor of RHBDD1 is provided, comprising: a) adding RHBDD1 protein or its truncated protein, fluorescently-labeled substrate short peptide, liposome transfection reagent and model buffer solution, and mixing; b) adding a test agent; c) reacting for 2-4 hours in a dark place; d) the change in fluorescence value is measured.

In a specific embodiment, the lipofection agent is Invitrogen^TMLipofectamine^TM3000。

In the above aspects, the tumor is a tumor that can be treated with RHBDD1 as a target, e.g., the tumor is selected from colorectal cancer and breast cancer.

Drawings

FIG. 1: the reaction system of the antitumor drug screening model with RHBDD1 as the target point is shown in the figure. The fluorescence labeled substrate short peptide emits light, the fluorescence of the fluorescence labeled substrate short peptide is quenched after the fluorescence labeled substrate short peptide is integrated into the liposome, and the fluorescence labeled substrate short peptide is cut and released after the RHBDD1 protein or the truncated body protein thereof is added.

FIG. 2: expression of different RHBDD1 truncate proteins. The RHBDD1-207, RHBDD1-237 and RHBDD1-247 which are constructed in the expression plasmid pcDNA3.1(-) and carry Strep tags can be expressed in 293T cells.

FIG. 3: selection of the substrate KEREN. Expression plasmids for four universal substrates of the Rhombiid family (Spitz, KEREN, Gurken, TatA) were constructed and co-transfected with RHBDD1 in 293T cells, and cleavage of the four substrates by RHBDD1 was examined, wherein KEREN was significantly cleaved as indicated by the arrows in the figure and the cleavage bands were identified. KEREN was thus determined to cleave the substrate for RHBDD 1.

FIG. 4: different RHBDD1 truncated body proteins RHBDD1-207, RHBDD1-237 and RHBDD1-247 can cut the substrate, the fluorescence value is changed (increased), and delta RHBDD1 after the RHBDD1 mutation inactivation cannot cut the substrate, and the fluorescence is not increased.

FIG. 5: after the serine protease inhibitor DCI is added into the anti-tumor drug screening model taking the RHBDD1 as the target, the cleavage activity of the RHBDD1 truncated protein is inhibited, and the anti-tumor drug screening model taking the RHBDD1 as the target establishes an anti-tumor drug screening platform taking the RHBDD1 as the target.

Detailed Description

The RHBDD1 truncated protein is obtained by expressing and purifying the membrane protein RHBDD1 protein truncated body by culturing HEK293F cells in vitro. Further discovers a substrate KEREN which can be cleaved by RHBDD1 in vivo and in vitro, and after the RHBDD1 truncated protein is proved to have cleavage activity in vivo, a KEREN short peptide is constructed in vitro, and a FITC fluorescent group is added to the N end of the short peptide, so that the activity of the RHBDD1 targeting molecule is verified. In the presence of liposome, purified RHBDD1 truncated protein and a substrate KEREN short peptide are added into the system, FITC labeled KEREN short peptide is wrapped and does not fluoresce in the presence of liposome, and RHBDD1 can cut the substrate KEREN short peptide when RHBDD1 truncated protein is added, so that FITC fluorescence is released, and the cutting of the substrate KEREN short peptide by the RHBDD1 truncated protein is verified. Adding DCI (3, 4-dichloroisocoumarin) serine protease inhibitor and other RHBDD1 targeting molecules to inhibit the cleavage activity of RHBDD1 truncated protein, and then inhibiting the effect of substrate cleavage. Therefore, the screening of small molecules or polypeptides with RHBDD1 as a target can be carried out through the model, so that the anti-tumor potential drug with RHBDD1 as a target can be obtained (see figure 1).

Example 1: construction of antitumor drug screening model with RHBDD1 as target

Preparation of RHBDD1 truncated protein

1. Buffer solution and culture medium preparation

(1) Preparation of LB culture medium

Weighing yeast powder 5g, tryptone 10g and NaCl 10g respectively, adding distilled water to 1L, sterilizing in autoclave at 121 deg.C for 30 min. The solid culture medium is prepared by adding 15g of agar powder based on the liquid culture medium, sterilizing with an autoclave at 121 deg.C for 15 min, cooling to 60-70 deg.C, adding antibiotics at a certain ratio into a super clean bench, cooling, standing upside down, and placing in a refrigerator at 4 deg.C.

(2) Antibiotic preparation

The antibiotics mainly used in the embodiment are ampicillin and kanamycin antibiotic, wherein ampicillin is used in the process of culturing the escherichia coli upgraded grains, the stock solution concentration of ampicillin is 80mg/ml, and ampicillin is diluted 1000 times in the use process.

(3) DNA electrophoresis reagent

a. Nucleic acid agarose gel

The conical flask is used, and the neck of the conical flask is wrapped with a heat insulating material. Weighing 1g of agarose, adding 100mL of 1 XTAE buffer solution, heating the agarose by using a microwave oven sufficiently, boiling the agarose twice, shaking the agarose sufficiently, cooling the agarose by using tap water along the wall of a conical flask, adding a dyeing agent Golden View dye according to the proportion of 1:1000 when the temperature is about 50-70 ℃, pouring the agarose and inserting a comb, standing the agarose for 20-30 minutes generally, and storing the agarose gel in a DNA gel electrophoresis buffer solution when the agarose gel is stored.

DNA gel electrophoresis buffer

Weighing Tris 242g and Na respectively₂EDTA·2H₂O37.2 g and glacial acetic acid 57.1mL, and ultrapure water was added to make up to 1L. This was a 50X stock solution of gel electrophoresis diluted 50-fold with water at the time of use.

(4) Reagent related to protein glue

a.1 XSDS electrophoresis buffer (running buffer)

3.03g of Tris, 14.4g of glycine and 1g of SDS powder were weighed out separately and made up to a volume of 1L with water.

b. Protein loading buffer

Taking preparation of 10ml of protein loading buffer as an example, 0.8g of SDS, 0.04g of bromophenol blue, 4ml of glycerol and 0.3g of Tris-HCl are respectively weighed and placed at normal temperature for storage, 500 mu l of each tube is recommended, and 20 mu l of beta-mercaptoethanol is added into each tube before use.

c. Coomassie brilliant blue R-250 staining solution

Taking preparation 1L as an example, weighing Coomassie brilliant blue R-2501 g, anhydrous ethanol 300ml and glacial acetic acid 100ml, wherein the volume can be determined to 1L by using tap water, fully and uniformly mixing by using a rotor, and standing at room temperature for storage.

d. Coomassie brilliant blue decoloring liquid

Taking preparation 1L as an example, respectively adding 300ml of absolute ethyl alcohol and 100ml of glacial acetic acid, and fixing the volume to 1L by using tap water, fully and uniformly mixing, and standing at room temperature for storage.

(5) Buffer solution of kit relevant to Western Blot (WB) experiment

a. Film transfer liquid

The membrane transferring liquid is pre-cooled about half an hour ahead of time. 3.03g of Tris, 150ml of methanol and 14.1g of glycine are weighed respectively, and distilled water is added to the mixture to reach the constant volume of 1L.

b. Membrane washing buffer 1 XTSST

3.03g of Tris-HCl, 8.78g of NaCl and 1ml of Tween-20 (Tween-20) are weighed respectively, and water is added to the mixture to achieve a constant volume of 1L.

c. Sealing liquid

2.5g of skim milk powder was weighed out and dissolved in 50mL of 1 XTSST buffer to a final concentration of 5% skim milk powder.

(6) Strep-Tag II affinity column buffer

Respectively weighing 2.5g Hepes and 8.7g NaCl, adding water to 950ml, adjusting pH to about 7.4, adding Dithiothreitol (DL-Dithiothreitol, DTT) to make DTT final concentration be 2mM, and adding water to constant volume to 1L.

(7) Protein purification and screening buffers

The buffer components of the RHBDD1 protein purified in the early stage are as follows: 20mM Hepes, 150mM NaCl in water, pH 7.4.

2. Gene cloning and protein purification method

2.1. Acquisition of related genes and vectors

The gene template encoding RHBDD1 protein used in this example was stored in the laboratory, and the pcDNA3.1(-) empty vector used was a gift from the Yangmaojun laboratory of Qinghua university.

RHBDD1 protein Gene sequence (Gene ID:84236, SEQ ID NO: 5):

ATGCAACGGAGATCAAGAGGGATAAATACTGGACTTATTCTACTCCTTTCTCAAATCTTCCATGTTGGGATCAACAATATTCCACCTGTCACCCTAGCAACTTTGGCCCTCAACATCTGGTTCTTCTTGAACCCTCAGAAGCCACTGTATAGCTCCTGCCTTAGTGTGGAGAAGTGTTACCAGCAAAAAGACTGGCAGCGTTTACTGCTCTCTCCCCTTCACCATGCTGATGATTGGCATTTGTATTTCAATATGGCATCCATGCTCTGGAAAGGAATAAATCTAGAAAGAAGACTGGGAAGTAGATGGTTTGCCTATGTTATCACCGCATTTTCTGTACTTACTGGAGTGGTATACCTGCTCTTGCAATTTGCTGTTGCCGAATTTATGGATGAACCTGACTTCAAAAGGAGCTGTGCTGTAGGTTTCTCAGGAGTTTTGTTTGCTTTGAAAGTTCTTAACAACCATTATTGCCCTGGAGGCTTTGTCAACATTTTGGGCTTTCCTGTACCGAACAGATTTGCTTGTTGGGTCGAACTTGTGGCTATTCATTTATTCTCACCAGGGACTTCCTTCGCTGGGCATCTGGCTGGGATTCTTGTTGGACTAATGTACACTCAAGGGCCTCTGAAGAAAATCATGGAAGCATGTGCAGGCGGTTTTTCCTCCAGTGTTGGTTACCCAGGACGGCAATACTACTTTAATAGTTCAGGCAGCTCTGGATATCAGGATTATTATCCGCATGGCAGGCCAGATCACTATGAAGAAGCACCCAGGAACTATGACACGTACACAGCAGGACTGAGTGAAGAAGAACAGCTCGAGAGAGCATTACAAGCCAGCCTCTGGGACCGAGGAAATACCAGAAATAGCCCACCACCCTACGGGTTTCATCTCTCACCAGAAGAAATGAGGAGACAGCGGCTTCACAGATTCGATAGCCAGTGA

2.2. construction of the relevant vectors

Primers for each truncation protein of RHBDD1 were designed as follows:

RHBDD1-207：

forward primer (SEQ ID NO: 7):

TGGATATCTGCAGAATTCGCCACCATGTGGAGCCAC

reverse primer (SEQ ID NO: 8):

ATTCCTCGACGCGGCCGCTCATTGAGTGTACATTAGTCCACC

RHBDD1-237：

forward primer (SEQ ID NO: 9):

TGGATATCTGCAGAATTCGCCACCATGTGGAGCCAC

reverse primer (SEQ ID NO: 10):

ATTCCTCGACGCGGCCGCTCATGAACTATTAAAGTAGTATTG

RHBDD1-247：

forward primer (SEQ ID NO: 11):

TGGATATCTGCAGAATTCGCCACCATGTGGAGCCAC

reverse primer (SEQ ID NO: 12):

ATTCCTCGACGCGGCCGCTCACGGATAATAATCCTGATATCC

a gene template of the delta RHBDD1 protein (144S of the RHBDD1 protein is mutated into A) is constructed and stored before the experiment, and the gene sequence is (SEQ ID NO: 6):

ATGCAACGGAGATCAAGAGGGATAAATACTGGACTTATTCTACTCCTTTCTCAAATCTTCCATGTTGGGATCAACAATATTCCACCTGTCACCCTAGCAACTTTGGCCCTCAACATCTGGTTCTTCTTGAACCCTCAGAAGCCACTGTATAGCTCCTGCCTTAGTGTGGAGAAGTGTTACCAGCAAAAAGACTGGCAGCGTTTACTGCTCTCTCCCCTTCACCATGCTGATGATTGGCATTTGTATTTCAATATGGCATCCATGCTCTGGAAAGGAATAAATCTAGAAAGAAGACTGGGAAGTAGATGGTTTGCCTATGTTATCACCGCATTTTCTGTACTTACTGGAGTGGTATACCTGCTCTTGCAATTTGCTGTTGCCGAATTTATGGATGAACCTGACTTCAAAAGGAGCTGTGCTGTAGGTTTCGCAGGAGTTTTGTTTGCTTTGAAAGTTCTTAACAACCATTATTGCCCTGGAGGCTTTGTCAACATTTTGGGCTTTCCTGTACCGAACAGATTTGCTTGTTGGGTCGAACTTGTGGCTATTCATTTATTCTCACCAGGGACTTCCTTCGCTGGGCATCTGGCTGGGATTCTTGTTGGACTAATGTACACTCAAGGGCCTCTGAAGAAAATCATGGAAGCATGTGCAGGCGGTTTTTCCTCCAGTGTTGGTTACCCAGGACGGCAATACTACTTTAATAGTTCAGGCAGCTCTGGATATCAGGATTATTATCCGCATGGCAGGCCAGATCACTATGAAGAAGCACCCAGGAACTATGACACGTACACAGCAGGACTGAGTGAAGAAGAACAGCTCGAGAGAGCATTACAAGCCAGCCTCTGGGACCGAGGAAATACCAGAAATAGCCCACCACCCTACGGGTTTCATCTCTCACCAGAAGAAATGAGGAGACAGCGGCTTCACAGATTCGATAGCCAGTGA

in this example, PCR polymerase from the whole gold company was used, and a 50. mu.l preparation system was as follows: mu.l of 5 XPCR buffer, 5. mu.l of 2mM dNTP, 2. mu.l of template cDNA, 2. mu.l of upstream primer, 2. mu.l of downstream primer, 1. mu.l of enzyme, and 28. mu.l of sterile water were added, respectively.

The PCR reaction conditions were as follows:

pre-denaturation: 5 minutes at 98 ℃; denaturation: 30 seconds at 98 ℃; annealing: 30 seconds at 58 ℃; extension: 60 seconds at 72 ℃; then 34 cycles were performed; and then extending: 5 minutes at 72 ℃.

After completion of PCR, 10. mu.l of 6 XDNA sample addition buffer was added, and DNA gel electrophoresis was performed to separate the correct DNA gel band and to perform gel cutting recovery using a commercial rapid gel recovery kit.

The enzyme digestion reaction conditions are as follows:

fragment cleavage after PCR: mu.l of the fragment, 2. mu.l of ECOR1 enzyme, 2. mu.l of Kpn1 enzyme, and 5. mu.l of digestion buffer were added thereto, and the mixture was digested at 37 ℃ for 3 hours.

And (3) carrying out enzyme digestion on the vector: 1500ng of the uncut vector, 2. mu.l of ECOR1 enzyme, 2. mu.l of Kpn1 enzyme, and 5. mu.l of digestion buffer were added thereto, and the mixture was digested with sterile water to 40. mu.l, at 37 ℃ for 3 to 5 hours.

And (3) carrying out agarose gel electrophoresis on the fragments and the vector after enzyme digestion, and then respectively carrying out gel recovery.

The fragment and vector were subsequently recovered using T4 ligase, 15. mu.l fragment, 2. mu.l vector, 1. mu.l T4 ligase and 2. mu.l T4 buffer were added. Then placed at 16 ℃ for 2 hours for ligation, after which transformation experiments were performed using DH5 alpha competence, plated, and placed in a 37 ℃ incubator overnight.

The next morning, the single colony is placed in an EP tube for culture, 200 mul of culture medium containing antibiotics is added, a shaking table at 37 ℃ is used, the culture is carried out for 3-5 hours at 220rpm, when the bacterial liquid is turbid, the PCR of the bacterial liquid is carried out by using Taq enzyme, and if the PCR of the bacterial liquid is correct, the sequencing is carried out.

2.3. Preparation of large quantity extracted plasmid and transfection reagent PEI (polyimide)

Due to the use of transient transfection technology, large amounts of plasmid and transfection reagent PEI are required to meet the subsequent protein purification work.

Mass extraction of plasmids: the plasmid inserted into each RHBDD1 truncated protein and RHBDD1 mutant obtained in the last step through gene cloning is transformed into DH5 alpha competent cells, and passes through a constant temperature incubator at 37 ℃ for overnight culture for about 12 hours, a single colony is picked up in a clean bench and added into a 50mL LB vial, the concentration of ampicillin is maintained at 80 mug/L, overnight culture is carried out at 220rpm, the single colony is transferred into a 4L LB vial culture medium, the culture is continued for about 6 hours, thalli are collected, and plasmids are extracted by using a Kangji plasmid extraction kit.

Preparing PEI: in this example, 50mg of PEI was weighed using a precision balance using PEI solid powder purchased from Polysciences, added to a clean 50mL centrifuge tube, double distilled water was added to 45mL, the centrifuge tube was sealed and placed in a 85 ℃ water bath, and heated for 20 minutes in a hot water bath, and the dissolution of the PEI solid powder was observed, after which the centrifuge tube was taken out and cooled to room temperature, the pH was adjusted to 7.2 using a pH meter, the volume was made to 50mL using double distilled water, filtered through a 0.22 μm filter in a clean bench and dispensed, and then frozen in a-20 ℃ refrigerator. When the PEI solid is used again, if the PEI solid is separated out, the PEI solution can be placed in a water bath kettle at the temperature of 65 ℃, the water bath is used for heating for 5 minutes, and the PEI solution can be continuously used after being cooled to the normal temperature.

Cell culture of HEK293F

HEK293F cells used in this example were from the university of qinghua, baopenjun laboratory gift. During the initial culture of the cells, the cryopreservation and recovery of mammalian cells are involved. The HEK293F cell is domesticated by human embryo kidney cell, and the cell line rarely expresses the endogenous ligand needed by the extracellular ligand, is easy to transfect and operate, and is the most common exogenous expression system.

Reviving HEK293F cells: frozen HEK293F cells were removed from liquid nitrogen and placed in a 37 ℃ water bath for about 1-2 minutes, the cells were lysed and immediately transferred to a clean bench, the lysed cells were added to about 10ml of cell culture medium (293Ti medium, available from Yinqiao), the cells were resuspended, and centrifuged at 600g for 5 minutes using a centrifuge, and the supernatant was discarded, followed by addition of 10ml of medium for a second resuspension and centrifugation. Finally, after about 25ml of culture medium is used for heavy suspension, and the streptomycin double antibody is added, and the mixture is placed in a carbon dioxide incubator for culture.

Cryopreservation of HEK293F cells: in this example, it is generally necessary to freeze cells with a small number of culture generations and cells with a good growth state. Collecting cells with good growth state at a density of about 1.5-2.5 × 10⁶Taking about 100ml of each cell/ml, centrifuging for 5 minutes by using a centrifuge 600g, discarding the supernatant, resuspending by using a freezing buffer solution (10% DMSO, 40% FBS and 50% culture medium), subpackaging into a freezing tube, placing into a freezing box, placing in a refrigerator at minus 80 ℃ for overnight, and transferring the frozen cells to liquid nitrogen the next dayStoring in a jar.

Passage of HEK293F cells: the mammalian cells need to be added with streptomycin during the culture process to inhibit the contamination of gram positive/negative bacteria. At the beginning of the culture, the cells were cultured using a vial, and for HEK293F suspension cells, the growth cycle was typically 24 hours until the cell density increased to 2-3X 10⁶At individual cells/ml, the vials were passaged to large bottles and diluted to a concentration of about 0.5X 10⁶Individual cells/ml, theoretically, the concentration of the culture medium should not be too high to exceed 4X 10⁶The growth state of the cells is deteriorated and aggregation phenomenon is generated per ml; the passage concentration is not too low and is not less than 0.3-0.5 × 10⁶Individual cells/ml. Too low a concentration of cells is less likely to grow.

2.5. Transient transfection of mammalian cells and optimization of transfection conditions

The transient transfection technology (transient transfection) is a simple, fast and efficient mammalian expression method, and is a way to introduce exogenous DNA into eukaryotic cells, PEI as a cationic liposome can wrap plasmid DNA with negative charges and enter the cell body through the endocytosis process, so that the transient but high-level expression of a target gene is obtained. In the operation process, the plasmid to be transfected does not need to be introduced into a host chromosome, and the transfected protein can be obtained in a short time. Has the advantages of simple operation, short period, high expression efficiency, safety, high efficiency, no need of screening genes, capability of operating on complete cell strains and the like.

For example, 500ml of HEK293F cells were transfected: when the cells are in logarithmic growth phase (1.5-2.5X 10)⁶One cell/ml) in a clean bench, two sterile centrifuge tubes were opened, and 25ml of antibiotic-free medium were added, one of the centrifuge tubes containing 0.5mg of plasmid and the other centrifuge tube containing 2mg of PEI. And placing the mixture in an oven at 37 ℃, standing and incubating for 3-5 minutes, slowly and dropwise adding the PEI solution into a plasmid buffer solution in a super clean bench, fully and uniformly mixing, then incubating in the oven at 37 ℃ for 20-30 minutes to form PEI-coated plasmid liposome, and then dropwise adding the PEI-coated plasmid liposome into cells in logarithmic phase. Culturing for 48 hours in generalThereafter, the transfection process is completed.

In addition, different transfection conditions were optimized for different proteins.

Four time gradients were set up as optimized for transfection time (24 hours, 36 hours, 48 hours, 60 hours), and cells were harvested at the expected time points and tested by Western Blot experiments. Transfection ratio optimization, cells used in this example: plasmid: the proportion of PEI is 1:1:4, different mixing proportions are respectively set in the process of optimizing conditions, and in a simple way, the proportion of fixing cells and plasmids is 1:1, and then the optimal proportion of PEI is screened and added. And then, according to the proportion of the screened PEI, changing the proportion of the plasmid, and screening out a proper proportion of the plasmid. Finally, combining with transfection time screening, the final optimized conditions are cell: plasmid: PEI 1:1:4, cells were harvested 48 hours after transfection.

Strep-Tag protein purification of RHBDD1 truncated protein

Strep-Tag II is an affinity purified protein Tag containing eight amino acids, which can help better obtain protein with higher purity, especially for membrane protein.

The transfected cells were harvested, typically in 1L extraction units, and HEK293F cells were resuspended in resuspension buffer (20mM Hepes pH 8.0, 150mM NaCl, 1mM PMSF, 2mM DTT), noting that protein purification was typically performed at 4 ℃ or ice temperature. The resuspended cells were crushed twice under high pressure using a high pressure crusher, and the resulting cell crushed solution was centrifuged for 5 minutes at 13000rpm using a high-speed low-temperature centrifuge to remove cell debris, and then the supernatant was collected and centrifuged using ultra-high-speed centrifugation of Beckmann, using a 70Ti rotor at 41000rpm for 1 hour. The supernatant was then gently discarded and the pellet in the tube was stored on ice. The bottom of the tube was resuspended using a rubber-tipped pipette and a buffer (25mM Tris pH 8.0, 200mM NaCl, 2mM DTT), and the resuspended suspension was disrupted using a homogenate disrupter. Adding DDM detergent to obtain a final concentration of 1% DDM, placing on a four-degree shaker, slowly shaking, and extracting for 2-3 hr.

After completion of the extraction, the extract was centrifuged at 41000rpm for 30 minutes using an ultra high speed centrifuge, and the supernatant was retained and filtered using a 0.45 μm filter.

The affinity column was pre-equilibrated with Strep column buffer (20mM Hepes pH 8.0, 150mM NaCl, 2mM DTT), typically around 2 column volumes. The protein supernatant was then hung onto the column twice, after which the contaminating proteins were washed off using wash buffer (20mM Hepes pH 8.0, 150mM NaCl); during the period, Coomassie brilliant blue G250 can be used for detecting whether the hybrid protein is washed clean, and the washing volume is about 10 column volumes generally. Finally, the target protein was eluted using an elution buffer (20mM Hepes pH 8.0, 150mM NaCl, 5mM desthiobiotin, 2mM DTT). After protein collection, the protein was concentrated using a Millipo protein concentration tube to facilitate the next experimental procedure. The concentration of the concentrated protein is 0.2-0.4 mg/ml, and the final protein is dissolved in elution buffer (20mM Hepes pH 8.0, 150mM NaCl, 5mM desthiobiotin, 2mM DTT) for later use.

It is noteworthy that the column needs to be treated each time after the Strep tag affinity column has been used. The method comprises the following steps:

washing the affinity column with reconstitution buffer (100mM Tris pH 8.0, 100mM NaCl, 1mM EDTA, 5mM HABA), typically for 5-10 column volumes;

washing the affinity column with affinity column buffer (25mM Tris pH 7.8, 150mM NaCl, 2mM DTT) for 5-10 column volumes;

washing the affinity column with 0.5M NaOH solution, washing away residual HABA, typically 5 column volumes;

washing the affinity column with double distilled water, washing NaOH, generally washing 5 column volumes;

the affinity column was washed with an affinity column buffer (25mM Tris pH 7.8, 150mM NaCl, 2mM DTT) for 10-20 column volumes and stored in a refrigerator at 4 ℃.

SDS-PAGE and Western Blot protein detection

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis. The prepared protein sample is completely opened in hydrogen bonds and hydrophobic bonds in the protein under the action of SDS, the SDS is used as an anionic surfactant, can be combined with the surface of the protein and enables the protein to be negatively charged, the charge quantity is far more than the original charge quantity of protein molecules, and further the charge difference among different proteins is covered, and at the moment, the protein molecule mobility of different lanes is mainly determined by the molecular weight. If the protein has a larger molecular weight, the mobility of the molecules is slower, and it is located above the lanes on SDS-PAGE. Generally, a protein sample is mixed with a protein sample-adding buffer solution in advance, and the mixture is heated in a heating block at 100 ℃ to sufficiently denature the protein sample, centrifuged, applied to SDS-PAGE gel, electrophoresed at 180V for about 60 to 80 minutes, stained with a staining solution for half an hour, and then decolorized with a decolorizing solution for 1 hour, thereby allowing observation of a protein band.

The Western Blot protein detection experiment steps are as follows:

(1) carrying out electrophoresis on a sample to be detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and generally using a prestained protein marker;

(2) after the electrophoresis is finished, transferring the protein gel sample to a PVDF membrane by using a wet-to-membrane converter for about 1.5 hours;

(3) after the membrane conversion is finished, sealing the PVDF membrane by using a sealing liquid (PBST + 5% skim milk), generally about 30 minutes;

(4) a first antibody: in the experiment, a mouse Strep-tag primary antibody is used, added into a sealing solution according to the proportion of 1:5000, and incubated for 1 hour at room temperature;

(5) washing the membrane: washing the membrane by using a TBST solution for three times, wherein each time lasts for about 5 minutes;

(6) secondary antibody: adding a goat anti-mouse secondary antibody according to the proportion of 1:5000, and incubating for 1 hour at room temperature;

(7) washing the membrane with PBST solution for about 5 minutes three times;

(8) a developer solution was added and exposure was performed using a bio-rad machine.

In the embodiment, 5 different RHBDD1 truncated bodies are constructed, wherein the RHBDD1-207, the RHBDD1-237 and the RHBDD1-247 are well expressed in HEK293F cells, so that a screening model is constructed after the RHBDD1-207, the RHBDD1-237 and the RHBDD1-247 are purified in a subsequent experiment. Wherein Strep is a tag antibody (see FIG. 2).

The purified truncated proteins RHBDD1-207, RHBDD1-237, RHBDD1-247 and the mutant delta RHBDD1 constructed previously are finally obtained by the method and are used for subsequent experiments, and the amino acid sequences are respectively as follows:

RHBDD1-207(SEQ ID NO:1)：

RHBDD1-237(SEQ ID NO:2):

RHBDD1-247(SEQ ID NO:3):

preparation of substrate KEREN short peptide

Because the protein of the Rhombioid family is highly conserved, the inventor constructs four substrates KEREN, Spitz, Gurken and TatA of the Rhombioid family and expresses the four substrates in 293T cells (cell resource center of Beijing Council medical college), after the RHBDD1 is co-transfected, WB verifies the cleavage effect of the RHBDD1 on the four substrates, and the cleavage effect on the KEREN is found to be the best, so the KEREN is selected as the substrate for in vitro screening.

The results are shown in FIG. 3, and the cleavage effect is detected by WB after different substrates are transfected in 293T cells and RHBDD1 is transfected at the same time, the cleavage effect is obvious after KEREN is transfected, and the cleavage band is also shown. KEREN was therefore selected as substrate for in vitro screening.

Substrate KEREN short peptide: the sequence of amino acids between 122-144 positions of the transmembrane region of KEREN protein is reserved, and a plurality of amino acid sequences at two ends are reserved to synthesize a short peptide sequence as follows (SEQ ID NO: 4): RNRVMLEKASIVSGATLALLFMAMCCVVLYLRHEKLQ and, the substrate KEREN is conjugated with FITC, the FITC-Ahx synthesis process is the dehydration condensation process of amino Acid, the synthesis process is connected by amido bond, Ahx is 6-Aminocaproic Acid (amino Acid), and the synthesis process obtains:

FITC-Ahx-RNRVMLEKASIVSGATLALLFMAMCCVVLYLRHEK LQ-NH2，

it was synthesized by Beijing Zhongke Sudoku Biotech Co., Ltd, and dissolved to 50. mu.M with a protein purification buffer (20mM Hepes pH 8.0, 150mM NaCl) for use.

Preparation of model buffer solution

The concrete formula of the model buffer solution is as follows:

20mM Hepes, pH7.4, 150mM NaCl in ddH₂O。

Using preparation 1L as an example, 4.766g Hepes, 8.766g NaCl were weighed out and about 800ml ddH was added₂Adjusting the pH of the solution to 7.4 after O, and then metering to 1L.

IV, liposome

The liposome used in this example was Invitrogen^TM Lipofectamine^TM 3000。

The experimental materials and instruments used in this example are shown in the following table:

example 2: validation of the cleavage of FITC-labeled KEREN by the different RHBDD1 proteins

The RHBDD1 truncated protein obtained in example 1, FITC-labeled KEREN, and model buffer were combined with commercially available liposomal Invitrogen^TM Lipofectamine^TM3000 can be used for constructing an anti-tumor drug screening model taking RHBDD1 as a target.

The corresponding reagents were added to different wells in black 384-well plates (3 multiple wells per set):

reacting for 2-4 hours at 37 ℃ in a dark place; the Synergy H1 fully functional microplate detector measures changes in fluorescence values (relative fluorescence units, RFU) at 485/528 nm. The data in this example are presented as mean value standard deviations, and all statistical analyses were performed using Prism version 5.0.

Control group (null), among others: no RHBDD1 protein was added, but an equal volume of solution dissolving RHBDD1 protein was added. Mutant delta RHBDD1 protein, RHBDD1-207, RHBDD1-237 and RHBDD1-247 truncated protein are added into the experimental group respectively.

Experimental results (see fig. 4): null is a control, the fluorescence of the delta RHBDD1 is not obviously different from that of a control group, and the fluorescence value is obviously increased after the addition of different truncations of RHBDD1, RHBDD1-207, RHBDD1-237 and RHBDD 1-247. The fluorescence of the delta RHBDD1 is not obviously different from that of a control group, which shows that the delta RHBDD1 has no cutting effect on a substrate KEREN (Krn) short peptide, and the fluorescence value of the RHBDD1 different truncations RHBDD1-207, RHBDD1-237 and RHBDD1-247 is obviously increased after the addition, thereby proving that the RHBDD1 truncate has the cutting effect on the substrate KEREN.

Example 3: verification of antitumor drug screening model with RHBDD1 as target

The system used in this example was substantially the same as in example 2, except that DCI was additionally added at a concentration of 20. mu.M. When drug screening is carried out, the adding amount of the drug to be detected can be tried at 2-300 mu M until proper concentration is determined, the concentration can be found from high concentration to low concentration, and the drug concentration can be tried at 200 mu M at the beginning.

Experimental results (see fig. 5): null is a control, the fluorescence of the delta RHBDD1 has no obvious difference with the control group, the fluorescence of the RHBDD1-207+ DCI has no obvious difference with the control group, and the fluorescence value of the RHBDD1 truncated protein RHBDD1-207 is obviously increased after the addition.

Discussion: DCI is a serine protease inhibitor, RHBDD1 belongs to serine protease, delta RHBDD1 is an RHBDD1 mutant, RHBDD1 is inactivated after mutation and cannot cut a substrate KEREN, so that the fluorescence value is not increased, and has no obvious difference with a control group (null), the DCI inhibitor is added, the DCI can inhibit the activity of RHBDD1, when RHBDD1 is inhibited, the substrate is not cut, the fluorescence is not increased, only when the RHBDD1 is not mutated, the activity is realized, the inhibitor corresponding to the RHBDD1 does not inhibit the substrate KEREN, and the substrate KEREN can be cut.

Reference documents:

1.Wu,Q.,et al.,Development of a novel multi-layer microfluidic device towards characterization of drug metabolism and cytotoxicity for drug screening.Chem Commun(Camb)；2014；50(21):2762-2764.

2.Upadhyay,J.,et al.,Combined Ligand-Based and Structure-Based Virtual Screening Approach for Identification of New Dipeptidyl Peptidase4Inhibitors.Curr Drug Discov Technol.2018.doi:10.2174.

3.Simonato,M.,et al.,Finding a better drug for epilepsy:preclinical screening strategies and experimental trial design.Epilepsia.2012.53(11):1860-1867.

4.Enna,SJ.,et al.,Phenotypic drug screening.J Peripher Nerv Syst.2014；2:S4-5.

5.Mundade,R.,et al.,Genetic pathways,prevention,and treatment of sporadic colorectal cancer.Oncoscience.2014；1(6):400-4066.

6.Waters,C.M.et al.,Quorum sensing:cell-to-cell communication in bacteria.Annu Rev Cell Dev Biol,2005；21:319-346.

7.Cohen,S.J.,et al.,Isolation and characterization of circulating tumor cells in patients with metastatic colorectal cancer.Clin Colorectal Cancer,2006；6(2):125-132.

8.Ioannidis,I.,et al.,Comparative study of the immunohistochemical expression of metalloproteinases 2,7 and 9 between clearly invasive carcinomas and"in situ"trophoblast invasion.Neoplasma.2010；57(1):20-28.

9.Song,W.,et al.,Rhomboid domain containing 1 promotes colorectal cancer growth through activation of the EGFR signalling pathway.Nat Commun,2015；(6):8022.

10.Zhang，M.,et al.,RHBDD1 promotes colorectal cancer metastasis through the Wnt signaling pathway and its downstream target ZEB1.J EXP CLIN CANC RES，2018；(37):22.

11.Miao,F.,et al.,RHBDD1 upregulates EGFR via the AP-1 pathway in colorectal cancer.Oncotarget,2017；8(15):25251-25260.

sequence listing

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Claims

1. An antitumor drug screening model taking RHBDD1 as a target comprises RHBDD1 protein or its truncated protein, a fluorescence labeled substrate short peptide, a liposome transfection reagent and a model buffer solution, wherein the amino acid sequence of the truncated protein of the RHBDD1 protein is shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3, and the fluorescence labeled substrate short peptide is a fluorescence labeled KEREN short peptide.

2. The RHBDD1 targeted antitumor drug screening model of claim 1, wherein the KEREN short peptide has the sequence shown in SEQ ID NO. 4.

3. The RHBDD 1-targeted antitumor drug screening model according to claim 1, wherein the lipofectin reagent is Invitrogen-Lipofectamine-3000.

4. The model for screening antitumor drug targeting RHBDD1, according to claim 1, wherein the model buffer is an aqueous solution containing 20mM Hepes and 150mM NaCl.

5. A method for establishing an anti-tumor drug screening model taking RHBDD1 as a target comprises the following steps:

a) adding RHBDD1 protein or its truncated protein, fluorescently-labeled substrate short peptide, liposome transfection reagent and model buffer solution, and mixing;

b) adding a drug to be tested;

c) reacting for 2-4 hours in a dark place;

d) measuring the change in fluorescence value;

the amino acid sequence of the truncated protein of the RHBDD1 protein is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, and the fluorescence-labeled substrate short peptide is fluorescence-labeled KEREN short peptide.

6. The method of claim 5, wherein,

in the step a), 0.4-1.2 mu g of RHBDD1 protein or its truncated protein, 0.5-3.5 mu M of fluorescence labeled substrate short peptide and 1-3 mu l of liposome transfection reagent are added, model buffer solution is supplemented to 20-80 mu l, and the mixture is mixed evenly;

in the step b), 2-300 mu M of the drug to be detected is added into every 20-80 mu l of the screened model.

The application of the RHBDD1 truncated protein in preparing a model for screening antitumor drugs taking RHBDD1 as a target point is disclosed, wherein the amino acid sequence of the RHBDD1 truncated protein is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, the model further comprises a fluorescence-labeled substrate short peptide and a liposome transfection reagent, and the fluorescence-labeled substrate short peptide is a fluorescence-labeled KEREN short peptide.