CN110558279B - Drug screening model, drug screening method and application thereof - Google Patents

Drug screening model, drug screening method and application thereof Download PDF

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CN110558279B
CN110558279B CN201910871510.9A CN201910871510A CN110558279B CN 110558279 B CN110558279 B CN 110558279B CN 201910871510 A CN201910871510 A CN 201910871510A CN 110558279 B CN110558279 B CN 110558279B
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dhx33
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zebrafish
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CN110558279A (en
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王鹍
魏天资
杨选军
仲寒冰
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Southwest University of Science and Technology
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Abstract

The invention provides a drug screening model, a drug screening method and application thereof, wherein the drug screening model is dhx33 gene defective zebra fish. The invention adopts dhx33 gene defective zebra fish as a drug screening model, the dhx33 gene can not normally express, the p53 gene and the downstream p21 and caspase8 genes thereof are both expressed and increased, the number of apoptotic cells is obviously increased, the head and eye phenotypes are obviously reduced compared with the head and eye phenotypes of wild zebra fish embryos, the phenotype change can be recovered by the treatment of a p53 inhibitor, and the dhx33 gene defective zebra fish has the potential of the drug screening model as the p53 inhibitor.

Description

Drug screening model, drug screening method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a drug screening model, a drug screening method and application thereof, in particular to a p53 inhibitor screening model, a p53 inhibitor screening method and application thereof.
Background
p53 is one of the most important cancer suppressor genes, and many clinical antitumor drugs take p53 as a target to inhibit tumor formation by activating the activity of p 53. However, recent studies have shown that p53 can kill tumor cells and damage cells with active proliferation such as lymph, hematopoiesis, and intestinal epithelial cells to produce side effects during tumor therapy, so that it is necessary to reduce the activity of p53 in normal cells. Due to the current few varieties of p53 inhibitors, more p53 inhibitors need to be searched by means of drug screening.
Currently, the commonly used drug screening methods mainly include virtual screening, molecular level screening and cell level screening. The virtual screening is to screen out a possibly effective drug by simulating the interaction between a target spot and a drug structure through a computer under the condition that the target spot and the drug structure are known, and has the advantages of low cost and high speed, but the problem that the screened drug has no effect or little effect exists, and further verification needs to be carried out through biological experiments; the molecular level screening is to mix target protein and candidate drug, detect the interaction between the protein and the drug by methods such as enzyme linked immunization, fluorescence color development, nuclear magnetic resonance and the like, and the screened drug has strong binding force with the target protein but may not play a role in the cell environment; the cell level screening refers to treating target cells with candidate drugs, and then examining the effect of the drugs on the target cells through biochemical experiments, wherein the screened drugs can effectively act on the target cells, but the experimental process is complex, and whether the drugs have toxic or side effect on the whole organism can not be evaluated. The drugs obtained by screening by the three methods have different problems, and the clinical application of the drugs is limited.
CN106699897A discloses a fusion protein for screening for or testing for MdmX inhibitors for inhibitory activity, comprising a test sequence, a binding sequence and a linker arm sequence; wherein the test sequence is a MdmX sequence or a MdmX fragment comprising a p53 binding domain in the MdmX sequence; the binding sequence is the p53 sequence or a p53 fragment comprising the MdmX binding domain in the p53 sequence; the linker arm sequence is a peptide fragment which is linked upstream and downstream to one of said test sequence and said binding sequence, respectively, and which has an amino acid sequence which does not participate in the formation of the secondary structure of the fusion protein, and which has a length and flexibility sufficient to allow any surface portion of the steric structure of said test sequence to come into steric contact with any surface portion of the steric structure of said binding sequence; the fusion protein is analyzed for inhibition by fluorescence spectroscopy, but may not function in a cellular environment.
CN106701996A discloses a method for screening an inhibitor of the interaction of p53 and MDM2 protein, comprising the following steps: 1) constructing a surface plasmon resonance sensing chip for the interaction of p53 and MDM2 protein; 2) screening inhibitors by using the chip; the method has the advantages of simplicity, rapidness, no mark, high specificity, real-time online and the like, is a molecular level screening method, and has the problem that the screened medicine cannot play a role in a cell environment.
Therefore, a novel drug screening model is established, is used for high-throughput rapid screening of the p53 inhibitor, and has important clinical significance and wide application value.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a drug screening model, a drug screening method and application thereof, and the invention can evaluate the influence of a candidate drug on the growth development and survival of a complete organism individual by using an organism with a dhx33 gene knocked out as a screening model of a p53 inhibitor, thereby improving the application potential of the candidate drug in clinical treatment and ensuring that the drug screening process is simple and rapid.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a drug screening model which is dhx33 gene deficient zebrafish.
In the invention, the dhx33 gene defective zebra fish constructed by the applicant by using the gene knockout technology has the characteristic of high expression of the p53 gene, the phenotype is obviously changed compared with the wild zebra fish, and the method can be used for quickly judging whether the small molecule drug can restore the mutant phenotype by inhibiting the activity of p53, thereby screening and obtaining the p53 inhibitor with the effect on the whole organism.
Preferably, the dhx33 gene-deficient zebrafish is overexpressed from the p53 gene.
Preferably, the dhx33 gene-deficient zebrafish is overexpressed from the p21 gene.
Preferably, the dhx33 gene-deficient zebrafish is overexpressed from the caspase8 gene.
In the invention, the apoptosis of the dhx33 gene defect type zebra fish embryo is at a high level, and the number of apoptotic cells is obviously increased compared with that of the wild zebra fish embryo.
Preferably, the dhx33 gene deficient zebrafish is phenotypically altered.
Preferably, the phenotypic change comprises a change in the size of the zebrafish head and/or eyes.
In the present invention, the head and eye of the dhx33 gene-deficient zebrafish embryo are significantly smaller than those of the wild-type embryo, probably due to apoptosis caused by the increased activity of p 53.
Preferably, the dhx33 gene-deficient zebra fish is obtained by knocking out dhx33 gene of wild zebra fish by a gene knockout method.
Preferably, the gene knockout method comprises any one of homologous recombination, ZFN, TALEN or CRISPR-Cas9 or a combination of at least two thereof, preferably CRISPR-Cas 9.
In a second aspect, the invention provides an application of a dhx33 gene-deficient zebra fish in preparation of a drug screening model.
Preferably, the dhx33 gene-deficient zebrafish is overexpressed from the p53 gene.
Preferably, the dhx33 gene-deficient zebrafish is overexpressed from the p21 gene.
Preferably, the dhx33 gene-deficient zebrafish is overexpressed from the caspase8 gene.
Preferably, the dhx33 gene deficient zebrafish is phenotypically altered.
Preferably, the phenotypic change comprises a change in the size of the zebrafish head and/or eyes.
Preferably, the dhx33 gene-deficient zebra fish is obtained by knocking out dhx33 gene of wild zebra fish by a gene knockout method.
Preferably, the gene knockout method comprises any one of, or a combination of at least two of, ZFNs, TALENs, or CRISPR-Cas9, preferably CRISPR-Cas 9.
In a third aspect, the present invention provides a method for constructing a drug screening model according to the first aspect, the method comprising the following steps:
(1) CRISPR-Cas9 target site design:
designing gRNA by using a 20bp sequence on the 3 rd exon of the dhx33 gene as a target site;
(2) RNA introduction:
introducing a gRNA and Cas9 mRNA into a zebrafish single cell embryo;
(3) and (3) mutant identification:
culturing the zebra fish embryo obtained in the step (2) to adult fish, and selecting dhx33 gene-deficient mutant.
Preferably, the nucleic acid sequence of the target site in the gRNA of step (1) is shown in SEQ ID NO: 1;
SEQ ID NO:1：
GGCCGCGCAGCGCAGACGCT。
preferably, the ratio of the gRNA and the Cas9 mRNA in the step (2) is 1 (2-3).
Preferably, the selection mode in the step (3) is genotype identification.
Preferably, the amino acid sequence of the dhx33 protein of the mutant in the step (3) is shown as SEQ ID NO: 2;
SEQ ID NO:2：
MPHEPDPPPAKRFKPGSPFFRLDKKPGMLLPRAGHAAAGVEAQRRHLPIYQSRTQVISQLRQLHSAVFIGETGSGKTTQIPQYLYEAGIGRQGIIAITQPRRVAAISLAGRVAEEKKVQLGKLVGYTVRFEDVTSPETKLKFMTDGMLLREAIGDPLLLRYTVVILDEAHERTVHTDVLFGVVKAALGAE。
in the present invention, the zebrafish mutant lacks 11 bases at the target site of the dhx33 gene, and forms a codon for premature termination of translation at exon 3 of the dhx33 gene, and in this mutant, the dhx33 protein has only 190 amino acids (680 amino acids in the full length of the wild type) from the N-terminus, and thus it is considered that gene knockout of dhx33 was successfully achieved.
In a fourth aspect, the present invention provides a drug screening composition comprising a drug screening model according to the first aspect.
Preferably, the drug screening composition further comprises zebrafish embryo culture fluid.
In a fifth aspect, the present invention provides a method of drug screening, the method comprising:
adding a candidate drug to the drug screening model according to the first aspect and/or the drug screening composition according to the fourth aspect, and observing phenotypic changes of zebrafish after culture.
In the invention, the small head and eyes of the mutant zebrafish embryo are recovered after being treated by the p53 inhibitor, so the zebrafish deficient in the dhx33 gene is an effective p53 inhibitor screening model.
Preferably, the concentration of the candidate drug is 1-5 μ M, and may be, for example, 1 μ M, 2 μ M, 3 μ M, 4 μ M or 5 μ M.
Preferably, the temperature of the culture is 27 to 29 ℃, for example, 27 ℃, 28 ℃, 28.5 ℃ or 29 ℃, preferably 28 to 28.5 ℃.
Preferably, the culture time is 24-48 h, for example, 24h, 27h, 30h, 33h, 36h, 39h, 42h, 45h or 48 h.
In a sixth aspect, the present invention provides a p53 inhibitor, wherein the p53 inhibitor is screened using the drug screening model of the first aspect and/or the drug screening composition of the fourth aspect.
Preferably, the p53 inhibitor comprises any one of pfta, PFT β or PFT μ, or a combination of at least two.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising an inhibitor of p53 according to the sixth aspect.
Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
In an eighth aspect, the present invention provides a drug screening model according to the first aspect and/or a drug screening composition according to the fourth aspect for use in screening for p53 inhibitors.
In a ninth aspect, the present invention provides a p53 inhibitor according to the sixth aspect and/or a pharmaceutical composition according to the seventh aspect, for use in the preparation of a medicament for the treatment of side effects of an anti-tumor therapy.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts dhx33 gene defective zebra fish as a drug screening model, the dhx33 gene in the dhx33 gene defective zebra fish can not be normally expressed, the p53 gene and the downstream p21 and caspase8 genes are both expressed and increased, and the number of apoptotic cells is obviously increased;
(2) the head and eye phenotypes of the dhx33 gene-deficient zebrafish embryos are obviously smaller than those of wild zebrafish embryos, the phenotype change can be recovered through treatment of a p53 inhibitor, and the mutant zebrafish has the potential of serving as a drug screening model of the p53 inhibitor;
(3) the drug screening model has the advantages of high screening speed and high flux, and the screened drug can also generally evaluate the toxic and side effects of the drug on growth and development on the basis of ensuring the biological overall effectiveness of the drug, thereby improving the possibility of applying the candidate drug to clinical treatment.
Drawings
FIG. 1(A) is the expression of p53 gene in zebra fish mutant detected by the whole in situ hybridization technique, FIG. 1(B) is the expression of p53 gene in zebra fish mutant and downstream genes p21 and caspase8 detected by qPCR, and FIG. 1(C) is the apoptosis staining for detecting apoptotic cells in zebra fish mutant;
FIG. 2 shows phenotypic changes of zebrafish wild-type embryos, mutant embryos and PFT alpha-treated mutant embryos.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 construction of dhx33 Gene-deficient Zebra Fish mutant
(1) CRISPR-Cas9 target site design
In order to achieve the effect of gene knockout, a CRISPR-Cas9 target site is designed on the 3 rd exon of a dhx33 gene, the sequence of the target site is shown as SEQ ID NO:1, and a guide RNA (gRNA) is synthesized according to the sequence of the target site;
(2) introduction of RNA
Collecting zebra fish embryos in a single cell period, and introducing gRNA and Cas9 mRNA into the single cell embryos by using a Warner PLI-100A microinjection instrument, wherein the ratio of gRNA to Cas9 mRNA is 1:2, the specific dosage is 150-300 pg of gRNA and 300-600 pg of Cas9 mRNA;
(3) mutant identification
Culturing the embryo after injection to adult fish, continuing to breed F1 generation, carrying out genotype identification on F1 generation adult fish, searching for individuals with mutation of target sites, finally selecting a mutant, deleting 11 bases AGCGCAGACGC (SEQ ID NO:3) at the target sites, forming a codon for premature translation termination on the 3 rd exon of dhx33, wherein the dhx33 protein only has 190 amino acids (the whole length of the wild type is 680 amino acids) SEQ ID NO:2 from the N end, and thus the gene knockout of dhx33 is considered to be successfully realized.
Example 2 characterization of a mutant of zebrafish deficient in dhx33 Gene
(1) Whole in situ hybridization
Wild-type zebrafish embryos and mutant zebrafish embryos were hybridized in situ in bulk using digoxin-labeled RNA probes and imaged using a Leica DMi8 microscope and a Leica DFC 450C digital camera.
As shown in FIG. 1(A), the dark portion is an in situ hybridization staining signal, and the darker the color indicates the higher expression level of p53, and it can be seen that the expression level of p53 gene in the mutant embryo was significantly increased at both 24 hours and 48 hours after fertilization.
(2)qPCR
Extracting total RNA of the zebra fish mutant by using an RNA extraction kit, carrying out qPCR detection after carrying out reverse transcription to cDNA, and synthesizing a primer adopted in the experimental process by Life Technologies; the expression amounts of p53, p21 and caspase8 genes were measured by Δ Δ CTThe method is used for calculation, and the internal reference gene is GAPDH.
As a result, as shown in FIG. 1(B), the expression of p53 gene, and the downstream p21 and caspase8 genes were all up-regulated in the mutant embryos compared to the wild embryos.
(3) Apoptosis assay
And (3) carrying out apoptosis analysis on the wild zebra fish embryo and the mutant zebra fish embryo by adopting a Roche apoptosis kit.
As a result, as shown in FIG. 1(C), the number of apoptotic cells in the dhx33 mutant embryo was significantly increased as compared with that in the wild type embryo.
Example 3 Effect of PFT alpha on dhx33 Gene deficient Zebra Fish mutants
PFT alpha is added into the culture solution when the embryo of the mutant zebra fish develops to 12 hours after fertilization, the final concentration is 5 mu M, the zebra fish is cultured for 48 hours at 28.5 ℃, and the phenotypic change of the zebra fish is observed under a microscope.
The results are shown in FIG. 2, where the head and eyes of the mutant embryos were significantly smaller than those of the wild-type embryos, and when the mutant embryos were PFTA-treated, the heads and eyes returned to normal, thus indicating that the smaller heads and eyes of the mutant embryos are likely due to apoptosis caused by the increased activity of p 53.
In conclusion, the invention adopts dhx33 gene defective zebra fish as a drug screening model, the dhx33 gene in the dhx33 gene defective zebra fish can not normally express, the p53 gene and the downstream p21 and caspase8 genes are both expressed and increased, and the number of apoptotic cells is obviously increased; the head and eye phenotypes of the dhx33 gene-deficient zebrafish embryos are significantly smaller than those of wild-type zebrafish embryos and the phenotypic changes can be restored by treatment with a p53 inhibitor; the mutant zebra fish has the advantages of high screening speed and high flux as a drug screening model, and the screened drug can also generally evaluate the toxic and side effects of the drug on growth and development on the basis of ensuring the biological overall effectiveness of the drug, thereby improving the possibility of applying the candidate drug to clinical treatment.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
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Claims (11)

1. A method for constructing a drug screening model, comprising the steps of:
(1) CRISPR-Cas9 target site design:
designing gRNA by using a 20bp sequence on the 3 rd exon of the dhx33 gene as a target site;
(2) RNA introduction:
introducing a gRNA and Cas9 mRNA into a zebrafish single cell embryo;
(3) and (3) mutant identification:
culturing the zebra fish embryo obtained in the step (2) to adult fish, and selecting dhx33 gene defect type mutant;
the nucleic acid sequence of the target site in the gRNA in the step (1) is shown as SEQ ID NO. 1;
the ratio of the gRNA to the Cas9 mRNA in the step (2) is 1 (2-3);
the amino acid sequence of the dhx33 protein of the mutant in the step (3) is shown as SEQ ID NO: 2.
2. The drug screening model obtained by the construction method according to claim 1, wherein the drug screening model is dhx33 gene-deficient zebrafish;
the dhx33 gene-deficient zebrafish is overexpressed from the p53 gene;
the dhx33 gene-deficient zebrafish is overexpressed from the p21 gene;
the dhx33 gene defect type zebra fish caspase8 gene is over-expressed;
the dhx33 gene-deficient zebrafish undergoes a phenotypic change;
the phenotypic change includes a change in the size of the zebrafish head and/or eyes.
3. A drug screening composition comprising the drug screening model of claim 2;
the drug screening composition also comprises a zebra fish embryo culture solution.
4. A method of drug screening, comprising:
adding a candidate drug to the drug screening model of claim 2 and/or the drug screening composition of claim 3, and observing phenotypic changes of zebrafish after culture.
5. The drug screening method according to claim 4, wherein the concentration of the drug candidate is 1 to 5 μ M;
the culture temperature is 27-29 ℃;
the culture time is 24-48 h.
6. The drug screening method according to claim 5, wherein the temperature of the culture is 28 to 28.5 ℃.
7. Use of a drug screening model according to claim 2 and/or a drug screening composition according to claim 3 for screening for p53 inhibitors.
8. A p53 inhibitor, wherein the p53 inhibitor is screened using the drug screening model of claim 2 and/or the drug screening composition of claim 3;
the p53 inhibitor comprises any one of PFT alpha, PFT beta or PFT mu or the combination of at least two of them.
9. A pharmaceutical composition comprising the p53 inhibitor of claim 8.
10. The pharmaceutical composition of claim 9, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.
11. Use of the p53 inhibitor according to claim 8 and/or the pharmaceutical composition according to claim 9 or 10 for the preparation of a medicament for the treatment of side effects of an anti-tumor therapy.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105981026A (en) * 2014-02-06 2016-09-28 因姆内克斯普雷斯私人有限公司 Biomarker signature method, and apparatus and kits therefor
CN109196114A (en) * 2016-04-04 2019-01-11 纳特诊断公司 Isothermal duplication component and technique
CN109811009A (en) * 2018-12-26 2019-05-28 首都医科大学 A method of lethal gene function is analyzed using CRISPR/Cas9 system building knock-out mice model

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016103269A1 (en) * 2014-12-23 2016-06-30 Ramot At Tel-Aviv University Ltd. Populations of neural progenitor cells and methods of producing and using same
CN107365804B (en) * 2017-08-13 2019-12-20 中国人民解放军疾病预防控制所 Method for packaging CRISPR-Cas9 system by using temperate phage vector
CN108192927A (en) * 2017-12-29 2018-06-22 上海海洋大学 A kind of gene editing of Oujiang Color Common Carp, Cyprinus carpio var. color is with being overexpressed operating method
CN109777837A (en) * 2018-12-26 2019-05-21 首都医科大学 A method of the systemic knock-out mice model of lethal gene is constructed using CRISPR/Cas9 system
CN110172481A (en) * 2019-04-08 2019-08-27 湖南师范大学 A kind of small fragment stat1a/stat1b Gene Double mutation deletion form zebra fish

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105981026A (en) * 2014-02-06 2016-09-28 因姆内克斯普雷斯私人有限公司 Biomarker signature method, and apparatus and kits therefor
CN109196114A (en) * 2016-04-04 2019-01-11 纳特诊断公司 Isothermal duplication component and technique
CN109811009A (en) * 2018-12-26 2019-05-28 首都医科大学 A method of lethal gene function is analyzed using CRISPR/Cas9 system building knock-out mice model

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DHX33 Transcriptionally Controls Genes Involved in the Cell Cycle;Baolei Yuan;《Molecular and Cellular Biology》;20161231;第2903-2917页 *

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