CN108531510B

CN108531510B - Application of transgenic zebra fish in preparation of animal model of chronic myelocytic leukemia

Info

Publication number: CN108531510B
Application number: CN201810464900.XA
Authority: CN
Inventors: 张文清; 黄志斌; 许梦畅; 刘伟; 叶志安; 叶茵; 徐进
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-05-16
Filing date: 2018-05-16
Publication date: 2020-05-22
Anticipated expiration: 2038-05-16
Also published as: CN108531510A

Abstract

The invention discloses application of transgenic zebra fish in preparation of an animal model of chronic granulocytic leukemia. The transgenic zebra fish is BCR/ABL transgenic zebra fish and is humanized BCR-ABL induced transgenic zebra fish. When the humanized BCR/ABL mRNA is microinjected into zebra fish embryos, the embryos show a phenotype similar to chronic myelocytic leukemia: the large increase of neutrophils suggests that the human BCR/ABL protein can play a role in the body of the zebra fish; the invention discovers that the transgenic zebra fish has a phenotype similar to human leukemia in the juvenile and adult periods for the first time, and proves that the transgenic zebra fish can be used for high-throughput screening of drugs for treating leukemia, namely, the invention successfully establishes an animal model of chronic granulocytic leukemia and provides an effective way for the research of the chronic granulocytic leukemia and the drug screening thereof.

Description

Application of transgenic zebra fish in preparation of animal model of chronic myelocytic leukemia

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of transgenic zebra fish in preparation of an animal model of chronic myelocytic leukemia.

Background

Chronic leukemia is commonly known as Chronic Myelogenous Leukemia (CML) and Chronic Lymphocytic Leukemia (CLL). BCR/ABL fusion genes can be found in leukemia cells of CML patients, and the disease course of CML is divided into a chronic stage, an accelerated stage and an acute stage. The biological behavior, clinical manifestations, therapeutic approaches and prognosis of tumor cells are distinct in different stages of the same disease. The chronic stage mainly shows that a large number of granulocytes are accumulated in bone marrow, peripheral blood and spleen, mainly mature middle and late juvenile granulocytes, the patient has good response to relative treatment of hydroxyurea, interferon, mild chemotherapy, tyrosine kinase inhibitor and the like at the stage, can be quickly and completely relieved, and can receive allogeneic hematopoietic stem cell transplantation after partial patients are relieved, so that long-term disease-free survival is achieved. The chronic stage lasts for about 3-5 years, CML patients enter an accelerated stage, the accelerated stage generally lasts for 4-6 months, the proportion of primitive cells and immature cells in the accelerated stage is increased, and finally the CML patients enter an acute transformation stage. The patients in the acute phase further worsen with massive accumulation of non-functional leukocytes in the bone marrow and peripheral blood, similar to the manifestations of AML (acute myeloid leukemia) and ALL (acute lymphocytic leukemia). CML can be changed suddenly to the medullary and lymphatic systems, and the acute change stage has poor response to various treatment effects and poor prognosis.

CML treatment is an ongoing exploratory process, undergoing radiotherapy, nuclides therapy, busulfan, hydroxyurea, other drugs such as cytarabine, homoharringtonine, indirubin, isoindigo, interferon use and combination chemotherapy, with a significant increase in efficacy over subsequent use of tyrosine kinase inhibitors (Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. effects of a selective inhibition of the Abl type kinase on the growth of Bcr-Abl reactive cells. Nature medicine 1996.2(5): p.561-566. (TKI)). Nevertheless, bone marrow transplantation, which is the only way to really cure CML at present, is to apply intensive chemotherapy to suppress tumor cells in CML patients and then transplant normal bone marrow into the patients, but the bone marrow transplantation is expensive and is difficult for general families to bear, and the bone marrow transplantation is difficult to find a suitable donor, which is even more difficult for china with relatively tight donor total. With the large number of TKIs applied, more and more CML resistant cases appear clinically, and drug resistance has become an important reason for clinical failure of CML treatment.

Although there are many mouse and cellular CML models and great breakthrough studies have been made, such as the traditional BCR/ABL1 transgenic mouse model and the transplanted mouse model and some targeted drugs found from these models. However, the existing CML model still has defects in mechanism research and drug screening, and a disease model at a cell level has a far difference from in-vivo physiological conditions, cannot reflect all physiological functions and cannot comprehensively evaluate adverse drug reactions and toxic and side effects. The mouse model can simulate the disease state and evaluate the effect of the medicine from the whole, and the obtained result has very important clinical value and application prospect. However, the vertebrate model of traditional mice and the like has obvious disadvantages of large size, high cost, long period, large required sample amount, incapability of large-scale targeted drug screening and the like (1, Hariharan IK, Harris AW, Crawford M, Abud H, Webb E, Cory S, Adams JM.A BCR-v-abl oncogene antigens in molecular Biol,1989.9(7) p.2798-805.2, Honda H, Fujii T, Takatoku M, Mano H, Witte ON, Yazaki Y, Hirai H.Express of p210BCR/abl by amyloid induced T-Cell leukemia gene transfer blood, blood of 1995.85(10) of science of bone of animal model of noise, scientific of biological of nucleic acids, scientific of nucleic acids, and biological of nucleic acids of scientific family, 1990.87(17): p.6649-53.4, Zhang, X.and R.ren, Bcr-Abl effective induced amino reactive distance and production of an expression interface in-3and gradient-collagen-stimulating factor in blood, a novel model for bacterial strains leukemia blood,1998.92(10): p.3829-40.). So far, a vertebrate model which is fast in modeling, small in size, low in feeding cost, convenient to administer and simple in phenotype reading is not established.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the transgenic zebra fish in the preparation of the animal model of Chronic Myelogenous Leukemia (CML).

The invention also aims to provide application of the transgenic zebra fish in preparing an animal model for screening a medicament effective on chronic myelogenous leukemia.

Still another object of the present invention is to provide a method for screening a drug effective for chronic myelogenous leukemia using transgenic zebrafish.

The purpose of the invention is realized by the following technical scheme: the application of transgenic zebra fish in preparing an animal model of Chronic Myelogenous Leukemia (CML), wherein the transgenic zebra fish is BCR/ABL transgenic zebra fish.

The BCR/ABL transgenic zebra fish is humanized BCR-ABL induced transgenic zebra fish; preferably, the construction is carried out by the following method: the recombinant plasmid pTol2hsp70, BCR/ABL and Tol2 transposase mRNA are together introduced into wild zebra fish to obtain BCR/ABL transgenic zebra fish.

The recombinant plasmid pTol2hsp70 BCR/ABL is constructed by the following steps:

(1) connecting the BCR/ABL fusion gene to a plasmid with a Tol2 transposon recognition site to obtain a recombinant plasmid pTol2 BCR/ABL;

(2) the hsp70 gene promoter is inserted into the recombinant plasmid pTol2BCR/ABL to obtain the recombinant plasmid pTol2hsp70: BCR/ABL.

The BCR/ABL fusion gene described in the step (1) is preferably a BCR/ABL fusion gene obtained by excision of the plasmid vector NGFR P210 with the restriction enzyme EcoRI.

The plasmid in the step (1) is preferably T2AL200R150G plasmid.

The hsp70 gene promoter in the step (2) is an hsp70 gene promoter sequence with Cla I, EcoR V and Age I recognition sites; the nucleotide sequence is shown as SEQ ID No: 1 is shown.

The recombinant plasmid pTol2hsp70, BCR/ABL and Tol2 transposase mRNA are introduced into wild type zebra fish preferably by the following steps: the recombinant plasmid pTol2hsp70: BCR/ABL and Tol2 transposase mRNA are introduced into the zebra fish embryo by microinjection method at 1 cell stage of the zebra fish embryo development.

The Tol2 transposase mRNA is Tol2 transposase mRNA transcribed in vitro from pCS2-Tol2 transposase vector by mMESSAGE mMACHINE system; the nucleotide sequence is shown as SEQ ID No: 2, respectively.

The chronic granulocytic leukemia is caused by the expression of a BCR/ABL fusion gene.

The chronic granulocytic leukemia is relieved after being treated by a medicament for blocking the action of tyrosine kinase or a medicament for blocking a TGF β R-I pathway.

The tyrosine kinase-breaking medicine is preferably at least one of imatinib, dasatinib and bosutinib.

The drug for blocking the function of the TGF β R-I pathway is preferably LY 364947.

Use of a transgenic zebrafish in the preparation of an animal model for screening for a drug effective in Chronic Myelogenous Leukemia (CML), wherein said transgenic zebrafish is a BCR/ABL transgenic zebrafish.

The Chronic Myelogenous Leukemia (CML) is caused by the expression of a BCR/ABL fusion gene.

The Chronic Myelogenous Leukemia (CML) is relieved after the treatment of drugs for blocking the action of tyrosine kinase or the TGF β R-I pathway.

The screening is preferably realized by the following steps:

(1) treating the transgenic zebrafish embryos and wild type sibling fish embryos at the first to two days after fertilization with the same concentration of the drug candidate:

(2) observing the blood phenotype of the embryonic tail hematopoietic tissue (CHT) region the second to five days after treatment to determine the therapeutic effect of the drug candidate on the Chronic Myelogenous Leukemia (CML).

The blood phenotype includes Sudan Black B (SB) staining positive granulocyte counts.

A method for screening a drug effective in Chronic Myelogenous Leukemia (CML) using transgenic zebrafish, wherein said transgenic zebrafish is a BCR/ABL transgenic zebrafish.

The general concept of the invention is as follows:

(1) in the embryonic stage, the detailed hematopoietic alteration of the myeloid lineage is detected by carrying out a Whole In Situ Hybridization (WISH) assay using gene probes that label the cells of the myeloid lineage at different stages, with lcp labeling the cells of the early myeloid lineage; mpo, lyz marking mature neutrophils; mfap4 to label mature macrophages. Elucidating the role of BCR-ABL induced differentiation of specific myeloid progenitor cells in chronic myelogenous leukemia zebrafish and subsequent selection of neutrophil and macrophage fate.

(2) During adult fish stage, the condition of kidney, marrow cell number, subtype and differentiation stage in blood circulation system of BCR-ABL induced chronic myelocytic leukemia zebra fish is analyzed by flow cytometry (FACS) and cytology method (3 months, 1 year) to see whether leukemia characteristics appear in these fish.

(3) According to the detailed comparison between the maturity degree and the natural course of the myeloid leukemia cells in the model and the clinic, the ratio of the transgenic zebra fish to develop AML respectively is counted.

(4) Pharmacological validation of known leukemia therapeutic drugs on leukemia models: and (3) hybridizing the BCR-ABL induced chronic myelocytic leukemia zebra fish with wild zebra fish AB, and enabling each mother fish to lay eggs for 200-300. And (4) picking out a BCR-ABL overexpression fertilized egg by green fluorescence. And (3) placing the BCR-ABL over-expressed fertilized eggs into a 96-well plate or a 384-well plate, wherein 3-5 fertilized eggs are placed in each well.

(5) There are clinical drugs or small libraries of compounds selected with potential biological activity (mainly CML drugs already in clinic). The different small molecules of choice were added to wells containing roe and water, with only one drug per well. After a period of incubation, large-scale analysis is carried out by methods such as SB staining and the like, and compounds capable of changing the phenotype of hematopoietic development of Bcr-ABL over-expressed roe are found.

Compared with the prior art, the invention has the following advantages and effects:

(1) because the homology between mice and human is high (the protein homology of BCR is 93.8%, and the protein homology of ABL is 87.8%), many plasmids and models of human origin are established on the basis of mice. The protein homology of BCR/ABL of zebrafish and human is relatively low compared with that of mice, and is 73.8%/69.5%, respectively. But the homology in the key domains is still high. So far, no report is made on a chronic myelocytic leukemia zebra fish model, and when the BCR/ABL mRNA of human origin is microinjected into the embryo of the zebra fish, the embryo of the zebra fish is found to have a CML-like phenotype: the neutrophil is greatly increased, which indicates that the BCR/ABL protein of human origin can play a role in the body of the zebra fish.

(2) Zebrafish are ideal model animals for drug development. Compared with a mouse, the zebra fish is more suitable for large-scale drug screening, and has the advantages of low cost, high efficiency and the like. The zebra fish used for drug screening can realize the following advantages: high throughput, high number of eggs laid by zebrafish, and 1 day after fertilization, with drug addition, 2-5 days later, during which time yolk can be used for embryo nutrition without feeding, allowing drug screening with 96-well or 384-well plates. The low cost, zebrafish feeding and consumption costs far less than mice, on average about 1/100 per mouse per day. The administration is convenient: the compound can be directly dissolved in water and diffused to the embryo, and the amount of the compound required is 1/100-1/1000 of mice. The invention overexpresses human BCR-ABL in zebra fish for the first time, and then causes the phenotype of abnormal medullary hematopoiesis; moreover, the invention discovers that the transgenic zebra fish has a phenotype similar to human leukemia in the early and adult periods for the first time, and proves that the transgenic zebra fish can be used for carrying out high-throughput screening on the drugs for treating leukemia.

(3) The zebra fish over-expressed BCR-ABL is constructed, and found to have the following phenotypes that ① early-stage myeloid progenitor cells are abnormally differentiated and granulocytes are obviously proliferated, ② adult fish is increased in granulocytes in kidney blood, leukemia cell systemic infiltration partially occurs in the zebra fish of ③ 1 years, part of the zebra fish can progress into Acute Myeloid Leukemia (AML), ④ CML drugs such as imatinib and the like which are human chemotherapy drugs can obviously improve hemogram change of the abnormality, and bone marrow transplantation experiments are utilized to determine that the zebra fish disease model has similar treatment effects with human diseases.

(4) Tg (hsp70: p 210) in the invention^BCR/ABL) Besides symptoms of the transgenic zebra fish in the embryonic period, symptoms of chronic granulocytic leukemia, even similar to acute granulocytic leukemia, also appear in the kidney bone marrow of adult zebra fish (equivalent to human bone marrow), which means that a chronic granulocytic leukemia model which can be used for researching the transition from the chronic stage to the acute stage is obtained, and the method cannot be realized in mice and cell models.

(5) The invention connects the cDNA gene sequence of human BCR/ABL with hsp70 promoter of hot start protein, so that people can control the expression quantity of BCR/ABL under different temperature and time, and simulate the cases of BCR/ABL expression quantity with different degrees in different patients. In addition, the invention utilizes the Tol2 transposon system to convert hsp70: p210^BCR/ABLIntegrated into zebra fish genome, constructed to be able to stably inherit and express p210^BCR/ABLTg of (hsp70: p 210)^BCR/ABL) Transgenic zebrafish. However, there is no model for performing different dosage control in the existing mice expressing humanized BCR/ABL, which cannot better simulate the heterogeneity of clinical patients, and an optimal scheme is selected for drug screening.

Drawings

FIG. 1 is a diagram showing the arrangement of elements of a transgenic vector pTol2hsp70: BCR/ABL according to the present invention; wherein "Tol 2" is a transposon recognition sequence, "hsp 70 promoter" is a promoter sequence of heat shock gene hsp70, "BCR" is a partial sequence of BCR gene in BCR/ABL fusion gene obtained from blood sample of chronic myelogenous leukemia patient, "ABL" is a partial sequence of ABL gene in BCR/ABL fusion gene obtained from blood sample of chronic myelogenous leukemia patient, and "pA" is a translation termination signal recognition sequence.

FIG. 2 is a wild typeZebra fish (WT) and Tg (hsp70: P210)^BCR/ABLA result graph of BCR/ABLmRNA expression level detection in transgenic zebra fish embryos (divided into four groups of samples according to gene types and embryo culture conditions, and each group of samples detects 160 embryos); wherein, the 'h' indicates that the embryo is subjected to heat shock treatment; "28.5" means that the embryos were continuously incubated and placed in a constant temperature incubator at 28.5 degrees (". about." P)<0.001；“****”P<0.0001)。

FIG. 3 is a graph of Tg detection using in situ hybridization (hsp70: p 210)^BCR/ABL) A BCR/ABL gene expression quantity result graph in the transgenic zebra fish embryo (the result of collecting transgenic zebra fish embryos at different development stages and wild zebra fish embryos and carrying out a BCR/ABL probe whole embryo in-situ hybridization experiment); wherein, the 'h' indicates that the embryo is heat-shocked, and the 'black arrow' indicates that the position is HSCs (hematopoietic stem cells) generation position.

FIG. 4 is the Tg (hsp70: p 210)^BCR/ABL) Abnormal expression pattern of myeloid markers (lcp, lyz, SB and mfap4) in transgenic zebrafish during embryonic hematopoietic development; wherein, panels A-D, I-J are lcp, lyz WISH results and counting statistics results for all positive signals (black box at the bottom right in the figure is PBI area signal enlargement, number marked at the top right of the figure is the number of zebrafish embryos with increased gene expression compared with the total number of zebrafish embryos); FIG. E, F, K shows the results of SB staining and counting of all positive signals (the lower right black box in the figure shows an enlarged view of the PBI zone signal, and the number marked on the right of the figure shows the number of zebrafish embryos with increased gene expression compared to the total number of zebrafish embryos); FIG. G, H, L shows the results of mfap4WISH and the counting statistics of all positive signals (the lower right black boxes in the figure are the head elevation and the enlarged view of PBI area signals, respectively, and the numbers marked on the right in the figure are the number of zebrafish embryos with increased gene expression compared to the total number of zebrafish embryos); the statistical method is t test, "+" p<0.05；“****”p<0.0001, "h" indicates that there is an embryo to be heat shock treated.

FIG. 5 is a plot of wild type zebrafish and Tg (hsp70: p 210)^BCR/ABL) A result graph of blood smear Giemsa staining of kidney blood and peripheral blood of adult transgenic zebrafish; it is composed ofIn the figure a, the result of Giemsa staining of WT adult fish peripheral blood smear (blood cell composition mainly including red blood cells, few fresh lymphocytes, myeloid cells and platelets); panel b shows Tg (hsp70: p 210)^BCR/ABL) The result of Giemsa staining of peripheral blood smears of adult zebrafish (the blood phenotype is mainly manifested by neutrophilia at various developmental stages); FIG. c shows the result of Giemsa staining on kidney blood smears of WT adult fish (each type of cells at different stages of development is in a certain proportion, red blood cells and neutrophils are abundant, lymphocytes are inferior, and other types of cells are in a smaller proportion); figure d is

Tg(hsp70:p210^BCR/ABL) The kidney blood smear Giemsa staining result of the transgenic adult zebrafish (the blood phenotype is mainly expressed by neutrophilia at each development stage); in the figure, the asterisk indicates primitive cells, the line arrows indicate myeloid precursors, the triangle arrows are immature neutrophils, and the lightning arrows are mature neutrophils.

FIG. 6 is the Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish progress to the hematologic phenotype of AML; wherein, the graph a is the result of Giemsa staining of WT adult fish peripheral blood smear (blood cell composition is mainly red blood cells, and few fresh lymphocytes, myeloid cells and platelets); panel b shows Tg (hsp70: p 210)^BCR/ABL) The result of the Giemsa staining of the peripheral blood smear of the adult zebrafish (the blood phenotype is mainly shown by the large increase of the primitive granulocytes); FIG. c shows the result of Giemsa staining of kidney blood smears of WT adult fish (each type of cells at different stages of development is in a certain proportion, red blood cells and neutrophils are abundant, lymphocytes are inferior, and other types of cells are in a smaller proportion); panel d is Tg (hsp70: p 210)^BCR/ABL) The kidney blood smear Giemsa staining result of the transgenic adult zebrafish (the blood phenotype is mainly shown by the large increase of primitive granulocytes); the lined arrows in the figure indicate primitive neutrophils.

FIG. 7 is the Tg (hsp70: p 210)^BCR/ABL) A reaction result graph of the transgenic zebra fish embryo to TKIs; wherein, Panel A is the Tg of placebo group (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryo 5dpf lcp WISH (whole in situ hybridization) results; panels B-D show Tg of TKIs (hsp70: p 210)^BCR/ABL) Transgenic zebraFish embryos, 5dpf lcp WISH results; the right hand side of the figure is marked with the number lcp⁺Reduced Tg of cells (hsp70: p 210)^BCR/ABL) Transgenic Zebra fish embryos compared to total Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryo number, the black square frame at the lower right corner is an enlarged view of a PBI area; in the figure, "h" indicates that the heat shock test treatment is performed.

FIG. 8 is the Tg (hsp70: p 210)^BCR/ABL) The result of the response of the transgenic zebra fish embryo to a TGF β R-I pathway inhibitor is shown in the graph, wherein the graph A, B shows the wild type wt zebra fish (A) and Tg (hsp70: p 210) in a placebo group^BCR/ABL) Transgenic zebrafish embryos (B)5dpf lcp WISH results, FIG. C, D TGF β R-I pathway inhibitor LY364947 treatment of wild type wt zebrafish (C) and Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryos (D)5dpf lcp WISH results; panel E shows the number of lcp positive neutrophils marked ("+" p) in statistics A-D<0.05)。

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The respective raw materials used in the following examples are commercially available except for those specifically mentioned.

The term "hpf" as used in the present invention refers to the number of hours after fertilization. The term "dpf" as used herein refers to the number of days after fertilization. For example, 3hpf refers to 3 hours after fertilization and 8dpf refers to 8 days after fertilization.

The terms "wild type" or "WT" as used herein both refer to wild type zebrafish.

The term "sib fish (sibling)" as used herein refers to an individual who is the offspring of the same parent.

Example 1

1. Materials and methods:

(1) zebra fish culture

Zebra fish are cultivated as described in The literature (Westerfield M: The zebrafish: guide for The laboratory use of zebrafish (Brachdanio relay.) Edition by Eugene, OR, M.Westerfield, 1993). The following lines were used in the present invention: AB wild type zebra fish and BCR-ABL transgenic zebra fish.

(2) Transgenic zebra fish establishment

The application adopts a transposon mediated transgenic technology to construct a transgenic zebra fish line. The BCR/ABL fusion gene sequence was obtained by cutting out the plasmid vector NGFR P210 with the restriction enzyme EcoRI. Among them, the plasmid vector of NGFR P210(Addgene, plasmid #27486) was obtained by committing Addgene, Inc., Synergestic-Progression limited company in Beijing, an exclusive agency in Addgene, China, to Addgene, USA (contract No.: QC 13120106). Our amplified NGFRP210 plasmid vector deliver Weiqi Weijie (Invitrogen)^TMI Thermo Fisher Scientific) company, and selecting a plasmid vector which has a completely consistent sequence with a BCR/ABL fusion gene obtained from a clinical human sample of chronic granulocytic leukemia to carry out subsequent experiments.

First, the BCR/ABL fusion gene is ligated into a T2AL200R150G plasmid having a Tol2 transposon recognition site by an enzyme digestion ligation system (the T2AL200R150G plasmid is disclosed in an article published by Koichi Kawakami laboratory of Japan as Functional discovery of the Tol2Transposable element Identified the minor cis-Sequence and a high reproducibility Sequence in th e subsidiary vector for transformation), thereby obtaining a pTol2BCR/ABL plasmid vector. Then, in order to obtain a transgenic zebra fish line which is stably inherited and conditionally expresses BCR/ABL fusion protein, we select a promoter of hsp70 gene which can be highly expressed only under the condition of 38.5 ℃, and a promoter of hsp70 gene is obtained by extracting the wild-type zebra fish genome and cloning the sequence of the wild-type zebra fish genome, wherein the nucleotide sequence is shown as SEQ ID No: 1 is shown. The expression of the BCR/ABL fusion protein in the zebra fish body is started. Aiming at the hsp70 promoter sequence, a front primer sequence (C la I-hsp 70-FP: 5'-CCATCGATTCAGGGGTGTCGCTTGGT-3') with a recognition site of restriction enzyme Cla I and a back primer sequence (EcoR V-Age I-hsp 70-RP: 5'-CCGATATCACCGGTCTGCAGGAAAAAAAAAC-3') with recognition sites of restriction enzymes EcoRV and Age I are respectively designed to amplify the hsp70 promoter sequence.

The hsp70 promoter sequence was inserted into the pTol2BCR/ABL plasmid vector described above by using an enzyme digestion ligation system, and the results are shown in FIG. 1,pTol2hsp70, BCR/ABL plasmid vector was obtained. Finally, a method of microinjecting Tol2 transposase mRNA (Tol 2 transposase mRNA transcribed in vitro from pCS2-Tol2 transposase vector by a mMESSAGE mMACHINE system, and having a nucleotide sequence shown in SEQ ID No: 2) and pTol2hsp70: BCR/ABL plasmid vector (the dosage of the two can be adjusted according to actual needs, for example, 50pg Tol2 transposase mRNA +50pg pTol2hsp70: BCR/ABL plasmid vector are injected into each zebra fish embryo), introducing the zebra fish embryo at the 1-cell stage of the zebra fish embryo development, and integrating the gene DNA fragment between the two Tol2 recognition site sequences on the transgenic vector onto the genome of the zebra fish under the action of transposase to obtain Tg (hsp70: P210P 70)^BCR/ABL) The transgenic zebra fish is the BCR/AB L transgenic zebra fish.

(3) Real-time fluorescent quantitative PCR detection of BCR/ABL expression

First we used fluorescent quantitative PCR to detect BCR/ABL expression in transgenic fish: the first and second groups were wild type AB strain zebrafish or Tg, respectively (hsp70: P210)^BCR/ABL) Transgenic zebrafish embryos are continuously placed in a constant-temperature incubator at 28.5 ℃ for incubation for 3 d; the third and fourth groups of samples were wild type AB strain zebrafish or Tg (hsp70: P210)^BCR/ABL) Transgenic zebrafish embryos were heat shocked 24h, 36h, 48h, 60h and 72h after fertilization. Total RNA from wild-type and transgenic fish was then extracted using the RNAeasy Kit (Qiagen, Maryland, USA; 74004), followed by cDNA synthesis using the reverse transcription Kit (Promega, Madison, USA, M1701). Real-time fluorescent quantitative PCR BCR/ABL mRNA expression detection was performed on a LightCycler N anosW1.0 machine using SYBR Green Real-time TIMEPC R Master Mix (Applied Biosystems, Austin, USA; 4472908) dye (BCR-qPCR-FP: 5'-GGCTCTATGGGTTTCTGAATGTC-3'; ABL-qPCR-RP: 5'-TTTCCTTGGAGTTCCAACG AG-3').

The results are shown in FIG. 2: tg (hsp70: P210)^BCR/ABL) The expression level of BCR/ABL mRNA in the offspring of the transgenic zebra fish is increased by thousands of times compared with that in the offspring of the wild zebra fish in the control group. The expression level of BCR/ABL mRNA in the transgenic offspring subjected to heat shock treatment is also remarkably increased compared with the transgenic offspring continuously incubated in a 28.5-degree constant temperature incubator. IntoOne step shows that the expression of BCR/ABL in the transgenic model established by us is regulated by the initiation of hsp70 and can be highly expressed under the condition of 38.5 degrees. Such a transgenic model helps us to study the relation between BCR/ABL expression and diseases. In order to avoid that the expression level of the BCR/ABL in the embryonic stage of the zebra fish is too high to influence the normal growth and development of the zebra fish or cause the zebra fish to be unable to survive to the adult, the transgenic fish can be continuously fed at low temperature, and then heat shock treatment is carried out according to the research requirement to induce the expression of the BCR/ABL, and the condition is controllable.

(4) In situ hybridization detection of expression of BCR/ABL

We designed human specific BCR/ABL probes with reference to the nucleic acid sequences of human and zebrafish BCR and ABL genes in NCBI and ENSEMBLE databases, wherein the nucleotide sequences are shown as SEQ ID No: 3, respectively. And carrying out a zebra fish embryo integral in-situ hybridization experiment by using a BCR/ABL probe to detect the space-time expression of BCR/ABL mRNA in the transgenic zebra fish embryo. Waiting for Tg (hsp70: P210)^BCR/ABL) Embryos were developed to 24hpf, heat shock experiments were performed on young fish, selecting 24hpf, 32hpf, 50hpf, 80hpf, 110hpf, 140hpf for heat shock, respectively, and 36hpf, 3dpf (85hpf) and 5dpf (145hpf) embryos were collected and whole embryo in situ hybridization experiments were performed with BCR/ABL probes (wild-type AB strain zebrafish was used as control).

The results are shown in FIG. 3: tg (hsp70: p 210)^BCR/ABL) The expression of BCR/ABL mRNA can be detected at each time point in the transgenic zebra fish embryo, and the expression mode is that all tissues are widely expressed; in contrast, BCR/ABL mRNA expression was not detected in the control wild-type AB line zebra fish embryos.

(5) Whole in situ hybridization

The Tg (hsp70: p 210) was collected at a size of 3dpf (about 78hpf)^BCR/ABL) Selecting a marrow cell specific marker gene (l-plastic, lyz, mpx) probe (the probe provides a commercially available source) to perform whole embryo in situ hybridization experiment and Sudan black B (SB) staining experiment, and detecting the transient expression of p210 in zebra fish^BCR/ABLInfluence on the development of myeloid cells during the process of embryo hematopoiesis. "l-plastin" marks all myeloid cell populations, including neutrophils and macrophages. Mpx,lyz' marks relatively mature late granulocyte cells and mature neutrophils. "SB" marks the mid-juvenile, late-juvenile, mature neutrophils.

As shown in FIG. 4, p210 was expressed in zebrafish^BCR/ABLThe number of l-plastin, mpx, lyz, SB-labeled cells of these myeloid lines increased significantly. It is known that the major hematological phenotype in patients with chronic phase of CML in the clinic is granulomatous hyperplasia. And p210 is transiently expressed in the zebra fish body^BCR/ABLIt also causes an increase in granulocytes, indicating that p210 is of human origin^BCR/ABLThe expression in zebra fish can also play similar biological functions in human body.

(6) Cytological analysis

To avoid p210^BCR/ABLHigh expression results in a Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryos are lethal and cannot normally develop to the adult, we will lose Tg (hsp70: p 210)^BCR/ABL) Breeding transgenic zebra fish in a circulating culture system at 28.5 ℃, and performing heat shock induction on p210 when the zebra fish grows to 8 months^BCR/ABLHigh expression, continuous heat shock treatment for 1 month, followed by normal feeding to 1 year size. Subsequently, we performed 100 Tg's (hsp70: p 210)^BCR/ABL) Dissecting the transgenic zebra fish and 55 wild zebra fish treated in the same batch, collecting renal blood and peripheral blood, staining blood smears with Giemsa, and observing and counting different types and different degrees of developed blood cell compositions in the renal blood and the peripheral blood. Hematopoietic cells were isolated as follows: zebrafish were anesthetized with tricaine and Peripheral Blood (PB) was obtained from gills by using heparinized microcytoplasmic pipettes. Renal blood (KM) cells were isolated from the kidney. The separated PB and KM were resuspended in 5% FBS, and then the resuspended cells were centrifuged onto a slide glass at 400 rpm for 3 minutes. Finally, the plate was fixed with Jiemsa (Merck, Germany; 1.09204.0500) for 5 minutes, washed with water, stained with Swiss Ji (Merck:1.01424.0500) for 40 minutes, and then washed with water for observation. PB and KM cells were counted according to cell morphology.

The results are shown in FIG. 5, and tables 1 and 2: 76 out of 100 fish showed an increase in myeloid lineage or myeloid precursor cells in peripheral blood or bone marrow blood. And can be classified into I-VI classes according to their degree, similar to human CML hemograms. And as shown in fig. 6: similar to the human CML catastrophe process, 1 of 100 transgenic zebrafish developed into AML catastrophe stage, becoming abundant progenitor cells in peripheral blood and bone marrow blood.

TABLE 1 Tg (hsp70: p210BCR/ABL) Zebra fish blood phenotype classification (peripheral blood)

TABLE 2 Tg (hsp70: p210BCR/ABL) Zebra fish blood phenotype classification (bone marrow blood)

Example 2

CML can relieve symptoms after being treated by a medicament for blocking tyrosine kinase, the medicament for blocking tyrosine kinase can be Imatinib (Imatinib), Dasatinib (Dasatinib) or Bosutinib (Bosutinib), and CML can relieve symptoms after being treated by a medicament for blocking TGF β R-I pathway (such as LY364947), and the specific experimental steps are as follows:

(1) tyrosine kinase inhibitor treatment experiments

a. The experimental group had a Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryos were treated with DMSO (dimethyl sulfoxide, placebo) after 39.5 degree heat shock. The control group is treated by BCR/ABL transgenic zebra fish embryo with tyrosine kinase inhibitor after 39.5-degree heat shock under the same condition.

b. Heat shock treatment: embryos were collected, heat shock treated for 30min at 10 hours post fertilization, 22hpf, 34hpf, 46hpf, 58hpf and 70hpf for 2h, respectively, and then transferred to 28.5 ℃ incubator for normal incubation.

c. Drug (Imatinib: 100 uM; dasatinib: 10 uM; bosutinib: 5uM) or DMSO at a concentration of 5-100 uM to Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryos were treated by continuous immersion for 48 hours.

d. Tg after 5dpf after treatment (hsp70: p 210)^BCR/ABL) Lcp in situ hybridization experiments were performed on transgenic zebrafish embryos and the neutrophil phenotype of the tail hematopoietic tissue region of the embryos was observed to determine the therapeutic effect of the drug candidate on the CML. The blood phenotype may also include the count and statistics of positive cells after in situ hybridization of the lcp gene. lcp A decrease in the number of positive granulocytes was considered to be symptomatic relief after drug treatment.

The results are shown in FIG. 7: compared with the placebo group, the number of lcp positive granulocytes is obviously reduced by adding a tyrosine kinase inhibitor (imatinib, dasatinib or bosutinib), and the symptom of granulocytosis in CML is relieved. It is suggested that consistent with clinical efficacy, tyrosine kinase inhibitors may not only alleviate symptoms of clinical CML, but also alleviate Tg (hsp70: p 210)^BCR/ABL) CML phenotype with abnormal increase of transgenic zebrafish granulocytes. The transgenic zebra fish provides a valuable platform for screening new drugs for treating CML.

(2) TGF β R-I pathway inhibitor treatment experiments

a. The experimental group had a Tg (hsp70: p 210)^BCR/ABL) Transgenic zebrafish embryos were treated by 39.5 degree heat shock. The control group was a 39.5 degree heat shock-treated group of wild-type WT zebrafish embryos under the same conditions.

c. LY364947 drug or DMSO vs Tg at 5uM concentration (hsp70: p 210)^BCR/ABL) Transgenic zebrafish or wild type embryos were treated by continuous immersion for 48 hours.

The results are shown in FIG. 8: addition was made relative to placebo (DMSO)TGF β R-I pathway inhibitor (LY364947) significantly reduces Tg (hsp70: p 210)^BCR/ABL) The number of lcp positive granulocytes in transgenic zebra fish embryos relieves the symptom of granulocytosis in CML, while the wild zebra fish group does not change obviously, the TGF β R-I pathway is one of the key pathways causing diseases of BCR/ABL, and no drug discovery of related pathways exists clinically at present, which indicates that Tg can be utilized (hsp70: p 210)^BCR ^/ABL) And (4) screening a new CML medicament by using the transgenic zebra fish.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of southern China's science

<160>7

<170>SIPOSequenceListing 1.0

<210>1

<211>1520

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hsp70 promoter sequence

<400>1

tcaggggtgt cgcttggtta tttccaaaaa tcaaattaat tttattaaac tattagaacg 60

agcatgtttt gtctatatgc tacagaagat aaaaaataat aggagttaac agttataaaa 120

caacacactt tgtttctatt gattgttgac cacactgggg tctcattaag ttagattaaa 180

gacacactaa ctgggtcaaa agcagcagat tgatttcata gcaccagggt aaactttcta 240

acacttttac ggcaatcata tacattaaaa ttaaatacag accacgactg aacaaggagg 300

atgatctcca atattaaaca aagagacttg tgcctatttc tctgagggta aacatgacct 360

ctcaagttag caagttgttt ttaacactac aaaaatagtt aagactgcaa tcccagaata 420

aagtattggt tttaaccaat caatatagta cagtaaacat ccatttgttt tgttgaaacg 480

ttaaacaaat ctgaccaaag ctattagctt atataaaaca ggtttgcctt ctatgtagct 540

gaaaacacca caggcccgat tttgctactg tgtaaaacat ttcagcaaga tttttttatt 600

gcattttttt ttactgaatc gttcaaacat tttatcattt tagtttgttc attcattgca 660

actggaaaaa caacacatca cacaaccgca catatttcag caataagtac aataaaacac 720

tcaaataaaa aaaaacattt taaatctctt tgtatttttg accgctgttt cgcgtaattt 780

cacggtaaaa ctctggaaat ctccactaca ttcctctcag cggctcctct caatgacagc 840

tgaagaagtg acgcggctgc ctgctgtgtt ttgattggtc gaattcactg gaggcttcca 900

gaacagtgta gagtctgaac gggtgcgcgc tctgctgtat ttaaagggcg aaagagagac 960

cgcagagaaa ctcaaccgaa gagaagcgac ttgacaaaga agaaaagagc agcctgacag 1020

gactttttcc ccgacgaggt gtttattcgc tctatttaag aatctactgt aaggtaagtc 1080

tcaatatatt gtactctatt ggctaatcag aattatatag agattatatg tacttaatgt 1140

caaaaaatca actttgtata tgtaatcttt ttacatgtgg actgcctatg ttcatcttat 1200

tttaggtcta ctagaaaatt atatttcccg ttttcacaat aaggattttt aaaaaaagca 1260

atgaacagac gggcatttac tttatgttgc tgacattatt ttatatgagc ataataacca 1320

taaatactag caaatgtcct aaatgaattt gtgttaatgt tgtctacaaa agaaaattag 1380

cgttttactt gtacaactaa taataacttg gttattaaga gaatttcact tgttgactag 1440

aaaaatcctt tcataatgaa acaattgcac cataaattgt ataaatataa aattaattct 1500

aattgttttt ttttcctgca 1520

<210>2

<211>6287

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCS2-Tol2 sequence

<400>2

cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag 60

ctacttgttc tttttgcagg atcccatcga ttcgaattca aggcctctcg aggtcgagtc 120

acatctatta ccacaatgca cagcaccttg acctggaaat tagggaaatt ataacagtca 180

atcagtggaa gaaaatggag gaagtatgtg attcatcagc agctgcgagc agcacagtcc 240

aaaatcagcc acaggatcaa gagcacccgt ggccgtatct tcgcgaattc ttttctttaa 300

gtggtgtaaa taaagattca ttcaagatga aatgtgtcct ctgtctcccg cttaataaag 360

aaatatcggc cttcaaaagt tcgccatcaa acctaaggaa gcatattgag agaatgcacc 420

caaattacct caaaaactac tctaaattga cagcacagaa gagaaagatc gggacctcca 480

cccatgcttc cagcagtaag caactgaaag ttgactcagt tttcccagtc aaacatgtgt 540

ctccagtcac tgtgaacaaa gctatattaa ggtacatcat tcaaggactt catcctttca 600

gcactgttga tctgccatca tttaaagagc tgattagtac actgcagcct ggcatttctg 660

tcattacaag gcctacttta cgctccaaga tagctgaagc tgctctgatc atgaaacaga 720

aagtgactgc tgccatgagt gaagttgaat ggattgcaac cacaacggat tgttggactg 780

cacgtagaaa gtcattcatt ggtgtaactg ctcactggat caaccctgga agtcttgaaa 840

gacattccgc tgcacttgcc tgcaaaagat taatgggctc tcatactttt gaggtactgg 900

ccagtgccat gaatgatatc cactcagagt atgaaatacg tgacaaggtt gtttgcacaa 960

ccacagacag tggttccaac tttatgaagg ctttcagagt ttttggtgtg gaaaacaatg 1020

atatcgagac tgaggcaaga aggtgtgaaa gtgatgacac tgattctgaa ggctgtggtg 1080

agggaagtga tggtgtggaa ttccaagatg cctcacgagt cctggaccaa gacgatggct 1140

tcgaattcca gctaccaaaa catcaaaagt gtgcctgtca cttacttaac ctagtctcaa 1200

gcgttgatgc ccaaaaagct ctctcaaatg aacactacaa gaaactctac agatctgtct 1260

ttggcaaatg ccaagcttta tggaataaaa gcagccgatc ggctctagca gctgaagctg 1320

ttgaatcaga aagccggctt cagcttttaa ggccaaacca aacgcggtgg aattcaactt 1380

ttatggctgt tgacagaatt cttcaaattt gcaaagaagc aggagaaggc gcacttcgga 1440

atatatgcac ctctcttgag gttccaatgt ttaatccagc agaaatgctg ttcttgacag 1500

agtgggccaa cacaatgcgt ccagttgcaa aagtactcga catcttgcaa gcggaaacga 1560

atacacagct ggggtggctg ctgcctagtg tccatcagtt aagcttgaaa cttcagcgac 1620

tccaccattc tctcaggtac tgtgacccac ttgtggatgc cctacaacaa ggaatccaaa 1680

cacgattcaa gcatatgttt gaagatcctg agatcatagc agctgccatc cttctcccta 1740

aatttcggac ctcttggaca aatgatgaaa ccatcataaa acgaggcatg gactacatca 1800

gagtgcatct ggagcctttg gaccacaaga aggaattggc caacagttca tctgatgatg 1860

aagatttttt cgcttctttg aaaccgacaa cacatgaagc cagcaaagag ttggatggat 1920

atctggcctg tgtttcagac accagggagt ctctgctcac gtttcctgct atttgcagcc 1980

tctctatcaa gactaataca cctcttcccg catcggctgc ctgtgagagg cttttcagca 2040

ctgcaggatt gcttttcagc cccaaaagag ctaggcttga cactaacaat tttgagaatc 2100

agcttctact gaagttaaat ctgaggtttt acaactttga gtagcgtgta ctggcattag 2160

attgtctgtc ttatagtttg ataattaaat acaaacagtt ctaaagcagg ataaaacctt 2220

gtatgcattt catttaatgt tttttgagat taaaagctta aacaagaaaa aaaaaaaaaa 2280

aaaaaaaaaa aagtactagt cgagcctcta gaactatagt gagtcgtatt acgtagatcc 2340

agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 2400

atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2460

taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2520

ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt 2580

tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg 2640

tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 2700

gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 2760

ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 2820

ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 2880

gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 2940

tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 3000

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 3060

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 3120

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 3180

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 3240

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 3300

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 3360

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 3420

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 3480

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 3540

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3600

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3660

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 3720

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 3780

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 3840

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 3900

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 3960

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 4020

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 4080

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 4140

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 4200

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 4260

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 4320

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 4380

tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 4440

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 4500

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 4560

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 4620

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 4680

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 4740

gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa 4800

aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 4860

aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 4920

acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 4980

agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 5040

gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 5100

cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 5160

caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 5220

agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 5280

cctcttcgct attacgccag tcgaccatag ccaattcaat atggcgtata tggactcatg 5340

ccaattcaat atggtggatc tggacctgtg ccaattcaat atggcgtata tggactcgtg 5400

ccaattcaat atggtggatc tggaccccag ccaattcaat atggcggact tggcaccatg 5460

ccaattcaat atggcggact tggcactgtg ccaactgggg aggggtctac ttggcacggt 5520

gccaagtttg aggaggggtc ttggccctgt gccaagtccg ccatattgaa ttggcatggt 5580

gccaataatg gcggccatat tggctatatg ccaggatcaa tatataggca atatccaata 5640

tggccctatg ccaatatggc tattggccag gttcaatact atgtattggc cctatgccat 5700

atagtattcc atatatgggt tttcctattg acgtagatag cccctcccaa tgggcggtcc 5760

catataccat atatggggct tcctaatacc gcccatagcc actcccccat tgacgtcaat 5820

ggtctctata tatggtcttt cctattgacg tcatatgggc ggtcctattg acgtatatgg 5880

cgcctccccc attgacgtca attacggtaa atggcccgcc tggctcaatg cccattgacg 5940

tcaataggac cacccaccat tgacgtcaat gggatggctc attgcccatt catatccgtt 6000

ctcacgcccc ctattgacgt caatgacggt aaatggccca cttggcagta catcaatatc 6060

tattaatagt aacttggcaa gtacattact attggaagta cgccagggta cattggcagt 6120

actcccattg acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta catccccatt 6180

gacgtcaatg gggaggggca atgacgcaaa tgggcgttcc attgacgtaa atgggcggta 6240

ggcgtgccta atgggaggtc tatataagca atgctcgttt agggaac 6287

<210>3

<211>1121

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BCR/ABL Probe sequence

<400>3

cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 60

ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 120

atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 180

gctttgaaga tcaagatctc ccagatcaag aatgacatcc agagagagaa gagggcgaac 240

aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 300

ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 360

ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 420

aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 480

tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 540

tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 600

agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 660

gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 720

cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 780

ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 840

aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 900

cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 960

atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 1020

ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 1080

tacgtctcct ccgagagccg cttcaacacc ctggccgagt t 1121

<210>4

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer Cla I-hsp 70-FP

<400>4

ccatcgattc aggggtgtcg cttggt 26

<210>5

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer EcoRV-Age I-hsp 70-RP

<400>5

ccgatatcac cggtctgcag gaaaaaaaaa c 31

<210>6

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer bcr-qPCR-FP

<400>6

ggctctatgg gtttctgaat gtc 23

<210>7

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer abl-qPCR-RP

<400>7

tttccttgga gttccaacga g 21

Claims

1. The application of the transgenic zebra fish in preparing the animal model of chronic granulocytic leukemia is characterized in that: the transgenic zebra fish is BCR/ABL transgenic zebra fish;

the BCR/ABL transgenic zebra fish is constructed by the following methodObtaining: the recombinant plasmid pTol2hsp70Introducing BCR/ABL and Tol2 transposase mRNA into wild zebra fish together to obtain BCR/ABL transgenic zebra fish; wherein,

the recombinant plasmid pTol2hsp70The BCR/ABL is constructed by the following steps:

(2) will be provided withhsp70The gene promoter is inserted into a recombinant plasmid pTol2BCR/ABL to obtain a recombinant plasmid pTol2hsp70:BCR/ABL；

The method for constructing the BCR/ABL transgenic zebra fish further comprises the steps of feeding and heat shock induction of the obtained BCR/ABL transgenic zebra fish, and specifically comprises the following steps: feeding the obtained BCR/ABL transgenic zebra fish in a 28.5 ℃ circulating culture system, performing heat shock induction when the BCR/ABL transgenic zebra fish grows to 8 months, continuously performing heat shock treatment for 1 month, and then continuing normal feeding to obtain the transgenic zebra fish;

the BCR/ABL transgenic zebra fish shows increase of marrow-line or marrow-line precursor cells in peripheral blood or marrow blood;

the plasmid in the step (1) is a T2AL200R150G plasmid;

the method described in step (2)hsp70The nucleotide sequence of the gene promoter is shown as SEQ ID No: 1 is shown.

2. Use of the transgenic zebrafish according to claim 1 for the preparation of an animal model of chronic myeloid leukemia, characterized in that:

3. Use of the transgenic zebrafish according to claim 1 for the preparation of an animal model of chronic myeloid leukemia, characterized in that:

4. Use of the transgenic zebrafish according to claim 3 for the preparation of an animal model of chronic myeloid leukemia, characterized in that:

the drug for blocking the tyrosine kinase from acting is at least one of imatinib, dasatinib and bosutinib;

the medicine for blocking the function of the TGF β R-I pathway is LY 364947.

5. The application of transgenic zebra fish in preparing an animal model for screening a medicament effective on chronic granulocytic leukemia is characterized in that: the transgenic zebra fish is BCR/ABL transgenic zebra fish;

the BCR/ABL transgenic zebra fish is constructed by the following method: the recombinant plasmid pTol2hsp70Introducing BCR/ABL and Tol2 transposase mRNA into wild zebra fish together to obtain BCR/ABL transgenic zebra fish; wherein,

(2) will be provided withhsp70The gene promoter sequence is inserted into a recombinant plasmid pTol2BCR/ABL to obtain a recombinant plasmid pTol2hsp70:BCR/ABL；

the plasmid in the step (1) is a T2AL200R150G plasmid;

6. Use of the transgenic zebrafish according to claim 5 for the preparation of an animal model for screening drugs effective against chronic myeloid leukemia, characterized in that:

7. Use of the transgenic zebrafish according to claim 5 for the preparation of an animal model for screening drugs effective against chronic myeloid leukemia, characterized in that:

8. Use of the transgenic zebrafish according to claim 7 for the preparation of an animal model for screening drugs effective against chronic myeloid leukemia, characterized in that:

the medicine for blocking the function of the TGF β R-I pathway is LY 364947.