CN101422617B

CN101422617B - Use of ZNF268 gene splicing isomer in treating leukaemia

Info

Publication number: CN101422617B
Application number: CN2007100536818A
Authority: CN
Inventors: 李文鑫; 郭明雄; 赵舟宙; 邵焕杰; 孙翀
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2007-10-30
Filing date: 2007-10-30
Publication date: 2012-01-11
Anticipated expiration: 2027-10-30
Also published as: CN101422617A

Abstract

The invention discloses an application of a ZNF268 gene splice variant in anti-leukemia, which aims at providing the application of one splice variant of the human zinc finger protein ZNF268 gene in preparing medicaments for curing or preventing leukemia. The splice variant of the human ZNF268 gene is named ZNF268a and is an expression product after that an mRNA precursor produced by transcribing the ZNF268 gene is spliced selectively to delete the integrated number six exon. The splice variant has the expression form with particularity in blood cells of leukemia patients, therefore, the expression marks can be detected by the RT-PCR method and can be used for clinical diagnosis of the leukemia. The application of the ZNF268 gene splice variant in preparing the medicaments for curing or preventing leukemia can quickly and correctly judge whether the leukemia happens.

Description

The application of a kind of ZNF268 gene splicing isomer in leukemia

Technical field

The present invention relates to the application of the splicing isomer of gene, particularly, the splicing isomer ZNF268a that the present invention relates to a kind of human zinc-finger protein ZNF268 gene is treating or is preventing the application in the leukemic medicine as preparation.

Background technology

Leukemia (Leukemia) is a malignant tumor of hematopoiesis system; It is characterized in that one or more hemocyte composition generation malignant proliferations in the hemopoietic systems such as bone marrow, lymph node; And each organs and tissues in the infiltration body, cause the normal hematopoiesis histiocyte to be suppressed, produce various symptoms.Leukemia is one of domestic ten big malignant tumor occurred frequently, and China has 4,000,000 leukaemics at present at least, and annual speed with 30,000 to 40,000 increases, and in the majority with the youngster below 35 years old, wherein 50% is the child, and with 2-7 year child patient be main.Increasing sign shows that in recent years, the child has become leukemic group of people at high risk.Now, leukemia serious harm the people's, particularly youngsters and children life security.Therefore, be necessary very much leukemic generation, diagnosis and control are furtherd investigate.

The ZNF268 gene is found a kind of typical KRAB/C2H2 type zinc finger protein genoid of calendar year 2001.Result of study shows: 1) this gene is positioned at the long-armed near-end grain district 12q24.34 of No. 12 chromosomes of people, and its ORFs is made up of 7 exons, on genome across the zone of about 40Kb; 2) the ZNF268 gene exists the alternative splicing phenomenon, and this alternative splicing mainly occurs on 3,4,5, No. 6 exons.Functional study is the result show, the ZNF268 gene is being brought into play certain function in human early embryo differentiation and development, hemopoietic system differentiation and development and leukemia incidence and development process.

Product (mRNA precursor) to genetic transcription in nucleus carries out various modifications, montage and editor, and the process that makes the exon of coded protein partly be connected to become a successive ORFs is called transcribes post-treatment.Wherein, In the montage process of mRNA precursor, through different montage modes (selecting different splice site combinations), the exon of participating in montage can carry out montage not according to its linear precedence; Intron can not kept by excision yet; Promptly whether exon or intron appear among the sophisticated mRNA and can select, thereby produce different mRNA splicing isomers, and this montage mode is called alternative splicing.The montage process receives multiple cis acting element and the interactional adjusting of trans acting factor.By from the mRNA precursor of a gene because of alternative splicing produces multiple mRNA (splicing isomer) form, and the different proteins that translates is called homology isomer (isoform).About 60% human gene can produce different protein isomers through alternative splicing under different tissues or physiological and pathological state, this is the basis of causing the eukaryote height heterogeneous.

In the research of genome times afterwards comprehensively; The eukaryotic gene expression regulation and control occupy crucial status; It will help further to illustrate important biosis, explain cell behavior and explore the disease genesis mechanism, thereby on molecular level, for the diagnosis of human diseases, treatment and prevention scientific basis and practical technique will be provided.Research shows; The expression of gene regulation and control are much carried out on the mRNA level; Therefore how precursor mRNA carries out montage and in the eukaryotic gene expression regulation and control, plays crucial effect; It has determined which sequence can be expressed and how to express under the different spatiotemporal modes, thereby for the variation of the phenotype of cell and protein structure and function ten minutes key.Be that the research of polygenes complex disease provides new clue particularly to the research association of alternative splicing Regulation Mechanism.

Summary of the invention

The objective of the invention is to be to provide a kind of application of splicing isomer ZNF268a in preparation treatment or prevention leukemia medicament of human zinc-finger protein ZNF268 gene.Utilize conventional RT-PCR method, adopt specific primer to detect the polynucleotide sequence that whether has the splicing isomer ZNF268a of ZNF268 gene in the peripheral blood sample, directly treat or prevent leukemic foundation as a kind of medicine.This splicing isomer all has expression in people's tissue that is detected and cell line, and the expression in the part leukemia human peripheral that is detected obviously reduces, to such an extent as in the sample that has, can't detect through PCR method.

In order to realize above-mentioned task, the present invention adopts following technical scheme:

One, the acquisition of ZNF268 gene splicing isomer

Design a series of nested primer clones and detect the ZNF268 expression of gene.Because zinc refers to that gene ZNF268 has 7 exons; Wherein No. 7 exons coding zinc refer to that spacer domain and the zinc of gene ZNF268 refer to the district; And PCR result shows that the deletion mutation of single exon part section does not take place the ZNF268 gene in No. 7 exon; And montage difference is mainly concentrated and is appeared on the 3rd, 4,5, No. 6 exon; Utilizing nested primer is that the cDNA of K562 etc. carries out augmentation detection to fetal liver and leukaemia, and gel electrophoresis analysis shows that the ZNF268 gene mainly expressed 7 amplified production bands (Fig. 2).

These amplified production bands are cut glue respectively, be cloned in the T-Easy carrier (available from Promega company), every product band send surveys three independent clonings; Sequencing result shows; The ZNF268 gene is altogether identified 8 kinds of splicing isomers by the clone, be respectively ZNF268a, ZNF268b, ZNF268c, ZNF268d, ZNF268e, ZNF268f, ZNF268g and KW-4 (Fig. 1,2) (referring to document Gou D etc., BBA; 2001,1518:306-310; KrackhardtAM etc., Blood, 2002,100:2123-2131; SunY etc., IUBMBLife, 2003,55:127-131; Sun C etc., heredity, 2006,28:513-517; Shao H etc., Int J Mol Med, 2006,18:457-463).

Two, the protein expressioning product ZNF268a albumen of the splicing isomer ZNF268a of ZNF268 gene and ZNF268b and ZNF268b1, the proteic functional analysis of ZNF268b2

In order to study the proteic transcriptional regulatory activity of protein expressioning product ZNF268a, ZNF268b1 and ZNF268b2 of ZNF268 gene, adopted the CAT reporting system.This system by reporter plasmid pG5SV-BCAT (referring to document Kakidani, H and Ptashne.M.1988, Cell 52:161-167; Sadowski, I., Ma J., Triezenberg S. and Ptashne.M.1988, Nature (London) 335:563-564.) and expression plasmid pBXG1 (referring to document Kakidani, H and Ptashne.M.1988, Cell 52:161-167; Sadowski, I., Ma J., Triezenberg S. and Ptashne.M.1988, Nature (London) 335:563-564.) constitute.PBXG1 is one and contains the GAL4 expression carrier, and pG5SV-BCAT is a plasmid that contains reporter gene CAT (chloromycetin acyltransferase), and it also contains five GAL4 that are cascaded and combines territories.Like this, the GAL4 expression product of pBXG1 can combine the territory to regulate and control the expression (Fig. 3) of its downstream reporter gene CAT through the GAL4 that is incorporated into pG5SV-BCAT.At first made up carrier for expression of eukaryon pBXG1/ZNF268a (open, the document Sun Y etc. that sees reference, IUBMB Life, 2003,55:127-131; Shao H etc., Int J Mol Med, 2006,18:457-463), pBXG1/ZNF268b1 (is pBXG1/ZNF268-KRAB among Fig. 3.Open, the document Sun Y etc. that sees reference, IUBMB Life, 2003,55:127-131; Shao H etc., Int J Mol Med, 2006,18:457-463) and pBXG1/ZNF268b2 (open, the document Sun Y etc. that sees reference, IUBMB Life, 2003,55:127-131; Shao H etc., Int J Mol Med, 2006; 18:457-463), ZNF268a, ZNF268b1 (pBXG1/ZNF268-KRAB) and ZNF268b2 are inserted in the pBXG1 carrier, the protein that express to produce the different fragments that has merged GAL4 is (open; Document Sun Y etc. sees reference; IUBMB Life, 2003,55:127-131; Shao H etc., Int J Mol Med, 2006,18:457-463).

Double digestion is identified and is shown that the structure of recombinant expression carrier is successful.

(source of pBXG1/rKid1 plasmid is referring to document Witzgall R, Volk R, Yeung RS, Bonventre JV.Genomics (1994) 20:203-209 with pBXG1 and pBXG1/rKid1-KRAB; Kim SS, Chem YM, O ' Leary E, Witzgall R, Vidal M, Bonventre JV.PNAs. (1996) 93:15299-15304.The pBXG1/rKid1-KRAB plasmid is open, the document Sun Y etc. that sees reference, IUBMBLife; 2003,55:127-131) level of regulation and control CAT transcriptional activity is blank and positive control, and it is (open to utilize CAT ELISA to systematically analyze pBXG1/ZNF268a, pBXG1/ZNF268b1 (pBXG1/ZNF268-KRAB), pBXG1/ZNF268b2; Document Sun Y etc. sees reference; IUBMB Life, 2003,55:127-131; Shao H etc., Int J Mol Med, 2006,18:457-463) to the transcriptional regulatory activity of CAT.The pBXG1 plasmid is an empty carrier, does not have any transcriptional control domain, only expresses saccharomycetic GAL4 albumen, and its expression to CAT has no influence, is the blank of test; PBXG1/rKid1-KRAB be before for experiment institute's proof can effectively suppress the plasmid of CAT expression, be that the positive inhibition test of this test contrasts.The result show pBXG1/ZNF268a and pBXG1/ZNF268b2 respectively with reporter plasmid pG5SV-BCAT cotransfection COS7 cell after; Can both activate the expression of reporter gene CAT; And its activity curve that activates the CAT expression is closely similar; All be certain plasmid consumption and rely on, there is transcriptional activation domain in this prompting in the ZNF268 gene; And the pBXG1/ZNF268b1 (pBXG1/ZNF268-KRAB) that contains ZNF268 gene KRAB zone separately has intensive depression effect (Sun Y etc., IUBMB Life, 2003, the 55:127-131 of transcribing to reporter gene CAT; Shao H etc., Int J Mol Med, 2006,18:457-463).

The proteic positioning result of ZNF268 shows that ZNF268a and ZNF268b2 albumen can go into to authorize to wave function, promptly as transcription regulatory factor work (Shao H etc., Int J Mol Med, 2006,18:457-463).Utilize CAT ELISA system, these two proteic transcriptional regulatory activities are studied, the result shows that ZNF268a and ZNF268b2 albumen can activate the expression of CAT on the report carrier, shows that these two albumen have certain transcriptional activation function.Because ZNF268a has the domain that inhibition is transcribed, but its transcriptional activation ability is identical with ZNF268b2 albumen, show that the core transcriptional activation function territory of ZNF268 gene is positioned the spacer domain of ZNF268 gene or/and zinc refers to the district.

Three, induce in the atomization in leukaemia system, the proteic expression of ZNF268a changes

In order to analyze the relation between ZNF268 gene and leukemia and the blood cell differentiation and development, collect leukaemic's bone marrow smear, utilize immunohistochemical method, the ZNF268 expression of gene is analyzed; Be that K562 cell strain etc. is a hemocyte differentiation model cell strain simultaneously with the leukaemia; Utilize derivant such as TPA to induce cell directional differentiation such as K562; Analyzing the ZNF268 gene is the expression variation in the atomization the leukaemia, studies the influence of ZNF268 gene pairs human blood cell's differentiation and development with this.

1, ZNF268 specific expressed analysis in the leukaemia

The bone marrow smear sample of marrow sexual cell leukemia patient is collected by hospital of Tongji University from Wuhan, is used for experimentation.All bone marrow smears all in 1 month, accomplish Wright's staining and SABC detects (Fig. 5-12).

Utilize the specific antibody (preparation method for antibody is the content of the patent of CN02115935.1 referring to application number) of ZNF268 gene that the SABC hybridization analysis is carried out in its expression in acute myeloblast leukemia undifferentiated type (M1); The result is as shown in Figure 5; Zinc refers to that gene ZNF268 does not express in original and immature granulocyte; And only in the granulocyte of the middle and advanced stage of only a few, expression is arranged; The obviously visible sophisticated nuclear of paging of this cell it should be noted that the ZNF268 protein expression mainly is positioned in the kytoplasm.

The detection case of ZNF268 gene in M2 type leukemia bone marrow smear is shown in Fig. 6-7.The expression of ZNF268 gene in M2a type leukaemic's bone marrow smear is similar with the expression of ZNF268 gene in acute myeloblast leukemia undifferentiated type (M1), and zinc finger protein ZNF268 presents strongly expressed in minority in than mature granulocyte; The cell number showed increased of the expression ZNF268 gene in M2b type leukaemic, ZNF268 albumen location, what have is positioned in the kytoplasm, and the kytoplasm, the karyon that have are located altogether.

The 3 routine patients' of M3b bone marrow smear is only collected in this experiment, and the result of its Wright's staining and SABC is as shown in Figure 8.The ZNF268 gene still only is expressed in the ripe marrow sexual cell of minority; And express the branch that power is obviously arranged; The hybridization signal disperse is in whole cell or be partial to a side of cell, further confirm the ZNF268 gene in original and promyelocyte low expression or do not express.

Showed by immune group result, in M5 type leukaemic cell, zinc refers to that gene ZNF268 also has expression in mononuclear cell, and in the mononuclear cell of different times its expressing quantity, protein expression location all demonstrates cell-specific.Specifically, ZNF268 gene expression in more sophisticated cell is high, is positioned in the kytoplasm; A little less than the ZNF268 gene is expressed in more primary mononuclear cell, and hybridization signal is scattered in (Fig. 9-10) in the whole cell.

Figure 11-12 has shown the expression of ZNF268 gene at four routine typical chronic myelocytic leukemia (CML) patient bone marrow smears respectively.The result shows ZNF268 gene great expression in middle metamyelocyte and band-cell, is in the different differentiation cell in period, and the ZNF268 expression of gene is strong and weak different, and this all detects in all patients' bone marrow smear.In addition, the localized difference of ZNF268 expression of gene, performance is particularly outstanding in Figure 12 (B).In the cell among the figure shown in the black arrow, nucleus presents tangible minute page status, and ZNF268 albumen then is positioned in the kytoplasm, and is rendered as strongly expressed; Cell shown in the red arrow among the figure, nucleus is bigger, has occupied the major part of whole cell, and ZNF268 albumen is dispersed in and is distributed in the nuclear.Be positioned to have in the karyon two kinds of possibilities for ZNF268 albumen, the one, cell is in different differentiation periods, and the inferior location of the cell of zinc finger protein ZNF268 is different; The one, cell is in different growth cycles, and nucleus is shown as big and looses, and cell possibly be in the active S phase of protein expression.

2, Hemin inducing leukemia cell line k562 breaks up to red system

Getting the leukaemia who is in exponential phase is that K562 is with 2 * 10 ⁵Cell/ml concentration is inoculated in the 50ml culture bottle; Divide into groups to add differentiation derivant Hemin (chlorhematin), at 37 ℃, 5%CO ₂2 bottles of cells are regularly taken out in following cultivation every day, the cell smear that takes a morsel behind the counting, and air drying is by conventional Rui Shi-Ji's nurse Sa dyeing.Microscopy, nucleus dyeing is for there being the positive cell of pink person, and several 200 cells calculate positive percentage, and the matched group positive rate is less than 10%, and test group induces differentiation agent effective greater than 50% explanation.1, the centrifugal 5mins of 000rpm/min abandons the supernatant collecting cell; All the other cells renew the bright RPMI1640 culture medium that contains Hemin and continue to cultivate.

For the cell that reaches incubation time; Extract total protein according to conventional method; Utilize the BioRad standard measure, and then through the colour developing of SDS-PAGE gel electrophoresis, comprehensive and quantitative; Make all samples total protein applied sample amount consistent, western blot method detects at the different phase ZNF268a proteic content (preparation method for antibody referring to application number be the content of the patent of CN02115935.1) of Hemin inducing leukemia cell line k562 to the differentiation of red system.Analysis result shows (Figure 13), and the expression of ZNF268a reduces along with the prolongation between K562 cell line differentiation phase gradually.

3, TPA inducing leukemia cell line U937 is to the differentiation of monokaryon system

Except that used derivant with cell line is respectively TPA, the leukaemia is the U937, other induces methods such as differentiation, ZNF268a Protein Detection with above-mentioned.Analysis result shows (Figure 14), and the expression of ZNF268a and ZNF268b2 increases along with the prolongation between differentiation phase gradually.

4, DMSO induces HL60 to the differentiation of grain system

Except that used derivant with cell line is respectively DMSO, the leukaemia is the HL60, other induces methods such as differentiation, ZNF268a Protein Detection with above-mentioned.Analysis result shows (Figure 15), and the expression of ZNF268a is along with the prolongation between differentiation phase increases gradually, and minimizing subsequently.

5, to induce HL60 be differentiation to monokaryon/huge biting to TPA

Except that used derivant with cell line is respectively TPA, the leukaemia is the HL60, other induces methods such as differentiation, ZNF268a Protein Detection with above-mentioned.Analysis result shows (Figure 16), and the expression of ZNF268a increases along with the prolongation between differentiation phase gradually.

Above result of study shows; The ZNF268 gene is expressed multiple this form of montage on rna level; The generation of these splicing isomers be the ZNF268 gene on rna level by the result of regulating and expressing, embodied the complexity and the function diversity of ZNF268 gene expression.It should be noted that zinc refers to that the splicing isomer of gene ZNF268 all is to realize through the exon skipping of gene.Can know that now a gene can produce splicing isomer through following several kinds of modes, like the different tailing signal site of different transcriptional start sites, alternative splicing, selection, rna editing etc.Alternative splicing comprises 3 types: 1, the reservation of intron; 2, the reservation of exon or excision; 3, the transfer of 3 ' and 5 ' splice site (shift) causes the growth or the shortening of exon.Alternative splicing also is multifarious to the influence of protein structure: as in the polypeptide chain one to having or not of hundreds of amino acid whose increases or minimizing, certain functional domain etc.If alternative splicing changes single open reading frame, then possibly can't effectively translate.Result of study shows, the ZNF268 gene mainly is to produce splicing isomer through the reservation of exon or excision, and this has at first increased ZNF268 expression of gene multiformity from rna level, thereby the basis on the rna level is provided for its function diversity.In these splicing isomers, the content of these two kinds of splicing isomers of ZNF268a and ZNF268b is maximum, and wide expression shows that it has important effect in various cell lines and histiocyte.

Research for many years shows, the aberrant splicing of gene is one of major reason of taking place of disease such as tumor.Owing to receive the influence of inside and outside various factors, gene occurs unexpected in the sophisticated course of processing of mRNA and causes occurring unusual splicing product, thereby causes the change of its translation product on structure and function, so the initiation heritability or the day after tomorrow acquired disease.In fact such gene instance have a lot (table 1) (Venables JP, Cancer Research, 2004,64,7647-7654).

Part aberrant splicing gene during table 1 tumor takes place.

Cancer?tissue	Gene	Function	Transacting?tactor
				Leukemia Leukemia Leukemia Thyroid Thyroid，colon Colorectal Gastric Gastric Pancreas Pancreas Liver Livcr Lung Lung Lung Lung Endometrium Endomtetrium Brcast Breast Breast Breast Breast Breast，brain Brain Brain Many Many Many Manby Many	Fyn Caspasc?8 PASG MUC1 Insulin?reccpor Rae1 KAl1/CD82 WISP1 Sccretin?rcceptor Gastrin?receptor DNMT3b4 SVH NRSF C-CAM VEGF Actinin-4 SHBG IntegrinβlC AIBI Androgen?receptor Estrogen?receptor Syk uPAR FGFRI Crk NF1 TSG101 MDM2 CD44 Tenascin-C Fthronectin	Tyrosine?kinase Apoptosis Chromatin?modelling Adhesion，metastasis Tyrosine?kinase Signalling?GTPase Metastasis Invasion Growth?inhibiton Proliferation Chromatin?modelling Novel Transcription?factor Adhesion Angiogenesis Adhesion，metastasis Honnone?signalling Adhesion Hormonc?signallino Transcription?Factor Transcriprion?Faetor Mclastasis Adhesion，proteolysis Growth?signalling Migration?invasion Signalling?GTPase Proteolysis Proteolysis Proliferarion?angiogenesis Adhesion?inhibior Angiogenesis	Rex U2AF PTB 9G8.SAM68 SRp40

The splicing isomer of the human zinc-finger protein ZNF268 of complete No. 6 exon of the above disappearance, called after ZNF268a, concrete polynucleotide sequence are referring to following network address: Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? Db=nuccore& Id=23308728(GeneBank number: NM_003415).

Splicing isomer ZNF268a codified such as the following network address of the human zinc-finger protein ZNF268 of No. 6 exon that said disappearance is complete: Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? Db=nuccore& Id=23308728Shown amino acid residue sequence.

The splicing isomer of the human zinc-finger protein ZNF268 of 7 exons that said reservation is complete, called after ZNF268b, concrete polynucleotide sequence are referring to following network address: Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? Db=nuccore& Id=14579578(GeneBank number: AF385187).The polynucleotide sequence of splicing isomer ZNF268b has 3782 bases, contains two complete ORFs.Read frame, a kind of polypeptide of forming by 193 amino acid residues of encoding, called after ZNF268b1 from the 181st to the 759th base of 5 ' end for first; Be second from the 770th to the 3127th base of 5 ' end and read frame, a kind of polypeptide of forming by 786 amino acid residues of encoding, called after ZNF268b2 (referring to: Shao Huanjie, Wuhan University's doctorate paper, 2006, p44-66).

The present invention is owing to disclose splicing isomer and the coded sequence thereof that has the human zinc-finger protein ZNF268 gene of unconventionality expression characteristic at the leukemia philtrum; Make it is in depth studied; Might make its quantitative or qualitative detection index, for further clinical expansion contributes as the leukemia diagnosis mark.

The present invention compared with prior art has the following advantages and effect:

Hemogram, bone marrow smear and cytochemistry inspection be diagnosis, typing, with the main foundation of examining the leukemia state of an illness and estimating prognosis.In existing leukemia diagnosis process, blood routine examination is absolutely necessary, but as need further making a definite diagnosis, then must carry out the bone marrow smear inspection.Result of study and document show that human zinc-finger protein ZNF268 gene is relevant with hematopoietic cell differentiation and development and leukemia incidence and development process.The present invention finds wherein a kind of splicing isomer ZNF268a of ZNF268 gene, in the part leukemia case of being investigated, does not express.Therefore, the inspection for this splicing isomer polynucleotide sequence can become a kind of effective leukemia diagnosis foundation.

The present invention relates to a kind of ZNF268 gene splicing isomer and treat or prevent the application in the leukemic medicine in preparation.Its objective is the application of a kind of splicing isomer in preparation leukemia diagnosis label that the ZNF268 gene is provided.Detection through to a kind of ZNF268 gene splicing isomer ZNF268a polynucleotide sequence provided by the invention can directly utilize blood sample, specific primer and conventional RT-PCR method, can judge whether to suffer from leukemia rapidly and accurately.Therefore, this splicing isomer very likely is used widely as novel leukemia diagnosis reagent.

Description of drawings

The multiple splicing isomer ideograph of Fig. 1 ZNF268 gene.

The PCR product analysis of the multiple splicing isomer of Fig. 2 ZNF268 gene.Result of study shows, in people's histoorgan and various kinds of cell system, has the ZNF268 expression of gene, detects multiple splicing isomer (Gou D etc., BBA, 2001, the 1518:306-310 of ZNF268 gene simultaneously; SunY etc., IUBMB Life, 2003,55:127-131; Sun C etc., heredity, 2006,28:513-517; Shao H etc., Int J MolMed, 2006,18:457-463).

Fig. 3 in the functional analysis of ZNF268 gene splicing isomer expression product, various expression vectors and reporter plasmid structural representation (Sun Y etc., IUBMB Life, 2003,55:127-131; Shao H etc., IntJ Mol Med, 2006,18:457-463).

Transcriptional regulatory activity analysis (Sun Y etc., IUBMB Life, 2003, the 55:127-131 of Fig. 4 ZNF268a, ZNF268b1 and ZNF268b2; Shao H etc., Int J Mol Med, 2006,18:457-463).

The expression analysis of Fig. 5 ZNF268 gene in acute myeloblast leukemia undifferentiated type (M1).A, Wright's staining result; B, SABC result.

The detection of expression of Fig. 6 ZNF268 gene in M2a.A, Wright's staining result; B, SABC result.

The detection of expression of Fig. 7 ZNF268 gene in M2b.A, Wright's staining result; B, SABC result.

The detection of expression of Fig. 8 ZNF268 gene in acute promyelocytic leukemia (M3b) patient.A, Wright's staining result; B, SABC result.

The detection of expression of Fig. 9 ZNF268 gene in acute monocytic leukemia (M5a).A, Wright's staining result; B, SABC result.

The expression of Figure 10 ZNF268 gene in acute monocytic leukemia (M5b).A, Wright's staining result; B, SABC result.

The detection of expression of Figure 11 ZNF268 gene in chronic myelocytic leukemia (CML).A, Wright's staining result; B, SABC result.

ZNF268 gene expression detects in Figure 12 3 routine CML patient's bone marrow smears.

Figure 13 Hemin inducing leukemia cell line k562 to red be in the atomization, western blot analyzes the proteic expression of ZNF268a and changes.

Figure 14 TPA inducing leukemia cell line U937 is in the atomization to monokaryon, and western blot analyzes the proteic expression of ZNF268a and changes.

Figure 15 DMSO inducing leukemia cell line HL60 is in the atomization to grain, and western blot analyzes the proteic expression of ZNF268a and changes.

Figure 16 TPA inducing leukemia cell line HL60 is in the atomization to monokaryon/huge biting, and western blot analyzes the proteic expression of ZNF268a and changes.

Figure 17 is through the differential expression spectrum of two kinds of splicing isomers (ZNF268a and ZNF268b) in normal person's hemocyte and leukemia human blood cell of RT-PCR checking ZNF268 gene; Wherein numeral number is represented leukemia people's numbering (totally 49 examples), and " n+ numeral " numbering expression normal person's numbering (totally 16 examples), M representes dna molecular amount marker.Wherein, ZNF268a and ZNF268b all have expression in normal person's hemocyte; And in 49 routine leukemia human blood cells, the expression of 8 routine samples wherein (being respectively the

leukemia people

24,41,42,54,63,64,65, No. 67) disappearance ZNF268a.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the concrete application process of the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted concrete experiment condition in the following example; Usually according to the normal experiment condition; Like volumes such as Sambrook; Experiment condition described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor laboratory Press, 2002), or the experiment condition of advising according to manufacturer.

The splicing isomer ZNF268a of 1 one kinds of ZNF268 genes of embodiment is used for preparing the application process of treating or preventing leukemic medicine

A, collection sample

Tissue samples among the present invention derives from the whole blood sample (whole blood sample of totally 49 routine leukemia patients and 16 routine healthy subjects) that obtains in leukemia patient and the normal healthy people health check-up blood sample.The diagnosis of leukemia patient is final foundation with pathological diagnosis all.

B, processing sample obtain a chain cDNA

One, the extracting of the total RNA of blood sample

The reagent of total RNA adopts TRIzol LS Reagent (available from Invitrogen company) in the extracting blood sample.Being used for used vessel of extracted total RNA and water does not all have the processing of RNA enzyme, to guarantee the environment of no RNA enzyme in the experiment.

Glass drying oven was done removal RNA enzyme, cooling roasting 4 hours in 150 ℃; Plastic ware was soaked in 0.5MNaOH 10 minutes, the water cleaning down, it is subsequent use to sterilize.

Institute's blood sampling is added to TRIzol LS Reagent in the blood sample of dilution through the dilution in 1: 1 of PBS buffer then, and making the ratio between TRIzol LS Reagent and the blood sample is 3: 1, mixing; Add 0.2 milliliter of chloroform, mixing, 4 ℃ of centrifugal layerings in per 1 milliliter of biased sample; Change the upper strata water centrifuge tube of a no RNA enzyme over to, add isopropyl alcohol, 4 ℃ of centrifugation RNA; Precipitate twice with 75% washing with alcohol; Deionized water dissolving deposition with no RNA enzyme.

The Quality Identification of extracting RNA: ultraviolet spectrophotometer is measured OD260/280 ratio (ratio is all between 1.8-2.0), and the extractive RNA of observation has or not degraded in MOPS denaturing formaldehyde glue.

-80 ℃ of cryopreservation.

Two, cDNA is synthetic

TaKaRa RNA PCR Kit method for using with reference to the precious biotech firm in Dalian.Get total RNA 0.5 microgram, Oligo dT-Adaptor primer 0.5 microlitre, MgCl ₂Each 1 microlitre of 2 microlitres, 10X buffer and dNTP mixture, AMV reverse transcriptase 0.5 microlitre, fully behind the mixing, 42 ℃ were reacted 2 hours.Template uses final concentration to be generally 1 microgram/100 microlitres.

Three, pcr amplification

Each composition of reactant mixture is: the ZNF268 forward primer (is positioned at No. four exon of ZNF268 gene; 5 '-GTA ATT CAT GGA TGT GTT TGT GG-3 '; Primer synthesizes gives birth to worker bio-engineering corporation in Shanghai) and the ZNF268 reverse primer (be positioned at No. seven exon of ZNF268 gene; 5 '-ATG TACCTG AAA CCC ATT AGG AT-3 ', primer synthesize give birth to worker bio-engineering corporation in Shanghai) each 1 microlitre, 10X buffer and MgCl ₂Each 2.5 microlitre, dNTP 0.5 microlitre, Taq archaeal dna polymerase 0.25 microlitre and an amount of cDNA template are replenished ddH at last ₂O, making reaction system is 25 microlitres.

As internal reference, its forward primer is with β-actin: 5 '-GCT CGT CGT CGA CAA CGGCTC-3 ', downstream primer is: 5 '-CAA ACA TGA TCT GGG TCA TCT TCT C-3 ' (be combined and be formed in Shanghai living worker bio-engineering corporation).

The reaction condition of PCR is: 95 ℃ of degeneration 10 minutes, and each circulation is 94 ℃ then, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 30 seconds; Totally 35 circulations, last 72 ℃ were extended 10 minutes fully.

C, RT-PCR technology for detection ZNF268 gene are at normal person's hemocyte and the differential expression in the disorders of blood human blood cell

The total RNA and the reverse transcription that obtain blood sample through above-mentioned each step obtain a chain cDNA.

Table 2

	Sample number	Express sample example number and the shared percentage ratio of ZNF268a	Do not express sample example number and the shared percentage ratio of ZNF268a	Express sample example number and the shared percentage ratio of ZNF268b
					The leukemia human blood sample	49	41(83.7％)	8(16.3％)	49(100％)
The normal health human blood sample	16	16(100％)	0	16(100％)

As template,, utilize corresponding primer to detect the differential expression of ZNF268 gene in 49 routine leukemia human blood cells and 16 routine normal health human blood cells with the chain cDNA that obtains through above-mentioned round pcr.The result shows that ZNF268a and ZNF268b have expression among the normal health human blood cell, and promptly the expression rate of ZNF268a is 100% among the normal health human blood cell; But in 49 routine leukemia human blood cells; Can both normal expression ZNF268b; And wherein lacked the expression of the splicing isomer ZNF268a of the total length ZNF268 zinc finger protein of can encoding in 8 routine patient's hemocytees fully, account for 16.3% (Figure 17, table 2) of the sample that detects.Therefore can directly judge whether to suffer from leukemia rapidly and accurately through detection to ZNF268a.

Claims

1. the application of human zinc-finger protein ZNF268 gene splicing isomer ZNF268a in the leukemic preparation of preparation diagnosis.