CN101712959A - Novel human cell growth inhibiting gene THAP11 and application thereof - Google Patents

Abstract

The invention provides a novel human cell growth inhibiting gene and an application of an encoded protein. The gene and the encoded protein are related to the regulation and control of a cell cycle, can inhibit the growth of tumor cells by inhibiting the expression of a proto oncogene c-Myc, and are down-regulated in various tumor tissues, thereby having a close relation with the generation of tumors. The gene and the encoded protein and derivatives thereof can be used for the diagnosis, treatment and drug screening for tumors caused by disordered regulation and control of cell cycles and neurodegenerative diseases and the like.

Description

A kind of new human cell growth inhibiting gene THAP 11 and application thereof

Technical field

The present invention relates to a kind of new human cell growth regulatory gene, also relate to the protein and the application in tumour, nerve degenerative diseases diagnosis, treatment of the DNA that forms this gene and this dna encoding.

Background of invention:

The cell growth is subjected to the complexity regulation and control of several genes.C-Myc occupies critical role as proto-oncogene in the signals-modulating network of regulating cell proliferation, growth, differentiation and apoptosis.A plurality of gene studies reports can be by the retarded growth of downward modulation c-Myc trigger cell (Roussel et al., 1991; He et al., 1998).As one of most important transcription factor, the c-Myc wide expression is each period of fetal development and have in the middle of the tissue of height multiplication capacity.The expression level of cell quiescent stage c-Myc is very low, under the somatomedin effect, c-Myc albumen is synthetic rapidly, and bHLHZ (basic-helix-loop-helix-zipper) structural domain and the MAX that pass through its C end form heterodimer, raise have acetylation of histone enzyme (HAT) function transcribe co-activation thing-TRRAP (transformation/transcription domain-associated protein), with the nucleosome acetylation of histone, thereby change chromosome structure, special regulating and controlling sequence one E-box (CACGTG) that makes the c-Myc-MAX mixture can be combined in target gene goes up (McMahon et al., 1998; Bouchard et al., 2001), activate or suppress transcribing of a series of genes involveds, inducing cell enters the G1 phase and begins propagation (Pelengaris et al., 2002).Discover, have the binding site of 25,000 c-Myc in the human genome.Can infer that c-Myc participates in regulating whole organic a large portion gene, it can activate eIF4 and eIF2 etc., and some are transcription initiation factors (Rosenwald et al., 1993) very important to growth; Also can activate a series of gene transcription relevant, thereby it is synthetic to influence DNA, albumen, finally promotes the cell growth with metabolism; And the genes of being regulated by c-myc that receive publicity are regulatory factors close with the cell cycle operational relation more.But c-Myc element activation cycle D (CyclinD), (CyclinD-Dependent Kinase, the CDK2) protein expression of Denging promote cell to cross restriction point to plain dependent kinase K2 of cycle, enter the cell cycle running, begin division (Steiner et al., 1995; Berns etal., 1997); C-myc also can suppress the supressor P21 (Seoane et al., 2002) of correlation factor such as CDK simultaneously, and the expression of P27 (Obaya et al., 2002) etc. is accelerated period run by the resistance of eliminating the cell cycle operation, promotes propagation.In addition, report is arranged also, c-Myc participates in apoptosis regulation.

C-Myc has regulated and control numerous and cell fission, gene transcription and activity that growth is relevant, and therefore, developing of kinds of tumors often followed in the unconventionality expression of c-Myc, imbalance, amplification etc.Can find that in～70% cancer directly amplification is relevant or lack of proper care with its regulatory factor and the c-Myc abnormal expression that causes is relevant with c-Myc family (c-myc, N-myc, and L-myc).At different tissues such as skin (Pelengaris et al., 1999), pancreas (Pelengaris et al., 2002), prostate gland (Ellwood-Yen et al., 2003) is crossed the transgenic mice of expressing c-Myc, can produce the malignant tumour of living lethality.Therefore, the activity that effectively suppresses c-Myc and downstream gene thereof often is used as the strategy of these tumours of interior therapeutic.A large amount of experiments have proved that suppressing c-Myc expresses, and propagation capable of inhibiting cell promotes apoptosis.And those genes that can suppress c-Myc performance transcriptional activity also receive much concern, and can suppress cell transition growth, propagation after they are expressed; On the contrary, lose or inactivation causes that probably the expression of the c-Myc tumour that finally causes out of control takes place, so these genes press down the cancer candidate gene often.Be positioned at karyomit(e) 16q22-23 as multi-functional zinc finger transcription factor CTCF, its wide expression is put into cancer suppressor gene by suppressing the c-Myc cell growth inhibiting.

THAP (Thanatos-associated protein) protein family is a newfound class functional protein, names (Roussigne et al., 2003) because of the N of its each member protein matter end contains the THAP structural domain of going up high conservative of evolving.(principal character is the DNA binding domains of this structural domain and fruit bat P element transposase: inner C2CH (Cys-Xaa for DNA binding domain, DBD) height homology _2-4-Cys-Xaa _35-50-Cys-Xaa ₂-His) zinc finger print preface and the AVPTIF box that is positioned at the domain C end.Having the research report to think that the THAP family protein originates from fruit bat P element, is protein has obtained P transposable element DBD structural domain (Quesneville et al., 2005) in very long evolutionary process result.Therefore, THAP albumen has with the similar distribution range of P element transposase, only limits to exist in the middle of the animal.Chromosomal localization between its each member does not have obvious dependency, is dispersed in to be distributed in the full genome.

Fewer for the understanding of THAP protein family function, a lot of clues all come from the research of THAP family homologue in the model animals.One of THAP albumen of fruit bat-DIP2 (Dorsal interacting protein) is a member (Bhaskar etal., 2000) of fruit bat transcription factor NF-KB family.Many THAP domain protein-HIM-17 of nematode (Reddy et al., 2004) has played the part of the key player in the process of reduction division chromosome segregation.LIN-36 in the nematode, LIN15A, LIN15-B also belong to THAP protein family (Ferguson et al., 1989).They all can interact with important tumor suppressor gene LIN35/Rb, participate in regulating the cell growth.LIN-36 also is proved the have FZR-1 function of (Fizzy related family-1), can regulate elegans cell propagation and ontogeny (Fay et al., 2002) on the whole.Can make cell under the situation that lacks CyD-1/CDK-4, still can enter the S phase even suppress the activity of LIN-36 or LIN-15A, thereby infer that these two albumen are inhibitions (Boxem et al., 2002) of changing the cell G1/S phase.

People's THAP protein family comprises 12 members (THAP0-THAP11), and 3 members that only limit to of research report are arranged at present.THAP0 screens when research IFN-γ induces the apoptotic genes involved of HeLa at first, the associated protein of called after death at that time (Deathassociated protein-4, DAP4/p52rIPK) (Deiss et al., 1995), back find it can by with p58 ^IPKInteract and remove this albumen PKR (interferon-induced serine/threonine protein kinase, PKR) inhibition, make PKR recover kinase activity, the phosphorylation associated protein, cause the cell protein biosynthesis block, cell proliferation is suppressed (Gale et al., 1998).THAP1 is a preceding antiapoptotic factors in the new nuclear, can interact with Par-4 (prostate-apoptosis-response-4) and co in PML-NBs (promyelocytic leukemianuclear bodies, PML NBs) in, participating in serum removes or TNF-α inductive apoptosis (Roussigne et al., 2002).External SELEX (Systematic Evolution of Ligands by Exponential Enrichment Assay) tests (Clouaire et al., 2005) find that also THAP1 can combine with specific dna sequence dna, confirm that THAP albumen has dna binding activity.Mainly concentrate on it and the pass of transcribing is fastened for the research of THAP7.THAP7 organizes wide expression more; it can combine with histone; promote the deacetylateization of histone H 3; raise NcoR (Nuclearhormone receptor corepressor) and HDAC3 (histone deacetylase 3) suppressor gene on the promotor of target gene simultaneously and transcribe (Macfarlanet al., 2005).Recently; there are some researches show that again THAP7 suppresses to transcribe also to have other mechanism: it can raise INHAT (Inhibitor ofacetyltrans-ferases) subunit TAF-I β, and ((template activating factor-I β) is on the promotor of gene; suppress to transcribe (Macfarlan et al., 2006) by the proteic acetylize of direct closed group.In sum, the THAP protein family is the SDBP that a class zine ion relies on, and may participate in the regulation and control of multiple vital movements such as cell proliferation, apoptosis widely by influencing processes such as cell cycle, chromosome modification, genetic transcription.

THAP11 is that the present invention screens the new gene that obtains from people's marrow cDNA library.It is last member of people THAP protein family, and chromosomal localization is at 16q22.1.THAP11mRNA total length 1903bp (the Genebank number of registration is BC012182), the open reading frame (sequence 1) that contains a 945bp, the protein (sequence 2) that 314 amino acid of encoding are formed, its principal character is held the localized THAP structural domain except containing N, and the molecule middle part also has the polyQ fragment of one section 28 glutamine series connection repeated arrangement and the nuclear localization signal of an information biology prediction.To the function of THAP11, do not see any research report so far as yet.

Summary of the invention

The present invention has detected tissue and the cell expressing spectrum of THAP11, shows that it is widely distributed, does not have tangible tissue specificity (Fig. 1).The GFP-THAP11 fusion protein technology shows that THAP11 is positioned at nucleus (Fig. 2).Detect discovery with many tissue cDNA of tumour micro-array chip, THAP11 is starkly lower than corresponding cancer beside organism at the kinds of tumors tissue expression, the endogenous expression level of THAP11 also very low (Fig. 3) in cancerous cell line.In addition, utilize Real-time PCR to detect the expression of THAP11 in the 12 routine hepatocarcinoma patients, show that THAP11 expression level detail in liver cancer tissue is higher than cancer beside organism (Fig. 4) in 9 routine patients.Thus, we infer that THAP11 may participate in the cell proliferation regulation and control.But utilize the HEK293 stable cell line (Fig. 5) of the abduction delivering THAP11 that makes up, we have studied the influence of THAP11 pair cell behavior, find to induce THAP11 to express, and can obviously suppress HEK293 cell proliferation (Fig. 6).In addition, 7721, cross expression THAP11 among the HeLa, MCF-7 cell and can cause that also colony forms ability obviously descend (Fig. 7).The The above results explanation, THAP11 is a growth suppressor gene.

Be the mechanism of research THAP11 inhibition cell proliferation, this has detected gene changing condition before and after THAP11 induces multiple and that cell is grown, division is relevant, finds the expression along with THAP11, and the c-myc protein level is reduced (Fig. 8) gradually.Luciferase reporter gene experiment confirm, THAP11 can negative regulation c-myc promotor transcriptional activity (Fig. 9).Further detect c-myc regulation and control downstream target gene, find that ODC, Nucleolin etc. and cellular metabolism, the genetic expression that the DNA building-up process is relevant obviously reduces (Figure 10), illustrate that THAP11 transcribes and changed this type of expression of gene by suppressing c-myc, causes the cell retarded growth.For estimating c-myc role in THAP11 performance biological function, we cross expression c-myc when expressing THAP11, repressed cell colony formation ability is obviously recovered, and shows that the c-myc downward modulation is the major reason (Figure 11) of THAP11 negative regulation cell proliferation.

THAP11 is positioned chromosomal region 16q22.1, this zone location a plurality of tumor suppressor genes, heterozygous deletion takes place in kinds of tumor cells, generally believed it is susceptible zone in the kinds of tumors generation evolution.In conjunction with THAP11 suppress that c-myc transcribes and in tumor tissues the phenomenon of down-regulated expression, monopolist of the present invention reaches a conclusion, this gene may be a kind of new tumor suppressor gene.

THAP11 has one section poly glumine zone in its 104-132 position, and propagation capable of inhibiting cell, promote also can to suppress transcription factor CREB activity (Figure 12), therefore by apoptosis, may be the Disease-causing gene of being selected of nerve degenerative diseases, can be used for the diagnosis and the treatment of this genoid.

The invention provides a kind of DNA that constitutes people's gene, have sequence 1 described nucleotide sequence or the nucleotide sequence of one or more base deletions, replacement or increase is arranged.

The invention provides a kind of protein, the aminoacid sequence that it has aminoacid sequence shown in the sequence 2 or one or more aminoacid deletion is arranged, substitute or increase with respect to described aminoacid sequence.

The invention provides a kind of probe, it comprises the DNA with the DNA hybridization of forming the foregoing nucleotide sequence composition.

The invention provides a kind of protein antibodies, comprise polyclonal antibody and monoclonal antibody, can specially detect at aforementioned protein expression.

The invention provides a kind of viral recombinant vectors, express and form the DNA that aforementioned a kind of nucleotide sequence is formed.

The invention provides a kind of dna fragmentation, this fragment can be used as primer, and is made up of the partial sequence of aforementioned a kind of nucleotide sequence.

The invention provides a kind of human diagnostic preparation, it comprises aforementioned probe and aforementioned proteinic antibody.Can be used for the inspection of genetic expression in the malignant tumours such as liver cancer, lung cancer, colorectal carcinoma, mammary cancer, leukemia, kidney, nervous system neoplasm.

The present invention also provides and has contained aforementioned proteic anti-tumor medicinal preparation, comprises the DNA. that expresses aforementioned aminoacid sequence

Therefore, constitute the DNA of gene of the present invention, the protein of their transcript mRNA and translation thereof can be used as treatment, diagnosis and the prophylactic of kinds of tumors.

The present invention is described in conjunction with the accompanying drawings better:

The distribution expression pattern of Fig. 1 THAP11.The result shows, in 8 kinds of grownup's tissues, is confidential reference items with house-keeping gene G3PDH, has the THAP11 of higher level to express in the heart, brain, the skeletal muscle, and remaining tissue all has low expression level.

Fig. 2 THAP11 is positioned nucleus.The pmTHAP11-GFP rotaring redyeing COS 7 cell of pTHAP11-GFP and sudden change nuclear localization signal, fluorescent microscope are observed the fluorescent signal distribution situation down.The result shows that THAP11 is positioned nucleus, and location, nuclear localization signal sudden change back is changed, and becomes full cell distribution.

Fig. 3 utilizes the expression of tumor tissues chip detection THAP11.Detect the expression of THAP11 by hybridizing method at 19 kinds of cancers and corresponding adjacent tissues and 9 kinds of tumor cell lines.

Fig. 4 Real-time PCR detects the expression of THAP11 in liver cancer tissue and cancer beside organism.The result shows that the expression of THAPi1 in liver cancer tissue is starkly lower than cancer beside organism in the most patients that is detected.

But the foundation of Fig. 5 THAP11 abduction delivering cell strain.(A) choose different mono-clonals after in the THAP11-pIND transfection 293-ECR cell, under 5 μ M PonA inductive conditions, cultivated 48 hours.Extract total protein of cell, utilize THAP11 antibody to carry out Western and identify the THAP11 expression.(B) No. 3 clone's various dose PonA induce 48 hours (left side) or induce different time (right side) through 5 μ M PonA for, extract total protein of cell, utilize THAP11 antibody to carry out Western and identify the THAP11 expression.β-actin is as reference.

Fig. 6 THAP11 can suppress the propagation of HEK293 cell.(A) 293EcR (on) and 293-THAP11 cell (descending) inoculation 24 orifice plates, cultivation in the presence of PonA, every day counting cells.The result shows, the propagation that the abduction delivering of THAP11 is capable of inhibiting cell, and have dose-effect relationship.(B) [ ³H] mix the DNA compound experiment and detect the influence of THAP11 pair cell DNA synthetic.Cell is handled with (A).

Fig. 7 colony forms experiment and shows that THAP11 can suppress 7721, HELA, the propagation of MCF-7 cell.Different tumour cell transfection pcDNA-THAP11 or pcDNA3.1 empty carrier.Inoculation 6 orifice plates cultivated for 2 weeks after 12 hours, detected colony number of cell.

Fig. 8 THAP11 can reduce the expression of c-Myc.There is (right side) in the 293-THAP11 cell or exist under (left side) condition at PonA cultivates different time, extracts total protein of cell, and Western detects the expression of THAP11.β-actin is as reference.

Fig. 9 THAP11 suppresses the promoter activity of c-Myc.(A) 293-THAP11 cell inoculation 24 orifice plates, transfection c-Myc promotor reporter gene carrier.PonA induced back 24 hours, collected cell, detected uciferase activity.The pRL-TK carrier of expressing the sea cucumber luciferase is demarcated transfection efficiency as confidential reference items.(B) corotation c-Myc promotor reporter gene carrier and pcDNA3.1-THAP11 carrier or pcDNA3.1 empty carrier in different tumour cells detected uciferase activity after 24 hours.No matter the expression of THAP11 has all suppressed the activity of c-Myc promotor in 293 cells or in tumour cell.

Figure 10 THAP11 reduces the c-Myc target gene expression.RT-PCR detects the changing conditions that THAP11 expresses c-Myc downstream, back target gene.The result shows that the expression of THAP11 can suppress ODC, the expression of hTERT and Nucleolin.CyclinA there is not influence.G3PDH is as confidential reference items.

Figure 11 turns round the inhibited proliferation that c-Myc can be alleviated THAP11.PIRES, pIRES-THAP11 and pIRES-Myc-THAP11 be the different tumour cells of transfection respectively, and the colony of 2 weeks back detection cell forms number.The result shows that the revolution of c-Myc can be alleviated the cell inhibitory effect effect that THAP11 causes.

Figure 12 THAP11 is inhibited to the CREB activity.293-THAP11 cell transfecting Gal4-CREB, Gal4-luci and pFC-PKA plasmid are after PonA induces.Collect cell after 24 hours, detect uciferase activity.The pRL-TK carrier of expressing the sea cucumber luciferase is demarcated transfection efficiency as confidential reference items.The result shows that the expression of THAP11 can suppress the transcriptional activation of PKA to CREB, shows that THAP11 has participated in the transcriptional regulatory process of CREB.

The acquisition of embodiment 1 people THAP11cDNA

With the EDAG gene is bait, utilizes the general yeast-two hybrid technique in this area to study its interaction protein, obtains the section of DNA sequence, adopts this dna sequence dna to do the BLAST retrieval, and the present invention finds to have higher homology with the Human genome THAP11 that infers.The total RNA of exhibitor medullary cell carries out reverse transcription by reverse transcription test kit (Promega company) specification sheets.Get the RNA of 1 μ g after quantitatively, add water and mend to 10.5 μ l, 70 ℃, 10min add 10 * buffer of 2 μ l, the MgCl of 2 μ l ₂, 2 μ l the AMV (15U) of rRNasin, 1.5 μ l of oligo dT, 1 μ l of dNTP, 1 μ l, reaction system totally 20 μ l; 42 ℃, 1h; 94 ℃, 5min; 4 ℃, 5min.RNA is that reverse transcription is cDNA.With 5 '-GGGGTACCATGCCTGGCTTTACGTGCTGCG-3 '; 5 '-CGGAATTCCACATTCCGTGCTTC TTGCGGATG-3 ' is primer PCR amplification purpose fragment.The PCR condition is:

Cycle?1(1x)

94℃?5min

Cycle?2(30x)

94℃?1min

55℃?30s

72℃?1min

Cycle?3(1x)

72℃?10min

The PCR product carries out 1% agarose gel electrophoresis, the result produces special DNA band, and this dna fragmentation is connected with PMD-18-T carrier (TAKARA biotech firm), connects product Transformed E .coli JM109, after bacterium colony PCR is accredited as positive colony, order-checking after enzyme is cut evaluation again.Sequence after the order-checking is shown in the sequence 1.Because dna sequence dna is confirmed its verity, therefore, can produce the specific hybrid probe with following method: make the coli strain growth of carrying plasmid, plasmid DNA purification digests it with suitable Restriction Enzyme, the electrophoretic separation dna fragmentation.

Bioinformatic analysis shows that the THAP11 protein interior has two clear and definite structural domains: be positioned at the THAP structural domain of 90 amino-acid residues compositions of protein molecular N end and be positioned at the PolyQ fragments that 28 or 29 the glutamine residue series connection in molecule middle part repeat to form; In addition, also have a prediction nuclear localization signal (Nuclear location sequence, NLS).The homology comparison shows, people THAP11 and mouse THAP11 cDNA sequence homology are up to 86.73%, with rat then be 88.11%, except the nucleotide difference of indivedual sites, its main difference be to encode (CAG) multiplicity difference to some extent of glutamine in the PolyQ fragment, the repetition number of mouse and P of Rats olyQ is respectively 18 and 24.

Embodiment 2THAP11 expresses in multiple tissue

With 5 ' GGGGTACCATGCCTGGCTTTACGTGCTGCG-3 '; 5 ' CGGAATTCCACATTCCGTGCTTCTTGCGGATG-3 is primer, adopts round pcr, is template amplification THAP11 with 8 kinds of adult's tissue cDNA, with G3PDH as confidential reference items.The result shows that THAP11 all has expression, the expression level in heart and skeletal muscle higher (Fig. 1) in the middle of 8 kinds of adult's tissues that detect.This shows that THAP11 is widely distributed in becoming human body, does not have obvious specific tissue distribution.

Embodiment 3THAP11 is positioned nucleus

For observing the Subcellular Localization of THAP11, we form the THAP11-GFP integrative gene expression vector with the EcoRI/KpnI site that THAP11 cDNA is building up to the pEGFP carrier.With this expression vector rotaring redyeing COS 7 cell, laser scanning co-focusing microscope detects and shows that fluorescent signal is distributed as nucleus (Fig. 2), after the nucleus signal for locating sudden change with the prediction of THAP11 intramolecule, cellular localization changes, become full cell distribution, show that the nuclear localization signal of THAP11 is essential to it in nuclear location.

Embodiment 4THAP11 has notable difference in the expression of multiple cancer and corresponding adjacent tissues

With 5 ' GGGGTACCATGCCTGGCTTTACGTGCTGCG-3 '; 5 ' CGGAATTCCACATTCCGTGCTTCTTGCGGATG-3 is primer, adopts round pcr amplification cDNA segment as probe template, α- ³²With CancerProfiling ArrayII hybridization, detect its expression behind the p mark at 19 kinds of cancers and corresponding adjacent tissues and 9 kinds of tumor cell lines.154 couples of cDNA that detected come from 154 patients' cancer and corresponding adjacent tissues.Results of hybridization shows that THAP11 expression in the tumour of different tissues has very big-difference (Fig. 3).In Tiroidina (10/10), skin (6/10), pancreas (4/7), vulva (5/4), testis cancerous tissues such as (4/10), THAP11 expresses and is suppressed, and significantly is lower than corresponding cancer beside organism; Similar difference is arranged in tumor tissues such as small intestine, uterus, but not obvious; Then opposite in the middle of lung cancer (7/10), ovarian cancer tissue (4/10), THAP11 expression level showed increased.Though above result shows that it may be different that the THAP11 distribution does not have tissue specificity, its function in the middle of different tissues.In addition,, arranged the cDNA of 9 kinds of tumor cell lines in the lower right of film, except lung cancer cell line A549, the hybridization signal of THAP11 and other tumor cell line generally a little less than.

We have further detected the expression level of THAP11 in the hepatocarcinoma patient.By liver cancer and the cancer beside organism total RNA of ordinary method extraction from 12 routine hepatocarcinoma patients.Obtain the first chain cDNA through reverse transcription.With 5 ' AGAAGACGTGAAGCCCA TCGA-3 '; 5 '-TGGTGCCTGACGACAAGGAGTA-3 ' is a primer, utilizes SYBRgreen reagent, carries out Real-time PCR.The PCR condition:

Cycle?1：(1X)

Step?1： 95.0℃ for?01:00.

Cycle?2：(40X)

Step?1： 95.0℃ for?00:10.

Step?2： 65.0℃ for?00:30.

Cycle?3：(1X)

Step?1： 95.0℃ for?01:00.

Cycle?4：(1X)

Step?1： 55.0℃ for?01:00.

Cycle?5：(81X)

Step?1： 55.0℃-95.0℃ for?00:10.

Use IQTM5 Multicolor Real-time PCR Detection System that report fluorescence dye institute emitted fluorescence is monitored in real time.The fluorescent emission amount reflects cycle number and is read by the software of sequential detection instrument that provide the significant cycle number threshold value of pcr amplification, the value of cycle number and genomic dna logarithm are linear.The result shows (Fig. 4), has in 9 routine patients' the liver cancer tissue THAP11 express in the 12 routine hepatocarcinoma patients and is starkly lower than cancer beside organism, shows THAP11 down-regulated expression in liver cancer takes place.

But the foundation of embodiment 5 abduction delivering THAP11 stable cell lines

According to above-mentioned experimental result, the endogenous expression level of THAP11 in tumor tissues is generally on the low side, points out this albumen negative effect to be arranged on cell proliferation.Because THAP11 expresses the stable cell line that back pair cell toxigenicity effect can't obtain the constructive expression, we have selected moulting hormone abduction delivering system-293EcR clone for use in order to avoid.

293EcR clone is cell-derived by HEK293, can express ecdysone receptor factor VpEcR (comprising a DNA land and a VP16 active region) and the homologue-RXR (retinoidX receptor) of native ligand USP (ultraspirale protein) in Mammals thereof through modifying, at moulting hormone (ecdysone) or its analogue pine sterone (PonasteroneA) when existing, the two can be combined into heterodimer VpEcR-RXR rapidly, activation contains the promotor of its effector (response element), and the guiding goal gene is transcribed.Because the no ecdysone receptor of Mammals itself, so this sole of this system induction reaction is low.

With 5 '-GGGGTACCATGCCTGGCTTTACGTGCTGCG-3 ' and 5 '-CGGAATTCCACATTCCGTGCTTCTTGCGGATG-3 ' is primer, through round pcr, the THAP11 CDNA segment of amplification is cloned into the pIND carrier.Recombinant vectors or pIND empty carrier transfection 293EcR cell, after G418 and Zeocin screening, limiting dilution assay picking mono-clonal, enlarged culturing.Identify through RT-PCR and Western, obtain a few strain positive cell mono-clonals (Fig. 5 A), picking clone 3 wherein, called after 293-THAP11.

Be the controllable express of realization THAP11, after we have detected interpolation various dose inductor respectively and have induced different time, the THAP11 changes of expression level.The result shows, induces under 24 hours conditions, and along with the increase of Ponasteron A (Pon A) concentration, the THAP11 expression amount also obviously raises, and when inductor was 5 μ M, the THAP11 expression amount reached the highest.Under the condition of using the same concentrations inductor, the expression of THAP11 then increases (Fig. 5 B) along with the prolongation of induction time.In the experiment afterwards, we can be according to the experiment needs, by adjusting inductor concentration or changing the controllable express that induction time is realized THAP11.

Embodiment 6THAP11 abduction delivering suppresses cell proliferation

2 * 10 ⁴293-THAP11 cell inoculation 24 well culture plates adopt different concns PonA to induce THAP11 to express, and cell counting detects the propagation situation of cell.For getting rid of inductor itself may pair cell be that propagation is influential, and after we at first observed and add PonA, the growth curve of 293EcR clone changed.The result shows that the inductor cell growth does not have influence.From the 3rd day, 293-THAP11 cell line cell propagation began to slow down but compared with the control,, along with the THAP11 prolongation of expression time, suppress obvious all the more, and this retarding effect strengthens along with the increase of inductor concentration, present dose-dependently (Fig. 6 A).Illustrate that THAP11 expresses propagation capable of inhibiting cell.

Adopt [ ³H] mix the DNA compound experiment and detect the influence of THAP11 pair cell DNA synthetic.Discovery is in the 293-THAP11 cell, and it is synthetic that the THAP11 abduction delivering can obviously suppress cell DNA, and along with the increase of inductor concentration, retarding effect strengthens gradually, has dose-effect positive correlation (Fig. 6 B); On the contrary, we do not detect inductor itself influences 293EcR cell DNA synthetic.

Embodiment 7THAP11 crosses the colony formation that expression can suppress tumour cell

Be further conclusive evidence THAP11 cell growth inhibiting whether in other clone, we adopt colony form experiment detected respectively THAP11 for 7721, the influence of HeLa and MCF-7 cell line proliferation.The result shows that the MCF-7 of transfection pcDNA-THAP11, HeLa and 7721 cells are with respect to the cell that changes empty carrier, and its colony forms ability and suppressed 75%, 80% and 60% (Fig. 7) respectively.

Embodiment 8THAP11 expresses and follows the c-myc down-regulated expression

For inquiring after the reason of THAP11 inhibition cell proliferation, we have detected before and after the THAP11 abduction delivering, with the variation of the more closely-related transcription factors of cell proliferation.Can see that the 293-THAP11 cell in the cultivation itself has higher c-myc to express, but be accompanied by the increase of THAP11 expression amount, the c-myc protein level presents obvious downward modulation (Fig. 8).

Embodiment 9THAP11 can suppress c-myc promoter transcription activity

For further research THAP11 is to the effect of c-myc, we have detected THAP11 to the active influence of c-myc promoter transcription with reporter gene.With connecting c-myc total length promotor (P2 promotor upstream～3kb, Dr.Kenneth W Kinzler gives) luciferase reporter gene carrier (Del-1) transfection 293-THAP11 cell, after inducing THAP11 to express 24 hours, the detection uciferase activity changes, the result shows that THAP11 expresses can suppress the c-myc promoter activity; And when inductor concentration was 5 μ M, the promoter activity inhibiting rate reached 52% (Fig. 9 A).

Further use pcDNA-THAP11 and c-myc promoter luciferase reporting gene co-transfection MCF-7, HeLa, clones such as 7721, with pcDNA3.1 in contrast, the result shows, in these clones, THAP11 can suppress the c-myc transcriptional activity equally, and dosage effect (Fig. 9 B) is arranged.

Embodiment 10THAP11 is to the influence of c-myc target gene

C-myc can be by activating or suppressing the numerous target gene expression in downstream and realize its regulating and controlling effect.For the influence of research THAP11 to c-myc regulation and control target gene, inquire into the mechanism that THAP11 suppresses cell proliferation, after we have detected the THAP11 abduction delivering with RT-PCR, the variation of the c-myc target gene that some are relevant with growth and cycle.As shown in figure 10, ornithine decarboxylase (the Orinithine decarboxylase that just regulated and control by c-myc, ODC), the human telomerase reverse transcriptase (Telomerase reverse transcriptase, hTERT) and the mRNA level of paranuclein (Nucleolin) present obvious downward modulation (Figure 10) after 60 hours at the THAP11 abduction delivering.

Embodiment 11 revolution c-myc can reverse the cell inhibitory effect that THAP11 causes

The C-myc down-regulated expression both may be the reason that suppresses cell proliferation, also may be the result (Pelengaris et al., 2003) of cell inhibitory effect.Be that assessment c-myc down-regulated expression suppresses role in the cell proliferation at THAP11, we with colony form experiment detected in the cell of expressing THAP11, cross simultaneously express c-myc after the propagation situation of cell what has change.

PIRES is a double gene expression vector, because it has introduced ribosome entry site(RES) (IRES) between two multiple clone site, therefore can express two goal gene simultaneously.We clone into carrier respectively with the coding region of c-myc and THAP11, have made up the pIRES-THAP11 that only expresses THAP11 and the pIRES-Myc-THAP11 carrier of two genes of coexpression.Western has confirmed this two proteic expressions.With the several clones of carrier difference transfection, detect and find that pIRES compares with empty carrier, pIRES-THAP11 expresses and makes plastidogenetic colony number obviously reduce (Figure 11).But after expressing c-myc simultaneously, this inhibition phenomenon obtains trivial solution and removes, and plastidogenetic colony number increases greatly.The HEK293 cell by 12% increase by 47%, 7721 by 39% increase to 69%, HeLa by 50% be increased to 76%, MCF-7 again 23% to 48%.Show that the c-myc downward modulation suppresses the major reason of cell proliferation at THAP11.

Embodiment 12THAP11 suppresses the transcriptional activity of PKA activated CREB

Then, we have detected the influence of THAP11 to several signal paths relevant with propagation in the born of the same parents with PathDetect signal transduction pathway trans-reporting systems.This system provides an expression vector pFA trans-activator who has merged transcription factor transcriptional activation domain and Gal4-DBD, by cotransfection goal gene and transcription factor, and the reporter gene carrier that contains the Gal4 binding site, can discern the information transmission whether a new gene participates in institute's detection signal path special relatively, apace, if participate in, be again to occupy which link.We have detected four included in system paths, are respectively the approach that transcription factor c-Jun, Elk, CREB and CHOP are regulated and control.Discovery changes THAP11 over to for the not influence of majority signal path, single expression THAP11 is to the also not influence of transcriptional activity of CREB, but if after changing the activator PKA of CREB over to simultaneously, can see that compared with the control the expression of THAP11 can obviously suppress PKA activated CREB transcriptional activity (Figure 12).

Sequence table

＜110〉INST OF EMISSION ﹠ RADIATION M

＜120〉a kind of new human cell growth inhibiting gene THAP 11 and application thereof

<160>2

<210>1

<211>945

<212>DNA

＜213〉ethnic group (Homo Sapiens.)

<400>1

atgcctggct?ttacgtgctg?cgtgccaggc?tgctacaaca?actcgcaccg?ggacaaggcg 60

ctgcacttct?acacgtttcc?aaaggacgct?gagttgcggc?gcctctggct?caagaacgtg 120

tcgcgtgccg?gcgtcagtgg?gtgcttctcc?accttccagc?ccaccacagg?ccaccgtctc 180

tgcagcgttc?acttccaggg?cggccgcaag?acctacacgg?tacgcgtccc?caccatcttc 240

ccgctgcgcg?gcgtcaatga?gcgcaaagta?gcgcgcagac?ccgctggggc?cgcggccgcc 300

cgccgcaggc?agcagcagca?acagcagcag?cagcagcaac?agcagcaaca?gcagcagcag 360

cagcaacagc?agcagcagca?gcagcagcag?cagcagtcct?caccctctgc?ctccactgcc 420

cagactgccc?agctgcagcc?gaacctggta?tctgcttccg?cggccgtgct?tctcaccctt 480

caggccactg?tagacagcag?tcaggctccg?ggatccgtac?agccggcgcc?catcactccc 540

actggagaag?acgtgaagcc?catcgatctc?acagtgcaag?tggagtttgc?agccgcagag 600

ggcgcagccg?ctgcggccgc?cgcgtcggag?ttacaggctg?ctaccgcagg?gctggaggct 660

gccgagtgcc?ctatgggccc?ccagttggtg?gtggtagggg?aagagggctt?ccctgatact 720

ggctccgacc?attcgtactc?cttgtcgtca?ggcaccacgg?aggaggagct?cctgcgcaag 780

ctgaatgagc?agcgggacat?cctggctctg?atggaagtga?agatgaaaga?gatgaaaggc 840

agcattcgcc?acctgcgtct?cactgaggcc?aagctgcgcg?aagaactgcg?tgagaaggat 900

cggctgcttg?ccatggctgt?catccgcaag?aagcacggaa?tgtga 945

<210>2

<211>315

<212>PRT

＜213〉ethnic group (Homo Sapiens.)

<400>2

Met?Pro?Gly?Phe?Thr?Cys?Cys?Val?Pro?Gly?Cys?Tyr?Asn?Asn?Ser?His 16

Arg?Asp?Lys?Ala?Leu?His?Phe?Tyr?Thr?Phe?Pro?Lys?Asp?Ala?Glu?Leu 32

Arg?Arg?Leu?Trp?Leu?Lys?Asn?Val?Ser?Arg?Ala?Gly?Val?Ser?Gly?Cys 48

Phe?Ser?Thr?Phe?Gln?Pro?Thr?Thr?Gly?His?Arg?Leu?Cys?Ser?Val?His 64

Phe?Gln?Gly?Gly?Arg?Lys?Thr?Tyr?Thr?Val?Arg?Val?Pro?Thr?Ile?Phe 80

Pro?Leu?Arg?Gly?Val?Asn?Glu?Arg?Lys?Val?Ala?Arg?Arg?Pro?Ala?Gly 96

Ala?Ala?Ala?Ala?Arg?Arg?Arg?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln 112

Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln 128

Gln?Gln?Gln?Gln?Ser?Ser?Pro?Ser?Ala?Ser?Thr?Ala?Gln?Thr?Ala?Gln 144

Leu?Gln?Pro?Asn?Leu?Val?Ser?Ala?Ser?Ala?Ala?Val?Leu?Leu?Thr?Leu 160

Gln?Ala?Thr?Val?Asp?Ser?Ser?Gln?Ala?Pro?Gly?Ser?Val?Gln?Pro?Ala 176

Pro?Ile?Thr?Pro?Thr?Gly?Glu?Asp?Val?Lys?Pro?Ile?Asp?Leu?Thr?Val 192

Gln?Val?Glu?Phe?Ala?Ala?Ala?Glu?Gly?Ala?Ala?Ala?Ala?Ala?Ala?Ala 208

Ser?Glu?Leu?Gln?Ala?Ala?Thr?Ala?Gly?Leu?Glu?Ala?Ala?Glu?Cys?Pro 224

Met?Gly?Pro?Gln?Leu?Val?Val?Val?Gly?Glu?Glu?Gly?Phe?Pro?Asp?Thr 240

Gly?Ser?Asp?His?Ser?Tyr?Ser?Leu?Ser?Ser?Gly?Thr?Thr?Glu?Glu?Glu 256

Leu?Leu?Arg?Lys?Leu?Asn?Glu?Gln?Arg?Asp?Ile?Leu?Ala?Leu?Met?Glu 272

Val?Lys?Met?Lys?Glu?Met?Lys?Gly?Ser?Ile?Arg?His?Leu?Arg?Leu?Thr 288

Glu?Ala?Lys?Leu?Arg?Glu?Glu?Leu?Arg?Glu?Lys?Asp?Arg?Leu?Lau?Ala 304

Met?Ala?Val?Ile?Arg?Lys?Lys?His?Gly?Met 314

Claims (10)

1. DNA who constitutes people's gene is with respect to nucleotide sequence shown in the sequence 1 or the nucleotide sequence of one or more base deletions, replacement or increase is arranged.

2. protein, the aminoacid sequence that it has aminoacid sequence shown in the sequence 2 or one or more aminoacid deletion is arranged, substitute or increase with respect to described aminoacid sequence.

3. probe, it is contained in the DNA of the DNA hybridization that the described nucleotide sequence of claim 1 forms.

4. dna fragmentation as primer, it is made up of the partial sequence of the described nucleotide sequence of claim 1.

5. viral recombinant vectors, it comprises the DNA that is made up of the described nucleotide sequence of claim 1.

6. human diagnostic preparation, it comprises the described probe of claim 3.

7. the described diagnostic preparation of claim 6 application that genetic expression is checked in malignant tumour such as liver cancer, lung cancer, colorectal carcinoma, mammary cancer, kidney and nerve degenerative diseases.

8. anti-tumor medicinal preparation, it contains the described protein of claim 2.

9. an energy and claim 2 described protein specific bonded polyclone and monoclonal antibody.

10. diagnostic pharmaceutical preparation, it contains the genetic expression that the described antibody of claim 9 is used for detecting tumour and nerve degenerative diseases patient.

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