CN100348614C - Idiosyncratic antigen protein, and antigen peptide of liver cancer orchis pellet - Google Patents

Abstract

The present invention relates to a liver cancer-testis specific antigen protein and an antigen peptide. The liver cancer-testis specific antigen protein is a protein with an amino acid sequence shown in figure 1 and a protein derived from the protein with the amino acid sequence. An amino acid sequence of the antigen peptide is from AA108 to AA191 in a sequence shown in figure 1. The liver cancer-testis specific antigen protein and the antigen peptide provided by the present invention can be used as a blocking agent of the activity of E2F, and perform an antagonistic function to an isogeny analogue TFDP-1. The protein or the peptide can be used as a CT antigen or an antigen gene to be made into vaccines. The tumor antigen protein is decomposed intracellularly, and can generate a peptide fragment combined with main histocompatibility compound(MHC)-I molecules, wherein the peptide fragment can be recognized by T cells in a combination state so as to achieve the goal of treating cancers by activating immune reaction. Consequently, the present invention can be applied to the preparation of medicine for treating cancers particularly for treating liver cancer.

Description

A kind of liver cancer orchis pellet specific antigen protein matter and antigen peptide

Technical field

The present invention relates to a kind of liver cancer orchis pellet specific antigen protein matter and antigen peptide.

Background technology

Tumor-testis (cancer-testis, abbreviation CT) antigen protein is a maximum class in the tumour antigen of identifying at present, the characteristics of their encoding genes are in the tumour of a lot of types expression to be arranged all, as expression is all arranged in melanoma, lung cancer, sarcoma and bladder cancer, but do not express in the healthy tissues except that testis, a certain amount of expression is arranged in placenta, ovary, pancreas.Because testis is immunity special permission position, so this class antigen is considered to the antigen of tumour-specific in treatment, is a class antigen that is hopeful to be used as the immunotherapy of tumors purposes most.Try out at present in clinically also mainly be exactly this class antigen, as MAGE-1 and NY-ESO-1 promptly is the expression that high positive rate is arranged in the tumour of certain type, the antigen protein that good immunogenicity is arranged again has good prospect when being used for the immunotherapy of tumour.

Primary hepatocellular carcinoma is in the human primary hepatocarcinoma modal one type, also is the whole world one of the malignant tumour of normal generation, and especially in South East Asia and Africa, its sickness rate is higher.On the Chinese side, there are every year 110,000 people to die from liver cancer approximately, account for 1/4th of the annual PLC mortality number in the whole world.This high mortality mainly is because early hepatocarcinoma lacks special clinical symptom, causes most of patient to go to a doctor late, and lacks due to effective treatment means and the easy recurrence of postoperative.Sickness rate height, grade of malignancy height, slow, few, the poor prognosis of methods of treatment of diagnosis are several big characteristics of liver cancer.Although the scientific research personnel has carried out many-sided research to liver cancer both at home and abroad, but the molecule mechanism of liver cancer genesis and development is also not clear at present, treatment to liver cancer also mainly is to take operative treatment and part or systemic chemotherapy, a few patients is aided with the knubble biological therapy, but shorter survival is still unsatisfactory.For this reason, research to liver cancer genesis and development mechanism becomes current an urgent demand, wishes to take this to illustrate early the molecular basis of liver cancer genesis and development, or with the closely-related molecule of liver cancer genesis and development, so that follow up a case by regular visits to the high risk population, effectively prevention, diagnosis early, complex therapy, minimizing recurrence.

Studies show that in the past, liver cancer be a multistage, the rapid process of multistep, be accompanied by the many cytology of body, genetic change in this process, the positive regulation and the relative equilibrium between the negative regulation that exist in cell are broken, the several genes abnormal expression appears, comprise oncogene active and/or cancer suppressor gene deactivation, excite anomalous signals transduction path, cause cell cycle and apoptosis unusual, thereby make malignant transformation of cells, and along with the accumulation of various molecule abnormalities, tumour progress occurs, shifts.And liver cancer is heterogeneous in the change of molecular level, may have a plurality of genes, the effect of a plurality of approach overlapping.Simultaneously, cell intrinsic self-protection mechanism also may be induced and be produced the antagonism molecule.So, the gene of clear and definite these differential expressions, for liver cancer genesis and development mechanism illustrate and all significant to the clinical diagnosis of liver cancer, treatment.

Summary of the invention

The object of the present invention is to provide a kind of tumour testes specificity antigen protein and tumour testes specificity antigen peptide that can be used for liver cancer treatment.

The objective of the invention is to realize by the following technical solutions:

The invention provides a kind of liver cancer orchis pellet specific antigen protein matter TFDP-3 (HCA661), it has following aminoacid sequence a or derived protein b:

Described aminoacid sequence a has aminoacid sequence shown in Figure 1;

Described derived protein b is with the replacement of the aminoacid sequence among the described aminoacid sequence a through one or several amino-acid residue, the derived protein that disappearance or interpolation sudden change are produced, this derived protein and described aminoacid sequence a have same or analogous function.

The invention provides a kind of gene of the above-mentioned liver cancer orchis pellet specific antigen protein matter of encoding.

Gene of the present invention belongs to DP family, and it has nucleotide sequence shown in Figure 2, and accession number is in gene library: AF219119.

The invention provides a kind of fusion rotein that comprises above-mentioned liver cancer orchis pellet specific antigen protein matter.

The invention provides a kind of liver cancer orchis pellet specific antigens peptide, it has following peptide section c or derived peptide d:

The specific DNA binding domains that described peptide section c is above-mentioned liver cancer orchis pellet specific antigen protein matter contain 84 amino acid whose peptide sections, this amino acid whose sequence is start-stop AA108-AA191 in sequence shown in Figure 1;

Described derived peptide d is with the replacement of the aminoacid sequence among the peptide section c through one or several amino-acid residue, the derived peptide that disappearance or interpolation sudden change are produced, and this derived peptide and described peptide section a have same or analogous function.

The invention provides a kind of gene of the above-mentioned liver cancer orchis pellet specific antigens peptide of encoding.

The invention provides a kind of fusion rotein that comprises above-mentioned liver cancer orchis pellet specific antigens peptide.

Liver cancer orchis pellet specific antigen protein matter provided by the invention, antigen peptide and fusion rotein in the pRB/E2F signal transduction, can be used as the active blocker of E2F, to its homology analogue TFDP-1 performance antagonistic action.This proteins/peptides and its DNA of encoding can be used as CT antigen or antigenic site thereby make vaccine, this tumor antigen protein matter is by decomposing in the cell, can produce the peptide fragment combine with major histocompatibility complex (MHC)-I quasi-molecule, can be discerned by the T cell at bonding state, challenge and reach the treatment cancer purpose, thereby can be applied in the medicine of preparation treatment cancer, particularly liver cancer.Use tumor antigen protein matter of the present invention the medicine of activation antineoplastic immune can be provided, diagnostic method of tumors can be provided.The antibody of anti-tumor antigen protein matter of the present invention can be used for the preparation of affinity chromatography, cDNA library screening, immunology diagnosis or medicine.

The present invention clone's new gene be liver cancer orchis pellet specific expressed, with the relevant gene of liver cancer generation, the research of function for it will help illustrating of liver cancer genesis and development mechanism.The new albumen TFDP-3 (HCA661) of this genes encoding is a new CT antigen, in primary hepatocellular carcinoma (hcc), TFDP-3 (HCA661) positive accounts for 1/3, has the potential clinical value that is applied to the hepatocellular carcinoma treatment as tumor vaccine.Simultaneously, different with other CT antigen, this albumen has tangible function, is the newcomer of DP family, and this family can form heterodimer with E2F, thereby strengthens the transcripting regulating function of E2F, is in the core position in the pRB/E2F signal path.PRB albumen is the target protein of the upright early gene product effect of multiple DNA tumour virus, as simian virus 40 large T antigen, papillomavirus E7 albumen, adenovirus E 1 a albumen etc.These oncoproteins can combine pRB albumen with E2F competition, the effect that destroys E2F and pRB with combine, active inhibition is eliminated to E2F to cause pRB, the transcribing of activation E2F dependency target gene.In most human tumors, the regulatory mechanism of E2F is lacked of proper care, and the imbalance of E2F function tends to be accompanied by transformation, even the generation of tumour.Many researchs in the past prove that all DP albumen or E2F albumen have the characteristic of proto-oncogene.The structural analysis of TFDP-3 (HCA661) shows that it has the structure similar to TFDP-1, promptly contains a DNA binding domains and a dimerization structural domain.Functional study shows, TFDP-3 (HCA661) can form heterodimer with the albumen that all of E2F family have a dimerization structural domain, and on the level of protein-interacting, TFDP-3 (HCA661) has the on all four biological characteristics with TFDP-1.But on albumen and the interactional level of DNA, TFDP-3 (HCA661) has greatly weakened E2F with the heterodimer that E2F albumen forms and has combined the ability of DNA, and can suppress the transcriptional activity of E2F, also can suppress the allosteric activation of TFDP-1 to E2F.TFDP-3 (HCA661) makes it be expected as the pRB/E2F signal path that causes when virus infection or pRB sudden change antagonist when out of control to the peculiar property of E2F function effect, thereby remedies the function of pRB, is partly to remedy at least.TFDP-3 (HCA661) is in the specificity of tissue expression and the inhibition E2F active characteristic similar to pRB that it had, can be considered the cancer suppressor gene of liver cancer testes specificity, this gene and albumen may provide a target spot preferably for developing in the future the liver cancer-specific treatment.

In addition, the present invention is a tumor-testis specific antigens gene with TFDP-3 (HCA661) gene identification first; When using immunization method treatment cancer, can induce body to produce a immune response with tumor antigen protein matter of the present invention at tumour cell, thereby do not killing and wounding under the Normocellular prerequisite the intravital tumour cell of specific killing and reach the effect that treatment or control tumour cell shift; Simultaneously, can be by to this tumor antigen gene or detection of antibodies, to generation, the development of tumour and whether have to shift and diagnose.

Description of drawings

Fig. 1 is the aminoacid sequence of liver cancer orchis pellet specific antigen protein matter provided by the invention;

Fig. 2 is the nucleotide sequence of coding liver cancer orchis pellet specific antigen protein matter provided by the invention;

Fig. 3 is for using the comparative analysis result of CLUSTAL W (1.82) routine analyzer to the aminoacid sequence of proteic aminoacid sequence of TFDP-3 (HCA661) and the homology analogue TFDP-1 of its DP family; Wherein, " * " refers to that the amino-acid residue in the sequence is in full accord; ": " refers to that the amino-acid residue in the sequence belongs to conservative and replaces; ". " refers to that the amino-acid residue in the sequence belongs to half conservative the replacement;

Fig. 4 is the Northem blot proof diagram of TFDP-3 (HCA661) mRNA in hepatocellular carcinoma (HCC); Wherein, Testis is a testis tissue; Adj is contiguous non-cancer tissue; HCC is a hepatocellular carcinoma; Normalliver is a normal liver tissue;

Fig. 5 is the expression and distribution of TFDP-3 (HCA661) in HCC clone; Wherein, L02 is a normal liver cell system, and Jurkat is that acute T lymphocytic leukemia cell is, all the other are HCC clone;

Fig. 6 is that TFDP-3 (HCA661) analyzes with the interactional GSTPull-down of E2F family protein; Wherein, A figure: the soluble g ST fusion rotein of E2F is induced in e. coli jm109 and is expressed, and the expression level of soluble proteins is assessed by coomassie brilliant blue staining; Arrow is depicted as the specificity migration band of soluble proteins; The GST albumen of amixis gene is as negative reference.B figure: combine with gsh-agarose microballon respectively as the GST albumen of negative reference and the gst fusion protein of E2F, then with TFDP-3 (HCA661) protein-interacting of in-vitro transcription, translation, and wash unconjugated albumen off, remaining and gsh-agarose microballon bonded albumen passes through electrophoresis, radioautographic analysis.The TFDP-3 of in-vitro transcription, translation (HCA661) albumen is as positive reference;

Fig. 7 is the co-immunoprecipitation analysis of TFDP-3 (HCA661) and E2F protein-interacting; Wherein, last figure is the co-immunoprecipitation albumen that adopts anti-HA polyclonal antibody to extract from the HeLa cell of cotransfection by immunoblotting assay; Middle figure adopts anti-HA polyclonal antibody by the proteic expression level of E2F in the HeLa cell lysate of immunoblotting assay cotransfection; Figure below is for adopting TFDP-3 (HCA661) the proteic expression level of anti-FLAG M2 monoclonal antibody by the HeLa cell lysate of immunoblotting assay cotransfection; Arrow is depicted as TFDP-3 (HCA661) or the proteic specificity migration of E2F band;

Fig. 8 is that TFDP-3 (HCA661) analyzes (EMSA) with the DNA binding ability of the heterodimer of E2F protein-interacting formation;

Fig. 9 is the analysis of TFDP-3 (HCA661) Subcellular Localization; Wherein, A, the Subcellular Localization during the independent transfection of E2F or DP albumen; B, the Subcellular Localization when E2F and DP cotransfection; All images is to absorb under 480 times the visual field in magnification by the immunofluorescence inverted microscope that has been equipped with CCD;

Figure 10 is the analysis of TFDP-3 (HCA661) to the influence of E2F transcriptional activity; Wherein, A is the transcriptional activity result that TFDP-3 (HCA661) suppresses E2F; B is the transcriptional activation result that TFDP-3 (HCA661) suppresses the E2F/TFDP-1 mediation; C is that the expression of TFDP-3 (HCA661) mistake can suppress the active result of endogenous E2F; Shown data are mean value and standard deviations thereof of three experiments;

Figure 11 is the diagram of TFDP-3 (HCA661) mutant; Wherein, A is the comparison of amino-acid residue of the DNA binding domains of the DNA binding domains of two known people DP family protein TFDP-1 and TFDP-2 and TFDP-3 (HCA661); " * " refers to that the amino-acid residue in the sequence is in full accord; ": " refers to that the amino-acid residue in the sequence belongs to conservative and replaces; ". " refers to that the amino-acid residue in the sequence belongs to half conservative the replacement; The employed routine analyzer of sequential analysis is CLUSTAL W (1.82); " △ " refers to that this amino-acid residue has participated in the interaction with the DNA base; " ▲ " refers to that this amino-acid residue has participated in the interaction with the DNA skeleton; " ■ " refers to that this amino-acid residue has participated in the interaction with proteinic different dimerization; B is the diagram of the replacement mutant that derives from the aminoacid sequence of total length TFDP-3 (HCA661) and TFDP-1; TFDP-3 (HCA661) and TFDP-1 can be divided into aminoacid sequence 4 parts according to the composition of structural domain, i.e. N end unknown function district (UNT), DNA binding domains (DBD), different dimerization structural domain (HD) and C end unknown function district (UCT); C is the diagram of the mutant that derives of replacement that TFDP-3 (HCA661) and TFDP-1 carry out in the DNA binding domains; The amino-acid residue shade mark of TFDP-3 (HCA661); Also marked the character of transcribing of this mutant simultaneously briefly on the right side of mutant;

Figure 12 is the molecular basis analysis that TFDP-3 (HCA661) unique function produces; Wherein, A is the protein expression of TFDP-3 (HCA661) and TFDP-1 mutant; B and C are the impact statement gene assessment of the replacement mutant of TFDP-3 and TFDP-1 to the transcriptional activity of E2F-4; Shown data are mean value and standard deviations thereof of three experiments.

Embodiment

Embodiment 1. genetic analysiss

TFDP-3 (HCA661) is the new gene that goes out with serum screening from patient's liver cancer tissue by the SEREX method.According to bioinformatic analysis, TFDP-3 (HCA661) assignment of genes gene mapping is a gene that single exon is formed on the X chromosome of people's gene.Its transcript size is 1,680bp, and wherein encoder block is 1,218bp, terminal is 90-1,307,405 the amino acid whose albumen (as shown in Figure 1) of encoding altogether.This albumen is the newcomer of transcription factor DP family, with the homology analogue TFDP-1 of its DP family similarity (75.2% amino acid consistence is highly arranged; 82.0% amino acid similarity).The comparative analysis of the aminoacid sequence of the two as shown in Figure 3, similar with TFDP-1, the aminoacid sequence of TFDP-3 comprises the DNA binding domains (AA108-AA191) of an evolution conservative and the different dimerization structural domain (AA192-AA264) of an evolution conservative, and, in the DNA binding domains, also contain a RRXYD DNA identification motif (AA162-AA166) that generally has in E2F family and DP family.In known E2F and DP family, the 3rd amino acids in the RRXYD DNA identification block is generally hydrophobic amino acid (Valine (V) or Isoleucine (I)), but in TFDP-3 (HCA661), this amino acid is hydrophilic Threonine (T), and the amino acid of this non-conservation is replaced the DNA combined function of TFDP-3 (HCA661) influential it be unclear that whether.In addition, 20 amino acid of the carboxyl terminal of TFDP-3 (HCA661) have 15 to be acidic amino acid, and TFDP-1 and TFDP-2 have only 13 and 11 acidic amino acids.It is the 3rd newcomer of people's DP family that these analytical resultss are pointed out TFDP-3 (HCA661) consumingly.

Embodiment 2.TFDP-3 (HCA661) mRNA Northern blot analyzes

Be to carry out Northem blot and analyze, from HCC sample and corresponding non-cancer tissue sample and testis tissue, extract total RNA.The integrity of RNA is by electrophoretic analysis.During electrophoresis, the applied sample amount of every total RNA in hole is 30mg, changes film then.Use specificity ³²The cDNA probe of P mark and nitrocellulose filter spend the night 65 ℃ of hybridization.With 0.1 * SSC/0.1%SDS solution washing three times, each 30 minutes ,-70 ℃ of radioautograph.The size of full length mRNA draws by the extent of migration contrast with the ribosome-RNA(rRNA) of 28S and 18S.

Because the high similarity of nucleotides sequence, Northern blot results of hybridization (as shown in Figure 4) show, have occurred two strong bands in the RNA of testis tissue, are respectively～1.7kb and 2.1kb.The band of an appearance～1.7kb in the RNA of HCC tissue, and non-cancer tissue and normal liver tissue have only detected the band of 2.1kb.Dna sequence analysis shows that the gene of 2.1kb is TFDP-1, and the gene of 1.7kb is TFDP-3 (HCA661).

The expression and distribution of embodiment 3.TFDP-3 (HCA661) in HCC and contiguous non-cancer tissue sample

The express spectra of TFDP-3 (HCA661) mRNA is analyzed by RT-PCR, the results are shown in table 1.Tissue comprises 16 kinds of healthy tissues cDNA that buy from Clontech: brain, the heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, colon, ovary, peripheral blood leucocyte, prostate gland, small intestine, spleen, testis and thymus gland become cDNA from total RNA reverse transcription of HCC tissue and the contiguous non-cancer tissue extraction of paired with it.Use Auele Specific Primer to amplification TFDP-3 (HCA661) fragment.Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA is as confidential reference items.The primer is as follows: TFDP-3, forward, 5 '-TACACT CGG CCT GGA AGA ATT G-3 '; Reverse, 5 '-TCT TCC TCC TCG ACT GCT G-3 ', size, 1,244 base pairs (bp).

In 16 kinds of healthy tissuess, have only testis to detect the high level expression of TFDP-3 (HCA661) mRNA, in normal pancreatic tissue, to express very weakly, all the other various healthy tissuess do not detect expression.In HCC tissue, positive sample has 5 parts in 17 parts of HCC tissue samples, and only has 2 parts of samples that faint expression is arranged in 17 parts of corresponding contiguous non-cancer tissues of HCC tissue sample, and 9 parts of none expression of normal liver tissue are a typical cancer-testis (CT) antigen.

The RT-PCR that table 1, TFDP-3 (HCA661) express in HCC and contiguous non-cancer tissue sample analyzes

	HCC	ADJ	Cirrhosis	Normal liver
					Case	17	17	5	9
+ve	5	2	0	0

The expression of embodiment 4.TFDP-3 (HCA661) mRNA in HCC clone

Adopt TRIZOL (GIBCO BRL) from L02 (people's normal liver cell system), CH-Hep-1, CH-Hep-2, CH-Hep-3, CH-Hep-4, CH-Hep-5, CH-Hep-6, Hep 3B, Bel-7402, Bel-7405, SSMC7721, Hep G2 and HLE HCC clone, SK-Mel-37 and Mel-526 K-1735, Jurkat (the acute T lymphocytic leukemia cell of people system) the total RNA of cell extraction.Total RNA reverse transcription also uses Auele Specific Primer to amplification TFDP-1, TFDP-2 and TFDP-3 (HCA661) fragment, and G3PDH mRNA is as confidential reference items.Use following parameters to carry out RT-PCR:TFDP-3 (HCA661), 35 circulations, 94 ℃ 30 seconds; 60 ℃ 30 seconds; 72 ℃ 90 seconds, 72 ℃ were extended 10 minutes; TFDP-1,35 circulations, 94 ℃ 30 seconds; 55 ℃ 30 seconds; 72 ℃ 90 seconds, 72 ℃ were extended 10 minutes; TFDP-2,35 circulations, 94 ℃ 30 seconds; 55 ℃ 30 seconds; 72 ℃ 60 seconds, 72 ℃ were extended 10 minutes; G3PDH, 25 circulations, 94 ℃ 15 seconds; 64 ℃ 20 seconds; 72 ℃ 20 seconds, 72 ℃ were extended 10 minutes.TFDP-3 (HCA661), TFDP-1, TFDP-2 and G3PDH primer sequence and PCR product sheet segment length are as follows: TFDP-3, forward, 5 '-TAC ACT CGG CCT GGA AGA ATT G-3 '; Reverse, 5 '-TCT TCC TCC TCGACT GCTG-3 ', size, 1,244 base pairs (bp); TFDP-1, forward, 5 '-ATG GCA AAA GAT GCC GGTCTA ATT G-3 '; Reverse 5 '-TCG TCC TCG TCA TTC TCG TTG-3 ', size, 1,229bp; TFDP-2, forward, 5 '-CCC TGC ACC AGC AAT GGT TAC TCA G-3; Reverse, 5 '-ACTGCT GGA CTG GTGACT GTT TGG G-3 ', size, 997bp; G3PDH, forward, 5 '-ACC ACAGTC CAT GCC ATC AC-3, reverse, 5 '-TCC ACC ACC CTG TTG CTG TA-3 ', size, 452bp.

We analyze at the tumor cell line especially expression level in HCC clone each member TFDP-1, TFDP-2 and the TFDP-3 (HCA661) of DP family by RT-PCR.Use G3PDH as confidential reference items, the quality of assessment mRNA.We have detected the expression of TFDP-3 (HCA661) in liver cancer tissue, in this experiment, it is right that we have designed Auele Specific Primer as implied above, TFDP-3 (HCA661) increases respectively, the fragment of TFDP-1 and TFDP-2 gene detects DP each member of family at the tumor cell line especially expression and distribution in HCC clone.TFDP-1 and TFDP-2 mRNA are expressed in the cell of all cells system, and being expressed in of TFDP-3 (HCA661) mRNA has very big difference in the different clone.In SMMC-7721, express higher relatively level, HepG2, then expression level is lower among HLE and the Jurkat, and all the other clones do not detect the expression (as shown in Figure 5) of TFDP-3 (HCA661) mRNA, and is consistent with the expression and distribution of TFDP-3 (HCA661) in liver cancer tissue.Different with the wide expression of TFDP-1 and TFDP-2, it is restricted that the expression of TFDP-3 (HCA661) has tangible histocyte.Therefore, TFDP-3 (HCA661) has the express spectra that is different from other member of DP family.

Embodiment 5.TFDP-3 (HCA661) albumen and E2F albumen are in external interaction

For the gst fusion protein that produces E2F-1～-6 is beneficial to protein purification and follow-up GSTPull-down analysis, E2F is gene constructed to go into pGEX-4T2, Transformed E scherichia coli (JM109) obtains the GST-E2F fusion rotein a large amount of with purifying (seeing Fig. 6 A) by traditional method then.TFDP-3 (HCA661) albumen is by the TNT of Promega ^Quick Coupled Transcription/Translation system test kit in 50 μ l reaction systems in radioactivity ³⁵The S-methionine existence is external down transcribes and translates and obtain.In the external association reaction, GST that the purifying of sufficient quantity is crossed or GST-E2F fusion rotein react with Triptide-gelose microballon earlier, add then and contain 50mM Tris (pH8.0), 150mM NaCl, 10mg/ml lysozyme, 0.5mM phenylmethylsulfonyl fluoride (PMSF), 50mg/ml leupeptin, 50mg/ml protease inhibitor is in TFDP-3 (HCA661) lysis buffer of 50mg/ml aprotinin and 50mM dithiothreitol.After 2.5 hours, centrifugal and collecting precipitation thing with lysis buffer washing four times, is removed unconjugated albumen in 4 ℃ of effects.Add the SDS sample-loading buffer, electrophoresis, dried glue on 12.5% polyacrylamide gel, radioautograph.The TFDP-3 of in-vitro transcription and translation (HCA661) albumen is as positive reference, and nonfused GST albumen is as negative reference.The result shows, TFDP-3 (HCA661) can with E2F-1～E2F-6 interact (seeing Fig. 6 B, lanes3 to 8).Interaction between TFDP-3 (HCA661) and the E2F albumen is high efficiency and is specific, because TFDP-3 (HCA661) and negative the interaction with reference to nothing between the GST albumen (are not seen Fig. 6 B, lane2).This external binding data supports that TFDP-3 (HCA661) is a newcomer of DP family.

Embodiment 6.TFDP-3 (HCA661) albumen and the interaction in vivo of E2F albumen

Human cervical carcinoma cell be the HeLa cell in 37 ℃, cultivate in the cell culture incubator of 5%CO2, used substratum is the DMEM substratum that contains 100units/ml penicillin, 100 μ g/ml streptomycin and 10% newborn calf serum.Before the transfection, the trysinization of HeLa cell, with the unparalleled anti-fresh DMEM substratum that contains 10% newborn calf serum with cell dilution in 6 orifice plates, be cultured to cell degree of converging and reach the required 90-95% of transfection.During transfection, the HA-tagged E2F plasmid DNA of 1.4 μ g and 1.4 μ gFLAG-tagged DP plasmid DNA, or the empty carrier DNA of 1.4 μ g, the DNA total amount adds to 2.8 μ g.LipofectAMINE 2000reagent (Invitrogen) is used for DNA is brought in the cell.Behind HeLa cell transfecting 24-36 hour, prepare non-degenerating cell lysate according to a conventional method.These proteic expression levels are monitored the expression of E2F albumen (as scheming among Fig. 7) or TFDP-3 (HCA661) albumen (as Fig. 7 figure below) by the immunoblotting assay cell lysate of anti-HA rabbit polyclonal antibody or anti-FLAG M2 mouse monoclonal antibody.In order to carry out the co-immunoprecipitation analysis, the HeLa cell of transfection is earlier with 1 * PBS washing secondary, add an amount of 20mM Tris (pH7.5) that contains again, 150mM NaCl, 1%Triton X-100,1mM EDTA, 5 μ g/ml aprotinin, 5 μ g/ml leupeptin, the lysis buffer lysing cell of and 2mM PMSF prepares non-degenerating cell lysate.By the proteic concentration of Bradford analyzing total.During co-immunoprecipitation, cell lysate acts on 2 hours at least in the cell lysis buffer solution of 4 ℃ of protein A-agarose that contain anti-FLAG antibody of 2 μ g/ml and 25 μ l.Precipitation after centrifugal is removed unconjugated albumen with the cell lysis buffer solution washing that contains 500mM NaCl three times.Washing centrifuged deposit thing adds sample-loading buffer, electrophoresis, commentaries on classics film on 12.5% polyacrylamide gel.Nitrocellulose membrane after electricity changes is earlier with the TBS sealing damping fluid sealing that contains 5% skim-milk 1 hour, then with the confining liquid incubation that contains the anti-HA antibody of 1 μ g/ml 1 hour, and at last with two anti-reactions of the anti-rabbit igg of HRP link coupled, the ECL colour developing.E2F albumen in all transfections detect in the co-immunoprecipitation thing of E2F DNA, but in a transfection empty carrier DNA or TFDP-3 (HCA661) dna immunization coprecipitate, do not detect.These data show that TFDP-3 (HCA661) can contain member's interaction of different dimerization structural domain and form heterodimer efficiently with in the E2F family, supports that further TFDP-3 (HCA661) is the newcomer's of DP family supposition.

Embodiment 7.TFDP-3 (HCA661) analyzes (EMSA, electrophoretic mobility shift assay) with the DNA binding ability of the heterodimer that the E2F protein-interacting forms.

Human cervical carcinoma cell be the HeLa cell in 37 ℃, cultivate in the cell culture incubator of 5%CO2, used substratum is the DMEM substratum that contains 100units/ml penicillin, 100 μ g/ml streptomycin and 10% newborn calf serum.Before the transfection, the trysinization of HeLa cell, with the unparalleled anti-fresh DMEM substratum that contains 10% newborn calf serum with cell dilution in 6 orifice plates, be cultured to cell degree of converging and reach the required 90-95% of transfection.During transfection, the E2F DNA of 1.4 μ g and 1.4 μ gDP DNA, or the empty carrier DNA of 1.4 μ g, the DNA total amount adds to 2.8 μ g.

IpofectAMINE 2000 reagent (Invitrogen) are used for DNA is brought in the cell.Behind HeLa cell transfecting 24-36 hour, prepare non-denaturing cell proteins extract according to a conventional method, the HeLa cell protein extract of equivalent adds and contains 10mM N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid (HEPES; PH7.9), 375mM KCl, 12.5mM MgCl2,0.5mM EDTA, 5mM DTT, 15%Ficoll, 10mg/ml BSA in the EMSA damping fluid of and 1mg/ml polydI/dC, adds the radioactivity of 0.1ng simultaneously ³²The oligonucleotide probe (ATTTAAGTTTCGCGCCCTTTCCAA) that P is isotope-labeled to contain specificity wild-type E2F binding site was room temperature incubation 10～15 minutes.Reaction mixture injects 5% non-sex change polyacrylamide gel then 4 ℃ of constant voltage 180V electrophoresis 2.25 hours, and dried glue was at-70 ℃ of radioautographic analysis 4-16 hours.In order to detect the specificity of protein binding wild-type dna probe, add 20 times of unlabelled double chain oligonucleotide probes that contain the E2F mutational site to the wild-type probe amount in the reaction solution as competition molecule (ATTTAAGTTTCGatCCCTTTCTCAA).

As shown in Figure 8, because the existence of endogenous E2F basal level is arranged, when single E2F or DP transfection, their DNA binding ability still is in basal level (strip-like developing pipe 2,3,6,9 and 1 relatively), shows that independent transfection E2F or DP can not improve its dna binding activity.When TFDP-1 and E2F cotransfection, the E2F/TFDP-1 heterodimer is compared significantly with independent transfection E2F in conjunction with the ability of DNA and has been strengthened (strip-like developing pipe 5 and 3,8 and 6,11 is compared with 9).On the contrary, when TFDP-3 (HCA661) and E2F corotation, the DNA binding ability of E2F/TFDP-3 (HCA661) heterodimer is not significantly improved (strip-like developing pipe 4 and 3,7 and 6,10 is compared with 9).This shows, may exist the still unidentified motif that destroys the DNA bonding force in TFDP-3 (HCA661) albumen, thereby causes the forfeiture of E2F/TFDP-3 heterodimer in conjunction with E2F DNA recognition site.

The Subcellular Localization of embodiment 8.TFDP-3 (HCA661)

The green monkey kidney cell that SV40 transforms be the COS-7 cell in 37 ℃, cultivate in the cell culture incubator of 5%CO2, used substratum is the DMEM substratum that contains 100units/ml penicillin, 100 μ g/ml streptomycin and 10% newborn calf serum.Before the transfection, the trysinization of COS-7 cell, with the unparalleled anti-fresh DMEM substratum that contains 10% newborn calf serum with cell dilution to 8 * 10 ⁴Cells/ml, each adds 1ml in 24 orifice plates, is cultured to cell degree of converging and reaches the required 90-95% of transfection.Separately during transfection, the empty carrier DNA of the EGFP-tagged E2F plasmid DNA of 280ng or 280ng FLAG-tagged DP plasmid DNA and 280ng, the DNA total amount adds to 560ng, and simultaneously, pEGFP-N1 is as negative reference.During common transfection, the EGFP-tagged E2F plasmid DNA of 280ng and 280ng FLAG-tagged DP plasmid DNA, the DNA total amount adds to 560ng.Lipofect AMINE 2000reagent (Invitrogen) is used for DNA is brought in the COS-7 cell.Washed a little with 1 * PBS in 24 hours after the transfection, the methyl alcohol that adds-70 ℃ of precoolings is then fixed 20 minutes at-20 ℃, adds the penetrating cytolemma of 1 * PBS 4 minutes that contains 0.2%Triton X-100.Behind the 1 * PBS confining liquid sealing non-specific antibody binding site that contains 1%BSA, fixed COS-7 cell is at room temperature incubation 1 hour in the 1 * PBS that contains 1 μ g/ml anti-FLAG M2 monoclonal antibody and 1%BSA, after 1 * PBS washing three times, the 1 * PBS of two anti-and 1%BSA that adds tetramethylrhodamineisothiocyanate (TRITC) the link coupled goat anti-mouse IgG that contains dilution in 1: 100 was in room temperature reaction 1 hour.The COS-7 nucleus dyes with 10 μ g/ml Hoechst33342 are contrary, and E2F adopts EGFP mark and detection.Gather the immunofluorescence image by the fluorescent microscope that has been equipped with CCD, all images is handled by Adobe Photoshop7.0.1 version.

Experimental result shows that when independent transfection, TFDP-3 (HCA661) and TFDP-1 all are positioned in the cytoplasm of COS-7 cell, and E2F-1 is positioned in the nucleus, and E2F-4 then is positioned in the cytoplasm (seeing Fig. 9 A).E2F-2, E2F-3 also are positioned in the nucleus, and E2F-5 is positioned (not shown) in the cytoplasm.

Bring into play function, the DP family member must form heterodimer with the E2F family member.Therefore, we have analyzed the influence of the Subcellular Localization of the interaction partners TFDP-3 (HCA661) between albumen and the albumen.As being inferred, similar during the common transfection of TFDP-3 (HCA661) and E2F-1 (seeing Fig. 9 B), E2F-2 or E2F-3 (not shown) with TFDP-1, all be positioned in the nucleus.Opposite with the location of first subfamily of E2F, when with E2F-4 (seeing Fig. 9 B) or the common transfection of E2F-5 (not shown), TFDP-3 (HCA661) and TFDP-1 still are positioned in the cytoplasm.These data show that the Subcellular Localization of TFDP-3 (HCA661) is consistent with TFDP-1.

The reporter gene analysis of embodiment 9.TFDP-3 (HCA661)

People's normal liver cell be L02 in 37 ℃, cultivate in the cell culture incubator of 5%CO2, used substratum is the DMEM substratum that contains 100units/ml penicillin, 100 μ g/ml streptomycin and 10% newborn calf serum.Before the transfection, the trysinization of L02 cell, with the unparalleled anti-fresh DMEM substratum that contains 10% newborn calf serum with cell dilution to 1 * 10 ⁵Cells/ml respectively adds 1ml in 24 orifice plates, and culturing cell makes it degree of converging and reaches the required 90-95% of transfection.During reporter gene was analyzed, the DNA total amount added to 560ng, wherein contains reporter gene 6 * E2F-firefly luciferase of 140ng.Carry out in the retarding effect experiment of TFDP-3 (HCA661) to the transcriptional activation of E2F/TFDP-1 mediation, the DNA total amount adds to 1,120ng.When execution TFDP-3 (HCA661) or TFDP-1 influenced endogenous E2F is active, the DNA total amount added to 840ng.LipofectAMINE 2000 reagent (Invitrogen) are used for DNA is brought in the cell.The L02 cell transfecting washs with 1 * PBS earlier after 24 hours a little, residual substratum is removed, add passive lysis buffer (PLB) then, after the room temperature cracking 15 minutes, the results lysate is also centrifugal, remove cell debris, the results supernatant, the operational manual that provides according to manufacturer uses dual specific luciferase analytical system assay products.For the influence to reporter gene 6 * E2F-firefly luciferase value of the difference of eliminating transfection efficiency, each transfection all adds the pRL-SV40 Renilla luciferase plasmid of 140ng as confidential reference items.

The reporter gene analytical results is shown in Figure 10 A, when showing the independent transfection of E2F gene and DP gene, the TFDP-1 of 140ng has produced about 2 times transcriptional activation to reporter gene 6 * E2F-firefly luciferase, equally, the E2F-1 of 20ng or E2F-3 have also produced the transcriptional activation of 10 times or 30 times respectively, and the transfection that the E2F-4 of 70ng or 140ngE2F-5 have also produced 17 times or 11 times activates.When E2F and TFDP-1 cotransfection, allosteric activation reporter gene 6 * E2F-fireflyluciferase, reached 30 to 70 times.Yet, be when E2F and TFDP-3 (HCA661) cotransfection, to have observed TFDP-3 (HCA661) transcriptional activation that E2F mediates has been produced significant retarding effect with TFDP-1 forms sharp contrast.Same experiment is triplicate (P＜0.01) at least.

TFDP-3 (HCA661) has the transcriptional activation ability that suppresses the E2F mediation, and therefore, we think whether the interaction of further analyzing TFDP-3 (HCA661) and E2F can suppress the transcriptional activation that the E2F/TFDP-1 heterodimer mediates effectively.In the experiment, the various combinations and 0,140 of E2F/TFDP-1,280 or TFDP-3 (HCA661) the cotransfection L02 cell of 560ng adopt 6 * E2F-firefly luciferase reporter gene analytical system to analyze the effect of TFDP-3 (HCA661) to the E2F/TFDP-1 heterodimer.

As mentioned above, the combination of all E2F/TFDP-1 has improved the transcriptional activity of 6 * E2F-firefly luciferase reporter gene significantly.When E2F/TFDP-1 is combined in TFDP-3 (HCA661) when existing, the transcriptional activity of E2F/TFDP-1 has been suppressed significantly, and is dose-dependently.Even can block the allosteric activation (as Figure 10 B shown in) of TFDP-1 fully to E2F.This discovery shows, although TFDP-3 (HCA661) is a newcomer of DP family, it suppresses the transcriptional regulatory of E2F dependency target gene as a supressor performance function.

At last, we have also assessed TFDP-3 (HCA661) to the active influence of endogenous E2F.Be willing to as scheduled, FLAG-tagged TFDP-1 has strengthened the transcriptional activity of 6 * E2F-firefly luciferase reporter gene, FLAG-tagged TFDP-3 (HCA661) has then suppressed the transcriptional activity of 6 * E2F-firefly luciferase reporter gene, and be (the seeing Figure 10 C) of dose-dependently, this shows that TFDP-3 (HCA661) has the ability to block the transcriptional activation of endogenous E2F mixture to 6 * E2F-firefly luciferase reporter gene.And we have also obtained similar (not shown) as a result from the Cyclin A-firefly luciferase reporter gene that makes up with the natural regulating and controlling sequence of the target gene Cyclin A of E2F.

TFDP-3 (HCA661) is consistent with the ability of TFDP-3 (HCA661)/E2F heterodimer forfeiture binding specificity E2F dna binding sequence row to the inhibition ability of transcribing of E2F mediation.And the retarding effect of TFDP-3 (HCA661) shows that also TFDP-3 (HCA661) may play the part of the role of antagonism TFDP-1 when TFDP-3 (HCA661) and TFDP-1 coexistence.

In a word, all The above results show, function difference between TFDP-3 (HCA661) and the TFDP-1 may come from the forfeiture of the DNA bonding force of E2F/TFDP-3 (HCA661) heterodimer, inhibiting molecular basis should be present in TFDP-3 (HCA661) molecule, because for some specific E2F albumen, the height of its transcriptional activity depends on the power of the DNA binding ability of the complete DNA binding domains of being made up of jointly E2F albumen and DP albumen.

The exploration of the molecular basis that embodiment 10.TFDP-3 (HCA661) unique function produces

For the forfeiture of the DNA binding ability of exploring the heterodimer that TFDP-3 (HCA661) and E2F albumen forms and TFDP-3 (HCA661) molecular basis to the inhibition of E2F transcriptional activity, we have made up the replacement mutant of TFDP-3 (HCA661) and TFDP-1, adopt 6 * E2F-luciferase reporter gene analytical system to analyze the character of transcribing of its mutant, assess the DNA binding ability of the heterodimer of TFDP-3 (HCA661) and E2F albumen formation indirectly.The replacement mutant of TFDP-3 (HCA661) and TFDP-1 is a template with corresponding plasmid, makes up by PCR method to form.Design forward mutation primer and inverse transition primer respectively in the replacement site earlier, primer contains the corresponding base that needs replacement, reverse primer (DP3CF-OA or DP1CF-OA) and forward primer (DP3CF-OS or DP1 CF-OS) with TFDP-3 (HCA661) or TFDP-1 ORF matches respectively, synthesize two small segments by PCR, and then two small segments are connected into a complete gene by connecting PCR.

Following mutant is a template with pCDNA3-TFDP-3-FLAG and pCDNA3-TFDP-1-FLAG, and used mutant primer is as follows: TFDP-3M, forward, 5 '-CGG CGC gtc TAC GAT GCC TTA AACGTG-3 '; Reverse, 5 '-ATC GTA gac GCG CCG TTT TAT GTT TTT C-3 '; TFDP-1M, forward, 5 '-CGG CGC acc TAC GAT GCC TTA AAC GTG-3 '; Reverse, 5 '-ATC GTA ggtGCG CCG TCT TAT GTT TTT C-3 '; TFDP-3MR, forward, 5 '-aag ggc cta cgg cat ttc tccatg aag gtc tgc gag aag GTG CAG AGG-3 '; Reverse, 5 '-ctt ctc gca gac ctt cat gga gaa atgccg tag gcc ctt GCC ATT CTTC-3 '; TFDP-1MR, forward, 5 '-atg ggc ctg tgc cgt ctt tcc atgaag gtc tgg gag acg GTG CAG AGG-3 '; Reverse, 5 '-cgt ctc cca gac ctt cat gga aag acg gcacag gcc cat GCC ATT CTT C-3 ' .3D-1H, forward, 5 '-ggt ctg acc acc AAC TCG GCTCAG-3 '; Reverse, 5 '-ctg agc cga gtt GGT GGT CAG ACC-3 '; 1D-3H, forward, 5 '-ggt ctgccc acc AAC TCG GCT CAG-3 '; Reverse, 5 '-ctg agc cga gtt GGT GGG CAG ACC-3 '.

It is template with 3D-1H and 1D-3H that the structural domain of TFDP-3 Δ H and TFDP-3 Δ D, TFDP-1 Δ H and TFDP-1 Δ D is replaced mutant, the used mutant primer of PCR is as follows: TFDP-3 Δ H, forward, 5 '-acc agcaag aag ACC GTC ATC AAC-3 '; Reverse, 5 '-gtt gat gac ggt CTT CTT GCT GGT G-3 '; TFDP-1 Δ H, forward, 5 '-agt agc aag aag ACG GTC ATC GAC-3 '; Reverse, 5 '-gtc gat gaccgt CTT CTT GCT AC-3 '; TFDP-3 Δ D, forward, 5 '-gga gag aag aat GGC AAG GGC CTAC-3 '; Reverse, 5 '-tag gcc ctt gcc ATT CTT CTC TCC-3 '; TFDP-1 Δ D, forward, 5 '-gga gagaag aat GGC ATG GGC CTG-3 '; Reverse, 5 '-cag gcc cat gcc ATT CTT CTC TCC-3 '.

TFDP-3 Δ DH is a template with TFDP-3 Δ D and TFDP-3 Δ H, and mutant primer is as follows: forward, 5 '-ggtctg ccc acc AAC TCG GCT CAG-3 '; Reverse, 5 '-ctg agc cga gtt GGT GGG CAG ACC-3 ' .TFDP-1 Δ DH is a template with TFDP-1 Δ D and TFDP-1 Δ H, mutant primer is as follows: forward, 5-ggt ctg accacc AAC TCG GCT CAG-3; Reverse, 5-ctg agc cga gtt GGT GGT CAG ACC-3 '.

It is template with the pCDNA3-TFDP-3-FLAG plasmid that TFDP-3MR2～TFDP-3MR5 replaces mutant, mutant primer is as follows: TFDP-3MR2, forward, 5 '-tac aac gaa gtg gca gac gag ctg gtt gcg gag ttc agtgct gcc gac AACC-3 '; Reverse, 5 '-gtc ggc agc act gaa ctc cgc aac cag ctc gtc tgc cac ttc gttgta GGAAG-3 '; TFDP-3MR3, forward, 5 '-atc tta cca aac gag tca gct tat gac cag aaa aac ataaga CGGCGC-3 '; Reverse, 5 '-tct tat gtt ttt ctg gtc ata agc tga ctc gtt tgg taa gatGTGGTTG-3 '; TFDP-3MR4 ', forward, 5 '-aag gag aag aag gag atc aag tgg att ggt ctg cccACCAACTCG-3 '; Reverse, 5 '-ggg cag acc aat cca ctt gat ctc ctt ctt ctc ctt GGAGATG-3 '; TFDP-3MR5, forward, 5 '-gca gac gag ctg gtt gcg gag ttc agt GCT GCC AGC-3 '; Reverse, 5 '-act gaa ctc cgc aac cag ctc gtc tgc CAC TTC CTG-3 '.

Following mutant is a template with the pCDNA3-TFDP-3-FLAG plasmid, and mutant primer is as follows: TFDP-3CY, forward, 5 '-G ACC ACT TCC tac CAG GAA GTG-3 '; Reverse, 5 '-CAC TTC CTG gtaGGAAGT GGT C-3 '; TFDP-3QN, forward, 5 '-C ACT TCC TGC aac GAA GTG GTG-3 '; Reverse, 5 '-CAC CAC TTC gtt GCA GGA AGT G-3 '; TFDP-3VA, forward, 5 '-CAG GAAGTG gca GGC GAG CTG-3 '; Reverse, 5 '-CAG CTC GCC tgc CAC TTC CTG-3 '; TFDP-3GD, forward, 5 '-GAA GTG GTG gac GAG CTG GTC-3 '; Reverse, 5 '-GAC CAGCTC gtc CAC CAC TTC-3 '; THDP-3KE, forward 5 '-CTG GTC GCC gag TTC AGAGC-3 '; Reverse, 5 '-GC TCT GAA ctc GGC GAC CAG-3 '; TFDP-3RS, forward, 5 '-C AAGTTC agt GCT GCC AGC AAC-3 '; Reverse, 5 '-GTT GCT GGC AGC act GAA CTT G-3 '; TFDP-3SD, forward, 5 '-C AGA GCT GCC gac AAC CAC GC-3 '; Reverse, 5 '-GC GTGGTT gtc GGC AGC TCT G-3 '.

After all mutant order-checkings were proofreaied and correct, behind Hind III-BamHI digestion with restriction enzyme, the clone advanced in the pCDNA3-FLAG expression vector, and Figure 11 has shown the aminoacid sequence of mutant.Figure 12 A has shown the protein expression of each mutant.Analyze of the influence of its mutant by 6 * E2F-luciferase reporter gene analytical system, assess the DNA binding ability of the heterodimer of TFDP-3 (HCA661) and E2F albumen formation indirectly the E2F-4 transcriptional activity.Concrete experimental arrangement is identical with method described in the embodiment 9.

At first, we had analyzed the difference of TFDP-3 (HCA661) with the amino acid composition of the DNA binding domains of TFDP-1 according to former result of study, in RRXYD DNA identification block, the 3rd amino acid has been carried out the single amino acids replacement, produced two and replace mutant (TFDP-3M:aa164T → V; TFDP-1M:aa169V → T), whether the non-conservation replacement of analyzing this amino acids is the reason that causes the unique function of TFDP-3 (HCA661).6 * E2F-firefly luciferase reporter gene analysis revealed, the function of the replacement of this amino acids and TFDP-3 (HCA661) is irrelevant.In addition, according to crystalline structure, in the DNA binding domains, other has 13 amino acid to participate in interaction with proteic different dimerization and DNA phosphoric acid skeleton.Wherein, in TFDP-3 (HCA661), have three amino acid different with TFDP-1 and TFDP-2, for this reason, we have designed 13 amino acid whose replacement (TFDP-3MR:aa109-121 MGL CRLSMKV WE T→ KGL RHFSMKV CE KTFDP-1MR:aa114-126 KGL RHFSMKV CE K→ MGL CRLSMKV WE T).Yet these replace the function that still can not reverse TFDP-3 (HCA661).

Use similar sequence replacement analysis, we have analyzed the full length amino acid sequence of TFDP-3 (HCA661) and TFDP-1, composition according to structural domain, be divided into 4 integral parts, it is aminoterminal unknown function district (UNT), DNA binding domains (DBD), different dimerization structural domain (HD) and carboxyl terminal unknown function district (UCT), and 3D-1H (containing the UNT of TFDP-3 (HCA661) and HD and the UCT of DBD and TFDP-1) and 1D-3H (containing the UNT of TFDP-1 and HD and the UCT of DBD and TFDP-3 (HCA661)) have been made up, the reporter gene analysis revealed, the level that the transcriptional activation of the E2F mediation that 1D-3H produces is on close level and is produced in wild-type TFDP-1 is not subjected to the influence of the AA192-405 of TFDP-3 (HCA661).On the contrary, 3D-1H shows the transcriptional activation obvious suppression effect to the E2F mediation.This shows that the molecular basis that TFDP-3 (HCA661) unique function produces should be positioned at the first half (AA1-191) of TFDP-3 (HCA661).

For the molecular basis that further analytic function produces, we have derived following replacement mutant by PCR method from 3D-1H and 1D-3H: TFDP-3 Δ D (DBD that contains TFDP-1), TFDP-1 Δ D (DBD that contains TFDP-3 (HCA661)), TFDP-3 Δ H (HD that contains TFDP-1), TFDP-1 Δ H (HD that contains TFDP-3 (HCA661)), TFDP-3 Δ DH (DBD and the HD that contain TFDP-1), TFDP-1 Δ DH (DBD and the HD that contain TFDP-3 (HCA661)).6 * E2F-firefly luciferase reporter gene is the result show, TFDP-3 Δ H and TFDP-1 Δ H produce transcribe suppress or activation level respectively with they corresponding wild type protein similars, on the contrary, replacement along with the DNA binding domains, TFDP-3 Δ DH and TFDP-3 Δ D have produced the allosteric activation that E2F is transcribed of similar TFDP-1, and TFDP-1 Δ DH and TFDP-1 Δ D have then produced the retarding effect that E2F is transcribed of similar TFDP-3 (HCA661).These data show, the functional zone that TFDP-3 (HCA661) suppresses the transcriptional activation of E2F mediation expeditiously are positioned at the DNA binding domains (seeing Figure 12 B) of TFDP-3 (HCA661).

Next, the strategy that our employing is identical with the TFDP-3M construction process with TFDP-3MR, to be divided into three parts with discrepant all the other 13 amino acid of TFDP-1 in the DNA binding domains of TFDP-3 (HCA661), replace TFDP-3 (HCA661) sequence with the corresponding aminoacid sequence of TFDP-1 respectively, produce TFDP-3MR2 (AA130-145 COEV VGELVA KFRAA S→ YNEV ADELVA EF SAA D), TFDP-3MR3 (AA148-161 ASPNESAYD VKNI K→ ILPNESAYD QKNI R) and TFDP-3MR4 (AA179-190 REKK KKIKWIGL T→ KEKK EIKWIGL P).The reporter gene analysis shows that TFDP-3MR3 and TFDP-3MR4 replace the transcriptional activity that mutant does not improve E2F, have produced the similar retarding effect to wild-type TFDP-3 (HCA661).The transfection activation that is produced when on the contrary, the transcriptional activation level that produces of TFDP-3MR2 is with E2F transfection separately is similar.This mutant also has the E2F/TFDP-1 of inhibition to be changeed when record activated ability (not shown).The replacement mutant TFDP-3MR5 (AA134-142 that another derives in TFDP-3MR2 inside VGELVA KF R→ ADELVA EF S) transcriptional activation of E2F mediation is had more weak restraining effect.Therefore, the AA130-145 sequence of TFDP-3 (HCA661) DNA binding domains is to produce retarding effect necessary (seeing Figure 12 C).In addition, in order to seek key amino acid, we have produced another group single amino acids and have replaced mutant TFDP-3CY (AA130C → Y) in this zone, TFDP-3QN (AA131Q → N), TFDP-3VA (AA134V → A), TFDP-3GD (AA135G → D), TFDP-3KE (AA140K → E), TFDP-3RS (AA142R → S) and TFDP-3SD (AA145S → D), but the result shows that the generation of TFDP-3 (HCA661) function may be the coefficient results of a plurality of amino acid.

In a word, the reporter gene analytical results shows, the DNA binding domains (AA108-AA191) of TFDP-3 (HCA661) is to cause the TFDP-3 (HCA661) and the heterodimer of E2F albumen formation to lose the basic reason of DNA binding ability, thereby the transcriptional activity that suppresses E2F, irrelevant with the space structure of proteic rest part, comprise the different dimerization structural domain.To the further analysis revealed of DNA binding domains, AA130-AA145 (CQEVVGELVAKFRAAS) is TFDP-3 (HCA661) to the DNA of E2F in conjunction with producing inhibiting prerequisite.

sequence-listing.txt

SEQUENCE LISTING

＜110〉Peking University

＜120〉a kind of liver cancer orchis pellet specific antigen protein matter and antigen peptide

<130>FPI05135

<160>2

<170>PatentIn version3.1

<210>1

<211>405

<212>PRT

<213>Homo sapiens

<400>1

Met Ala Lys Tyr Val Ser Leu Thr Glu Ala Asn Glu Glu Leu Lys Val

1 5 10 15

Leu Met Asp Glu Asn Gln Thr Ser Arg Pro Val Ala Val His Thr Ser

20 25 30

Thr Val Asn Pro Leu Gly Lys Gln Leu Leu Pro Lys Thr Phe Gly Gln

35 40 45

Ser Ser Val Asn Ile Asp Gln Gln Val Val Ile Gly Met Pro Gln Arg

50 55 60

Pro Ala Ala Ser Asn Ile Pro Val Val Gly Ser Pro Asn Pro Pro Ser

65 70 75 80

Thr His Phe Ala Ser Gln Asn Gln His Ser Tyr Ser Ser Pro Pro Trp

85 90 95

Ala Gly Gln His Asn Arg Lys Gly Glu Lys Asn Gly Met Gly Leu Cys

100 105 110

Arg Leu Ser Met Lys Val Trp Glu Thr Val Gln Arg Lys Gly Thr Thr

115 120 125

Ser Cys Gln Glu Val Val Gly Glu Leu Val Ala Lys Phe Arg Ala Ala

130 135 140

Ser Asn His Ala Ser Pro Asn Glu Ser Ala Tyr Asp Val Lys Asn Ile

145 150 155 160

Lys Arg Arg Thr Tyr Asp Ala Leu Asn Val Leu Met Ala Met Asn Ile

165 170 175

Ile Ser Arg Glu Lys Lys Lys Ile Lys Trp Ile Gly Leu Thr Thr Asn

180 185 190

Ser Ala Gln Asn Cys Gln Asn Leu Arg Val Glu Arg Gln Lys Arg Leu

sequence-listing.txt

195 200 205

Glu Arg Ile Lys Gln Lys Gln Ser Glu Leu Gln Gln Leu Ile Leu Gln

210 215 220

Gln Ile Ala Phe Lys Asn Leu Val Leu Arg Asn Gln Tyr Val Glu Glu

225 230 235 240

Gln Val Ser Gln Arg Pro Leu Pro Asn Ser Val Ile His Val Pro Phe

245 250 255

Ile Ile Ile Ser Ser Ser Lys Lys Thr Val Ile Asn Cys Ser Ile Ser

260 265 270

Asp Asp Lys Ser Glu Tyr Leu Phe Lys Phe Asn Ser Ser Phe Glu Ile

275 280 285

His Asp Asp Thr Glu Val Leu Met Trp Met Gly Met Thr Phe Gly Leu

290 295 300

Glu Ser Gly Ser Cys Ser Ala Glu Asp Leu Lys Met Ala Arg Asn Leu

305 310 315 320

Val Pro Lys Ala Leu Glu Pro Tyr Val Thr Glu Met Ala Gln Gly Thr

325 330 335

Phe Gly Gly Val Phe Thr Thr Ala Gly Ser Arg Ser Ash Gly Thr Trp

340 345 350

Leu Ser Ala Ser Asp Leu Thr Asn Ile Ala Ile Gly Met Leu Ala Thr

355 360 365

Ser Ser Gly Gly Ser Gln Tyr Ser Gly Ser Arg Val Glu Thr Pro Ala

370 375 380

Val Glu Glu Glu Glu Glu Glu Asp Asn Asn Asp Asp Asp Leu Ser Glu

385 390 395 400

Asn Asp Glu Asp Asp

405

<2l0>2

<2ll>1218

<212>DNA

<213>Homo sapiens

<400>2

atggcaaaat atgtcagtct cactgaagct aacgaagaac tcaaggtctt aatggacgag 60

aaccagacca gccgccccgt ggccgttcac acctccaccg tgaacccgct cgggaagcag 120

ctcttgccga aaacctttgg acagtccagt gtcaacattg accagcaagt ggtaattggt 180

atgcctcaga gaccagcagc atcaaacatc cctgtggtag gaagccc8aa cccacccagc 240

sequence-listing.txt

actcactttg cctctcagaa ccagcattcc tactcctcac ctccttgggc cgggcagcac 300

aacaggaaag gagagaagaa tggcatgggc ctgtgccgtc tttccatgaa ggtctgggag 360

acggtgcaga ggaaagggac cacttcctgc caggaagtgg tgggcgagct ggtcgccaag 420

ttcagagctg ccagcaacca cgcctcacca aacgagtcag cttatgacgt gaaaaacata 480

aaacggcgca cctacgatgc cttaaacgtg ctgatggcca tgaatatcat ctccagggag 540

aaaaagaaga tcaagtggat tggtctgacc accaactcgg ctcagaactg tcagaactta 600

cgggtggaaa gacagaagag acttgaaaga ataaagcaga aacagtctga acttcaacaa 660

cttattctac agcaaattgc tttcaagaac ctggtgctga gaaaccagta tgtggaggag 720

caggtcagcc agcggccgct gcccaactca gtcatccacg tgcccttcat catcatcagc 780

agtagcaaga agaccgtcat caactgcagc atctccgacg acaaatcaga atatctgttt 840

aagtttaaca gctcctttga aatccacgat gacacagaag tgctgatgtg gatgggcatg 900

acttttgggc tagagtccgg gagctgctct gccgaagacc ttaaaatggc cagaaatttg 960

gtcccaaagg ctctggagcc gtacgtgaca gaaatggctc agggaacttt tggaggtgtg 1020

ttcacgacgg caggttccag gtctaatggc acgtggcttt ctgccagtga cctgaccaac 1080

attgcgattg ggatgctggc cacaagctcc ggtggatctc agtacagtgg ctccagggtg 1140

gagaccccag cagtcgagga ggaagaggag gaggacaaca acgatgacga cctcagtgag 1200

aatgacgagg atgactga 1218

Claims (6)

1, a kind of liver cancer orchis pellet specific antigen protein matter has following aminoacid sequence (a) or derived protein (b):

Described aminoacid sequence (a) has aminoacid sequence shown in Figure 1;

Described derived protein (b) is with the replacement of the aminoacid sequence in the described aminoacid sequence (a) through one or several amino-acid residue, the derived protein that disappearance or interpolation sudden change are produced, this derived protein and described aminoacid sequence (a) have same or analogous function.

2, the application of the described antigen protein of a kind of claim 1 in preparation treatment liver-cancer medicine.

3, the gene of the described liver cancer orchis pellet specific antigen protein of a kind of claim 1 of encoding matter.

4, a kind of liver cancer orchis pellet specific antigens peptide, it has following peptide section (c) or derived peptide (d):

Described peptide section (c) is 84 the amino acid whose peptide sections that contain of the specific DNA binding domains of above-mentioned liver cancer orchis pellet specific antigen protein matter, and this amino acid whose sequence is start-stop AA108-AA191 in the sequence shown in Figure 1;

Described derived peptide (d) is with the replacement of the aminoacid sequence in the described peptide section (c) through one or several amino-acid residue, the derived peptide that disappearance or interpolation sudden change are produced, and this derived peptide and described peptide section (a) have same or analogous function.

5, the application of the described antigen peptide of a kind of claim 4 in preparation treatment liver-cancer medicine.

6, the gene of the described liver cancer orchis pellet specific antigens of a kind of claim 4 of encoding peptide.

Images