CN1872877A - Idiosyncratic antigen protein, and antigen peptide of liver cancer orchis pellet - Google Patents

Abstract

The invention relates to a liver cancer-testis specific antigen protein and antigen peptide. The liver cancer-testis specific antigen protein is a protein having the amino acid sequence shown in FIG. 1 and a derivative protein thereof. The amino acid sequence of the antigenic peptide is AA108-AA191 in the sequence shown in FIG. 1 . The liver cancer-testis specific antigenic protein and antigenic peptide provided by the invention can be used as an E2F activity blocker in pRB/E2F signal transduction, and exert an antagonistic effect on its homologous analogue TFDP-1. The protein/peptide can be used as CT antigen or antigen gene to make a vaccine. This tumor antigen protein can be decomposed in the cell to produce a protein that is combined with the major histocompatibility complex (MHC)-I class molecule and is in a combined state. The peptide fragments that can be recognized by T cells can stimulate the immune response to achieve the purpose of treating cancer, and thus can be used in the preparation of drugs for treating cancer, especially liver cancer.

Description

A liver cancer-testis specific antigenic protein and antigenic peptide

technical field

The invention relates to a liver cancer-testis specific antigen protein and antigen peptide.

Background technique

Tumor-testis (cancer-testis, referred to as CT) antigen protein is the most identified class of tumor antigens, and their coding genes are characterized by expression in many types of tumors, such as melanoma, lung cancer, sarcoma and It is expressed in bladder cancer, but it is not expressed in normal tissues except testis, and it is expressed in a certain amount in placenta, ovary and pancreas. Because the testis is an immune privileged site, this type of antigen is considered to be a tumor-specific antigen in therapy, and it is the most promising type of antigen for tumor immunotherapy. At present, this type of antigen is mainly used clinically, such as MAGE-1 and NY-ESO-1, which have a high positive rate in certain types of tumors and have good immunogenicity. , has a good prospect when used in tumor immunotherapy.

Primary hepatocellular carcinoma is the most common type of primary liver cancer in humans and one of the most common malignant tumors in the world, especially in Southeast Asia and Africa, where its incidence is higher. As far as China is concerned, about 110,000 people die from liver cancer every year, accounting for a quarter of the annual liver cancer deaths worldwide. This high mortality rate is mainly due to the lack of specific clinical symptoms of early liver cancer, which leads to the late treatment of most patients, lack of effective treatment methods and easy recurrence after surgery. High incidence, high degree of malignancy, late diagnosis, few treatment methods, and poor prognosis are the major characteristics of liver cancer. Although researchers at home and abroad have conducted various studies on liver cancer, the molecular mechanism of liver cancer development is still not clear. The treatment of liver cancer is mainly surgical treatment and local or systemic chemotherapy, and a small number of patients are supplemented by tumor biological therapy. therapy, but postoperative survival is still unsatisfactory. For this reason, the research on the mechanism of liver cancer development has become an urgent requirement at present. It is hoped that the molecular basis of liver cancer development or the molecules closely related to the development of liver cancer will be clarified as soon as possible, so as to follow up high-risk groups, effectively prevent, diagnose early, and comprehensively treatment to reduce recurrence.

Previous studies have shown that the occurrence of liver cancer is a multi-stage and multi-step process, which is accompanied by many cytological and genetic changes in the body, and the balance between positive regulation and negative regulation in cells The relative balance is broken, and a variety of gene expression abnormalities appear, including the activation of oncogenes and/or the inactivation of tumor suppressor genes, which stimulate abnormal signal transduction pathways, resulting in abnormal cell cycle and apoptosis, resulting in malignant transformation of cells, and Tumors progress and metastasize as various molecular abnormalities accumulate. Moreover, the changes at the molecular level of liver cancer are heterogeneous, and there may be multiple genes and overlapping pathways. At the same time, the cell's intrinsic self-defense mechanism may also induce the production of antagonistic molecules. Therefore, the identification of these differentially expressed genes is of great significance for the elucidation of the pathogenesis and development mechanism of liver cancer, and for the clinical diagnosis and treatment of liver cancer.

Contents of the invention

The purpose of the present invention is to provide a tumor-testis-specific antigen protein and a tumor-testis-specific antigen peptide that can be used in the treatment of liver cancer.

The purpose of the present invention is achieved through the following technical solutions:

The present invention provides a liver cancer-testis specific antigen protein TFDP-3 (HCA661), which has the following amino acid sequence (a) or (b):

(a) have the amino acid sequence shown in Figure 1;

(b) The amino acid sequence in (a) undergoes one or several amino acid residue substitution, deletion or addition mutations to produce a derivative protein, and the derivative protein has the same or similar function as the protein in (a).

The present invention provides a gene encoding the above liver cancer-testis specific antigen protein.

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

The present invention provides a fusion protein comprising the above liver cancer-testis specific antigen protein.

The present invention provides a liver cancer-testis specific antigen peptide, which has the following amino acid sequence (c) or (d):

(c) It is a peptide segment containing 84 amino acids of the specific DNA binding domain of the above-mentioned liver cancer-testis specific antigen protein, and its amino acid sequence is AA108-AA191 in the sequence shown in Figure 1;

(d) The amino acid sequence in (c) undergoes one or several amino acid residue substitution, deletion or addition mutations to produce a derivative peptide, and the derivative peptide has the same or similar function as the peptide in (a).

The present invention provides a gene encoding the above liver cancer-testis specific antigen peptide.

The present invention provides a fusion protein comprising the above liver cancer-testis specific antigen peptide.

The liver cancer-testis specific antigenic protein, antigenic peptide, and their fusion protein provided by the present invention can be used as an E2F activity blocker in pRB/E2F signal transduction, and exert an antagonistic effect on its homologous analogue TFDP-1. The protein/peptide and the DNA encoding it can be used as CT antigen or antigen gene to make a vaccine. This tumor antigen protein can be produced and combined with major histocompatibility complex (MHC)-I molecules through intracellular decomposition The peptide fragments that can be recognized by T cells in the combined state can stimulate the immune response to achieve the purpose of treating cancer, so that they can be used in the preparation of drugs for treating cancer, especially liver cancer. Using the tumor antigen protein of the present invention can provide medicines for activating anti-tumor immunity and methods for diagnosing tumors. The antibody against the tumor antigen protein of the present invention can be used in affinity chromatography, screening of cDNA library, immunological diagnosis or preparation of medicine.

The new gene cloned in the present invention is a liver cancer-testis-specific expression gene related to the occurrence of liver cancer. The study of its function will help to clarify the mechanism of liver cancer occurrence and development. The new protein TFDP-3 (HCA661) encoded by this gene is a new CT antigen. In primary hepatocellular carcinoma, TFDP-3 (HCA661) positive accounts for about 1/3. As a tumor vaccine, it can be applied to liver cells. Potential clinical application value in the treatment of chronic liver cancer. At the same time, unlike other CT antigens, this protein has obvious functions and is a new member of the DP family. This family can form heterodimers with E2F, thereby enhancing the transcriptional regulation function of E2F. It is in the pRB/E2F signaling pathway. central location. The pRB protein is the target protein of a variety of DNA tumor virus immediate gene products, such as the simian virus 40 large T antigen, papilloma virus E7 protein, adenovirus E1a protein, etc. These oncogene products can compete with E2F for binding to pRB protein, destroy the interaction and binding of E2F and pRB, lead to the elimination of pRB's inhibition of E2F activity, and activate the transcription of E2F-dependent target genes. In most human tumors, the regulatory mechanism of E2F is out of regulation, and the disorder of E2F function is often accompanied by cell transformation and even tumorigenesis. Many previous studies have proved that DP protein or E2F protein has the characteristics of proto-oncogene. Structural analysis of TFDP-3 (HCA661) showed that it has a similar structure to TFDP-1, that is, it contains a DNA-binding domain and a dimerization domain. Functional studies have shown that TFDP-3 (HCA661) can form heterodimers with all proteins of the E2F family that have a dimerization domain. At the level of protein interaction, TFDP-3 (HCA661) has a complete consistent biological properties. However, at the level of protein-DNA interaction, the heterodimer formed by TFDP-3 (HCA661) and E2F protein greatly weakens the ability of E2F to bind DNA, and can inhibit the transcriptional activity of E2F and TFDP-1 Allosteric activation of E2F. The unique nature of TFDP-3(HCA661)'s effect on E2F function makes it promising to act as an antagonist of the unregulated pRB/E2F signaling pathway caused by viral infection or pRB mutation, thereby compensating the function of pRB, at least partially. TFDP-3 (HCA661) can be regarded as a specific tumor suppressor gene for liver cancer testis due to its specificity in tissue expression and its ability to inhibit E2F activity similar to pRB. Treatment provides a better target.

In addition, the present invention identifies the TFDP-3 (HCA661) gene as a tumor-testis-specific antigen gene for the first time; when immunotherapy is used to treat cancer, the tumor antigen protein of the present invention can be used to induce the body to produce an immune response that only targets tumor cells , so that on the premise of not killing normal cells, it can specifically kill tumor cells in the body to achieve the effect of treating or preventing tumor cell metastasis; at the same time, through the detection of the tumor antigen gene or antibody, the tumor occurrence, development and whether Diagnosed with metastases.

Description of drawings

Fig. 1 is the amino acid sequence of liver cancer-testis specific antigen protein provided by the present invention;

Fig. 2 is the nucleotide sequence encoding liver cancer-testis specific antigen protein provided by the present invention;

Figure 3 is the result of comparative analysis of the amino acid sequence of the TFDP-3 (HCA661) protein and its DP family homolog TFDP-1 using the CLUSTAL W (1.82) analysis program; where "*" refers to the amino acid in the sequence The residues are completely identical; ":" means that the amino acid residues in the sequence are conservative substitutions; "." means that the amino acid residues in the sequence are semi-conservative substitutions;

Fig. 4 is a Northern blot verification diagram of TFDP-3 (HCA661) mRNA in hepatocellular carcinoma (HCC); wherein, Testis is testicular tissue; Adj is adjacent non-cancerous tissue; HCC is hepatocellular carcinoma; Normalliver is normal liver organize;

Figure 5 is the expression distribution of TFDP-3 (HCA661) in HCC cell lines; wherein, L02 is a normal liver cell line, Jurkat is an acute T lymphocytic leukemia cell line, and the rest are HCC cell lines;

Figure 6 is the GST Pull-down analysis of the interaction between TFDP-3 (HCA661) and E2F family proteins; among them, Figure A: the soluble GST fusion protein of E2F was induced and expressed in Escherichia coli JM109, and the expression level of the soluble protein was measured by Coomassie Blue staining evaluation; arrows indicate the specific migration band of soluble protein; GST protein without fusion gene was used as negative reference. Panel B: The GST protein used as a negative reference and the GST fusion protein of E2F are bound to glutathione-agarose microbeads, and then interact with the in vitro transcribed and translated TFDP-3 (HCA661) protein, and the unbound protein is washed away The remaining proteins bound to glutathione-agarose beads were analyzed by electrophoresis and autoradiography. The in vitro transcribed and translated TFDP-3 (HCA661) protein was used as a positive reference;

Figure 7 is the co-immunoprecipitation analysis of the interaction between TFDP-3 (HCA661) and E2F protein; wherein, the upper figure shows the co-immunoprecipitation protein extracted from co-transfected HeLa cells by Western blot analysis using anti-HA polyclonal antibody; The middle panel shows the expression level of E2F protein in co-transfected HeLa cell lysates analyzed by Western blot using anti-HA polyclonal antibody; the lower panel shows the co-transfected HeLa cell lysates analyzed by Western blot using anti-FLAG M2 monoclonal antibody The expression level of the TFDP-3 (HCA661) protein of the object; the arrow shows the specific migration band of TFDP-3 (HCA661) or E2F protein;

Figure 8 is the DNA binding ability analysis (EMSA) of the heterodimer formed by the interaction between TFDP-3 (HCA661) and E2F protein;

Figure 9 is the subcellular localization analysis of TFDP-3 (HCA661); wherein, A, the subcellular localization of E2F or DP protein transfection alone; B, the subcellular localization of E2F and DP co-transfection; all images are equipped with Immunofluorescence inverted microscope of CCD was taken under the field of view with a magnification of 480 times;

Figure 10 is the analysis of the influence of TFDP-3 (HCA661) on the transcriptional activity of E2F; wherein, A is the result of TFDP-3 (HCA661) inhibiting the transcriptional activity of E2F; B is the result of TFDP-3 (HCA661) inhibiting the transcriptional activity of E2F/TFDP-1 The transcriptional activation result of ; C is the result of TFDP-3 (HCA661) overexpression can inhibit endogenous E2F activity; the data shown are the average value and standard deviation of three experiments;

Figure 11 is a diagram of the TFDP-3 (HCA661) mutant; wherein, A is the DNA binding domain of two known human DP family proteins TFDP-1 and TFDP-2 and TFDP-3 (HCA661) Comparison of amino acid residues in the structural domain; "*" means that the amino acid residues in the sequence are completely consistent; ":" means that the amino acid residues in the sequence are conservative substitutions; "." means that the amino acid residues in the sequence are semi-conservative substitutions ; The analysis program used for sequence analysis is CLUSTAL W (1.82); "Δ" means that the amino acid residue participates in the interaction with the DNA base; "▲" means that the amino acid residue participates in the interaction with the DNA backbone; "■" means that the amino acid residue is involved in the interaction with the heterodimerization of the protein; B is a diagram of the substitution mutant derived from the amino acid sequence of full-length TFDP-3 (HCA661) and TFDP-1; The amino acid sequence of TFDP-3 (HCA661) and TFDP-1 can be divided into four parts according to the composition of the structural domain, namely, the N-terminal unknown functional region (UNT), the DNA binding domain (DBD), and the heterodimerization domain (HD ) and the C-terminal unknown domain (UCT); C is a schematic representation of mutants derived from substitutions in the DNA binding domain of TFDP-3(HCA661) and TFDP-1; amino acid residues of TFDP-3(HCA661) Marked by shading; the transcriptional nature of the mutant is also briefly marked on the right side of the mutant;

Figure 12 is the molecular basis analysis of the unique function of TFDP-3 (HCA661); wherein, A is the protein expression of TFDP-3 (HCA661) and TFDP-1 mutant; B and C are the replacement of TFDP-3 and TFDP-1 Reporter assessment of the effect of mutants on the transcriptional activity of E2F-4; data shown are the mean of three experiments and their standard deviations.

Detailed ways

Example 1. Gene Analysis

TFDP-3 (HCA661) is a new gene screened from the patient's liver cancer tissue with serum by SEREX method. According to bioinformatics analysis, the TFDP-3 (HCA661) gene is located on the X chromosome of the human gene and is a gene consisting of a single exon. The size of the transcript is 1,680bp, the coding frame is 1,218bp, the start and end points are 90-1,307, and it encodes a protein of 405 amino acids (as shown in Figure 1). This protein is a new member of the DP family of transcription factors, and has a high degree of similarity (75.2% amino acid identity; 82.0% amino acid similarity) to its DP family homologue TFDP-1. The comparative analysis of the amino acid sequences of the two is shown in Figure 3. Similar to TFDP-1, the amino acid sequence of TFDP-3 includes an evolutionarily conserved DNA binding domain (AA108-AA191) and an evolutionarily conserved heterodimerization structure domain (AA192-AA264), and, in the DNA-binding domain, also contains a RRXYD DNA recognition motif (AA162-AA166) common in E2F family and DP family. In the known E2F and DP families, the third amino acid in the RRXYD DNA recognition motif is usually a hydrophobic amino acid (Valine (V) or Isoleucine (I)), but in TFDP-3 (HCA661), this The amino acid is hydrophilic Threonine (T), whether this non-conservative amino acid substitution has an effect on the DNA binding function of TFDP-3 (HCA661) is unclear. In addition, 15 of the 20 amino acids at the carboxy-terminal of TFDP-3 (HCA661) are acidic amino acids, while TFDP-1 and TFDP-2 only have 13 and 11 acidic amino acids. The results of these analyzes strongly suggest that TFDP-3 (HCA661) is the third new member of the human DP family.

Example 2.TFDP-3 (HCA661) mRNA Northern blot analysis

For Northern blot analysis, total RNA was extracted from HCC specimens and corresponding noncancerous tissue specimens as well as testicular tissue. RNA integrity was analyzed by electrophoresis. During electrophoresis, the loading amount of total RNA per well was 30 mg, and then transferred to the membrane. Nitrocellulose membranes were hybridized with specific ³² P-labeled cDNA probes overnight at 65°C. Wash three times with 0.1×SSC/0.1% SDS solution, each time for 30 minutes, and perform autoradiography at -70°C. The size of the full-length mRNA is compared with the degree of migration of 28S and 18S ribosomal RNA.

Due to the high similarity of nucleotide sequences, the results of Northern blot hybridization (as shown in Figure 4) showed that two strong bands appeared in the RNA of testis tissue, which were ~1.7kb and 2.1kb respectively. Only a ~1.7 kb band appeared in the RNA of HCC tissues, while only a 2.1 kb band was detected in noncancerous and normal liver tissues. DNA sequence analysis showed that the 2.1 kb gene was TFDP-1, and the 1.7 kb gene was TFDP-3 (HCA661).

Example 3. Expression distribution of TFDP-3 (HCA661) in HCC and adjacent non-cancerous tissue samples

The expression profile of TFDP-3 (HCA661) mRNA was analyzed by RT-PCR, and the results are listed in Table 1. Tissues included 16 normal tissue cDNAs purchased from Clontech: brain, heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, colon, ovary, peripheral blood leukocytes, prostate, small intestine, spleen, testis, and thymus, from HCC tissue The total RNA extracted from the matched adjacent non-cancerous tissue was reverse transcribed into cDNA. The TFDP-3(HCA661) fragment was amplified using specific primer pairs. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA was used as an internal reference. The primers used are as follows: TFDP-3, forward, 5’-TAC ACT CGG CCT GGA AGA ATT G-3’; reverse, 5’-TCT TCC TCC TCG ACT GCT G-3’, size, 1,244 base pairs (bp).

Among the 16 kinds of normal tissues, only the testis detected the high level expression of TFDP-3(HCA661) mRNA, the expression was very weak in the normal pancreas tissue, and no expression was detected in other normal tissues. In HCC tissue, 5 of the 17 HCC tissue samples were positive, only 2 of the adjacent non-cancerous tissues corresponding to the 17 HCC tissue samples had weak expression, and none of the 9 normal liver tissues expressed. It is a typical cancer-testis (CT) antigen.

Table 1. RT-PCR analysis of TFDP-3(HCA661) expression in HCC and adjacent non-cancerous tissue specimens HCC ADJ Cirrhosis Normal liver case 17 17 5 9 +ve 5 2 0 0

Example 4. Expression of TFDP-3 (HCA661) mRNA in HCC cell lines

Using TRIZOL (GIBCO BRL) from L02 (human normal liver cell line), 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 cell lines, SK-Mel-37 and Mel-526 melanoma cell lines, Jurkat (human acute lymphoblastic leukemia cell line) cell extraction total RNA. Total RNA was reverse transcribed and TFDP-1, TFDP-2 and TFDP-3 (HCA661) fragments were amplified using specific primer pairs, and G3PDH mRNA was used as an internal reference. RT-PCR was performed using the following parameters: TFDP-3(HCA661), 35 cycles, 94°C for 30 seconds; 60°C for 30 seconds; 72°C for 90 seconds, 72°C extension for 10 minutes; TFDP-1, 35 cycles, 94°C 30 seconds at 55°C; 90 seconds at 72°C, 10 minutes at 72°C; TFDP-2, 35 cycles, 30 seconds at 94°C; 30 seconds at 55°C; 60 seconds at 72°C, 10 minutes at 72°C; G3PDH, 25 cycles, 94°C for 15 seconds; 64°C for 20 seconds; 72°C for 20 seconds, 72°C for 10 minutes. TFDP-3(HCA661), TFDP-1, TFDP-2 and G3PDH primer sequences and PCR product fragment lengths are as follows: TFDP-3, forward, 5'-TAC ACT CGG CCT GGA AGA ATT G-3'; reverse, 5' -TCT TCC TCC TCG ACT GCTG-3', size, 1, 244base 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 GTG ACT GTT TGG G-3' , size, 997bp; G3PDH, forward, 5'-ACC ACAGTC CATGCCATCAC-3, reverse, 5'-TCC ACC ACC CTG TTG CTG TA-3', size, 452bp.

We analyzed the expression levels of TFDP-1, TFDP-2 and TFDP-3 (HCA661), members of the DP family, in tumor cell lines, especially in HCC cell lines, by RT-PCR. The quality of mRNA was assessed using G3PDH as an internal reference. We detected the expression of TFDP-3(HCA661) in liver cancer tissues. In this experiment, we designed specific primer pairs as shown above to amplify TFDP-3(HCA661), TFDP-1 and TFDP-2 respectively. Gene fragments, detecting the expression distribution of each member of the DP family in tumor cell lines, especially in HCC cell lines. TFDP-1 and TFDP-2 mRNA were expressed in cells of all cell lines, while the expression of TFDP-3 (HCA661) mRNA was significantly different in different cell lines. It was expressed at a relatively high level in SMMC-7721, while the expression level was low in HepG2, HLE and Jurkat, and the expression of TFDP-3 (HCA661) mRNA was not detected in the remaining cell lines (as shown in Figure 5), and TFDP -3 (HCA661) expression distribution in liver cancer tissues was consistent. Different from the widespread expression of TFDP-1 and TFDP-2, the expression of TFDP-3(HCA661) has obvious histiocyte restriction. Therefore, TFDP-3 (HCA661) has an expression profile different from other members of the DP family.

Example 5. Interaction between TFDP-3 (HCA661) protein and E2F protein in vitro

In order to produce the GST fusion protein of E2F-1～-6 to facilitate protein purification and subsequent GST Pull-down analysis, the E2F gene was constructed into pGEX-4T2, and then transformed into Escherichia coli (JM109), and a large amount of GST was obtained and purified by traditional methods - E2F fusion protein (see Figure 6A). TFDP-3 (HCA661) protein was obtained by in vitro transcription and translation in the presence of radioactive ³⁵ S-methionine in a 50 μl reaction system using Promega's TNT ^(R) Quick Coupled Transcription/Translation system kit. In the in vitro binding reaction, an appropriate amount of purified GST or GST-E2F fusion protein was first reacted with glutathione-agarose microbeads, and then added with 50mM Tris (pH8.0), 150mM NaCl, 10mg/ml lysozyme , 0.5mM phenylmethylsulfonyl fluoride (PMSF), 50mg/ml leupeptin, 50mg/ml protease inhibitor, 50mg/ml aprotinin and 50mM dithiothreitol in TFDP-3 (HCA661) lysis buffer. After 2.5 hours at 4°C, the precipitate was collected by centrifugation and washed four times with lysis buffer to remove unbound protein. Add SDS loading buffer, electrophoresis on 12.5% polyacrylamide gel, dry gel, and autoradiography. The TFDP-3 (HCA661) protein transcribed and translated in vitro was used as a positive reference, and the non-fused GST protein was used as a negative reference. The results showed that TFDP-3 (HCA661) could interact with E2F-1～E2F-6 (see Figure 6B, lanes 3 to 8). The interaction between TFDP-3(HCA661) and E2F protein was efficient and specific, because there was no interaction between TFDP-3(HCA661) and the negative reference GST protein (see Figure 6B, lane2). This in vitro binding data supports that TFDP-3(HCA661) is a new member of the DP family.

Example 6. Interaction between TFDP-3 (HCA661) protein and E2F protein in vivo

Human cervical cancer cell line HeLa cells were cultured at 37°C in a 5% CO2 incubator, and the medium used was DMEM medium containing 100 units/ml penicillin, 100 μg/ml streptomycin and 10% newborn calf serum. Before transfection, HeLa cells were digested with trypsin, and the cells were diluted in 6-well plates with fresh DMEM medium containing 10% newborn calf serum without double antibody, and cultured until the cell confluency reached 90-95% required for transfection. %. For transfection, add 1.4 μg of HA-tagged E2F plasmid DNA and 1.4 μg of FLAG-tagged DP plasmid DNA, or 1.4 μg of empty vector DNA, to bring the total amount of DNA to 2.8 μg. LipofectAMINE 2000 reagent (Invitrogen) was used to bring DNA into cells. 24-36 hours after transfection of HeLa cells, non-denatured cell lysates were prepared according to conventional methods. The expression levels of these proteins were monitored by Western blot analysis of cell lysates with anti-HA rabbit polyclonal antibody or anti-FLAG M2 mouse monoclonal antibody to monitor E2F protein (Fig. 7 middle panel) or TFDP-3 (HCA661) protein (Fig. 7 below) expression. In order to perform co-immunoprecipitation analysis, the transfected HeLa cells were washed twice with 1×PBS, and then an appropriate amount of 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100, 1mM EDTA, 5μg/ml was added Aprotinin, 5μg/ml leupeptin, and 2mM PMSF lysis buffer lysed cells to prepare non-denaturing cell lysates. The concentration of total protein was analyzed by Bradford. For co-immunoprecipitation, the cell lysate was reacted for at least 2 hours at 4°C in cell lysis buffer containing 2 μg/ml anti-FLAG antibody and 25 μl protein A-agarose. The pellet after centrifugation was washed three times with cell lysis buffer containing 500mM NaCl to remove unbound protein. After washing and centrifuging, the precipitate was added to a sample buffer, electrophoresed on a 12.5% polyacrylamide gel, and transferred to a membrane. After electroporation, the nitrocellulose membrane was first blocked with TBS blocking buffer containing 5% skimmed milk powder for 1 hour, then incubated with blocking solution containing 1 μg/ml anti-HA antibody for 1 hour, and finally blocked with HRP-coupled anti-rabbit IgG Anti-reaction, ECL color development. E2F protein was detected in all co-immunoprecipitates transfected with E2F DNA, but not in co-immunoprecipitates transfected only with empty vector DNA or TFDP-3(HCA661) DNA. These data suggest that TFDP-3(HCA661) can efficiently interact with members of the E2F family containing heterodimerization domains and form heterodimers, further supporting the speculation that TFDP-3(HCA661) is a new member of the DP family .

Example 7. DNA binding ability analysis (EMSA, electrophoretic mobility shift assay) of the heterodimer formed by the interaction between TFDP-3 (HCA661) and E2F protein.

Human cervical cancer cell line HeLa cells were cultured at 37°C in a 5% CO2 incubator, and the medium used was DMEM medium containing 100 units/ml penicillin, 100 μg/ml streptomycin and 10% newborn calf serum. Before transfection, HeLa cells were digested with trypsin, and the cells were diluted in 6-well plates with fresh DMEM medium containing 10% newborn calf serum without double antibody, and cultured until the cell confluency reached 90-95% required for transfection. %. For transfection, add 1.4 μg of E2F DNA and 1.4 μg of DP DNA, or 1.4 μg of empty vector DNA, to bring the total amount of DNA to 2.8 μg.

ipofectAMINE 2000 reagent (Invitrogen) was used to bring DNA into cells. After 24-36 hours of HeLa cell transfection, non-denatured cell protein extracts were prepared according to conventional methods, and an equal amount of HeLa cell protein extracts were added containing 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, and 1mg/ml polydI/dC in the EMSA buffer, while adding 0.1ng of radioactive ³² P isotope-labeled Oligonucleotide probes (ATTFAAG TTTCGCGCCC TITCCAA) containing specific wild-type E2F binding sites were incubated at room temperature for 10-15 minutes. The reaction mixture was then poured into a 5% non-denaturing polyacrylamide gel for 2.25 hours at a constant voltage of 180V at 4°C, and the dried gel was analyzed by autoradiography at -70°C for 4-16 hours. In order to detect the specificity of the protein binding to the wild-type DNA probe, an unlabeled double-stranded oligonucleotide probe containing an E2F mutation site 20 times the amount of the wild-type probe was added to the reaction solution as a competitor molecule (ATTTAAG TTTCGatCCC TITCTCAA ).

As shown in Figure 8, because of the existence of the basal level of endogenous E2F, when a single E2F or DP is transfected, their DNA binding ability is still at the basal level (compared with 1 in the development zone 2, 3, 6, 9), It indicated that transfection of E2F or DP alone could not improve its DNA binding activity. When TFDP-1 and E2F were co-transfected, the ability of E2F/TFDP-1 heterodimer to bind DNA was significantly enhanced compared with transfection of E2F alone (developing bands 5 and 3, 8 and 6, 11 and 9 Compare). On the contrary, when TFDP-3(HCA661) was co-transformed with E2F, the DNA binding ability of E2F/TFDP-3(HCA661) heterodimer was not significantly improved (developing bands 4 and 3, 7 and 6, 10 and 9 compared to). This suggests that there may be an unidentified motif in the TFDP-3(HCA661) protein that disrupts DNA binding, resulting in the loss of E2F/TFDP-3 heterodimer binding to the E2F DNA recognition site.

Example 8. Subcellular localization of TFDP-3 (HCA661)

SV40-transformed green monkey kidney cell line COS-7 cells were cultured in a cell incubator at 37°C with 5% CO2, and the medium used was DMEM culture containing 100 units/ml penicillin, 100 μg/ml streptomycin and 10% newborn calf serum base. Before transfection, COS-7 cells were digested with trypsin, and the cells were diluted to 8×10 ⁴ cells/ml with fresh DMEM medium containing 10% newborn calf serum without double antibody, and 1 ml of each was added to a 24-well plate. Grow until cells are 90-95% confluent for transfection. When transfected alone, 280ng of EGFP-tagged E2F plasmid DNA or 280ng of FLAG-tagged DP plasmid DNA and 280ng of empty vector DNA were added to the total amount of DNA to 560ng. Meanwhile, pEGFP-N1 was used as a negative reference. For co-transfection, 280ng of EGFP-tagged E2F plasmid DNA and 280ng of FLAG-tagged DP plasmid DNA were added, and the total amount of DNA was added to 560ng. LipofectAMINE 2000 reagent (Invitrogen) was used to bring DNA into COS-7 cells. 24 hours after transfection, wash slightly with 1×PBS, then add -70°C pre-cooled methanol to fix at -20°C for 20 minutes, add 1×PBS containing 0.2% Triton X-100 to permeabilize the cell membrane for 4 minutes. After blocking non-specific antibody binding sites with 1×PBS blocking solution containing 1% BSA, fixed COS-7 cells were incubated in 1×PBS containing 1 μg/ml anti-FLAG M2 monoclonal antibody and 1% BSA for 1 hour at room temperature, After washing three times with 1xPBS, a secondary antibody containing goat anti-mouse IgG conjugated to tetramethylrhodamineisothiocyanate (TRITC) diluted 1:100 and 1% BSA was added in 1xPBS for 1 hour at room temperature. COS-7 nuclei were counterstained with 10 μg/ml Hoechst 33342, and E2F was labeled and detected with EGFP. Immunofluorescence images were collected by a fluorescence microscope equipped with a CCD, and all images were processed by Adobe Photoshop version 7.0.1.

The experimental results showed that when transfected alone, both TFDP-3 (HCA661) and TFDP-1 were localized in the cytoplasm of COS-7 cells, E2F-1 was localized in the nucleus, and E2F-4 was localized in the cytoplasm (See Figure 9A). E2F-2, E2F-3 were also localized in the nucleus, while E2F-5 was localized in the cytoplasm (not shown).

To function, DP family members must form heterodimers with E2F family members. Therefore, we analyzed the effect of protein-protein interactions on the subcellular localization of TFDP-3(HCA661). As speculated, TFDP-3 (HCA661 ), when co-transfected with E2F-1 (see FIG. 9B ), E2F-2 or E2F-3 (not shown), localized in the nucleus similar to TFDP-1. In contrast to the localization of the first subfamily of E2F, TFDP-3 (HCA661) and TFDP-1 remained cytoplasmically localized when co-transfected with E2F-4 (see Figure 9B) or E2F-5 (not shown) Inside. These data suggest that the subcellular localization of TFDP-3 (HCA661) is consistent with that of TFDP-1.

Example 9. Reporter gene analysis of TFDP-3 (HCA661)

Human normal liver cell line L02 was cultured in a cell incubator at 37°C with 5% CO2, and the medium used was DMEM medium containing 100 units/ml penicillin, 100 μg/ml streptomycin and 10% newborn calf serum. Before transfection, L02 cells were digested with trypsin, and the cells were diluted to 1×10 ⁵ cells/ml with fresh DMEM medium containing 10% newborn calf serum without double antibodies, and 1 ml was added to each 24-well plate, and the cells were cultured Bring to 90-95% confluency required for transfection. In reporter gene analysis, the total amount of DNA was added to 560ng, which contained 140ng of reporter gene 6xE2F-firefly luciferase. In the experiment of the inhibitory effect of TFDP-3 (HCA661) on E2F/TFDP-1-mediated transcriptional activation, the total amount of DNA was increased to 1,120ng. When performing effects of TFDP-3 (HCA661) or TFDP-1 on endogenous E2F activity, the total amount of DNA was added to 840 ng. LipofectAMINE 2000 reagent (Invitrogen) was used to bring DNA into cells. After 24 hours of transfection, L02 cells were washed with 1xPBS to remove the residual medium, then added passive lysis buffer (PLB), and lysed at room temperature for 15 minutes, harvested the lysate and centrifuged to remove cell debris, and harvested the supernatant , analyze the product using the bispecific luciferase analysis system according to the operation manual provided by the manufacturer. In order to eliminate the influence of the difference in transfection efficiency on the value of the reporter gene 6xE2F-firefly luciferase, 140ng of pRL-SV40Renilla luciferase plasmid was added to each transfection as an internal reference.

The results of the reporter gene analysis are shown in Figure 10A, indicating that when the E2F gene and the DP gene were transfected alone, 140ng of TFDP-1 produced approximately 2-fold transcriptional activation of the reporter gene 6xE2F-firefly luciferase, and similarly, 20ng of E2F-1 or E2F-3 also produced 10-fold or 30-fold transcriptional activation, respectively, and 70 ng of E2F-4 or 140 ng E2F-5 also produced 17-fold or 11-fold transfection activation. When E2F was co-transfected with TFDP-1, the reporter gene 6xE2F-firefly luciferase was allosterically activated by 30- to 70-fold. However, in stark contrast to TFDP-1, when E2F was co-transfected with TFDP-3(HCA661), a significant inhibitory effect of TFDP-3(HCA661) on E2F-mediated transcriptional activation was observed. The same experiment was repeated at least three times (P<0.01).

TFDP-3(HCA661) has the ability to inhibit E2F-mediated transcriptional activation, therefore, we wanted to further analyze whether the interaction between TFDP-3(HCA661) and E2F could effectively inhibit E2F/TFDP-1 heterodimer-mediated Transcriptional activation. In the experiment, various combinations of E2F/TFDP-1 and 0, 140, 280 or 560ng of TFDP-3 (HCA661) were co-transfected into L02 cells, and the 6xE2F-firefly luciferase reporter gene analysis system was used to analyze the effect of TFDP-3 (HCA661) on Role of E2F/TFDP-1 heterodimers.

As mentioned above, all E2F/TFDP-1 combinations significantly increased the transcriptional activity of the 6xE2F-firefly luciferase reporter gene. When the E2F/TFDP-1 combination was present in TFDP-3(HCA661), the transcriptional activity of E2F/TFDP-1 was significantly inhibited in a dose-dependent manner. It can even completely block the allosteric activation of E2F by TFDP-1 (as shown in Figure 10B). This finding suggests that, despite being a novel member of the DP family, TFDP-3 (HCA661) functions as a repressor, repressing the transcriptional regulation of E2F-dependent target genes.

Finally, we also assessed the effect of TFDP-3(HCA661) on endogenous E2F activity. As expected, FLAG-tagged TFDP-1 enhanced the transcriptional activity of the 6xE2F-firefly luciferase reporter gene, while FLAG-tagged TFDP-3(HCA661) inhibited the transcriptional activity of the 6xE2F-firefly luciferase reporter gene in a dose-dependent manner (see Figure 10C), suggesting that TFDP-3(HCA661) has the ability to block the transcriptional activation of the 6xE2F-firefly luciferase reporter gene by the endogenous E2F complex. Moreover, we also obtained similar results from the CyclinA-firefly luciferase reporter gene constructed with the natural regulatory sequence of CyclinA, the target gene of E2F (not shown).

The ability of TFDP-3(HCA661) to suppress E2F-mediated transcription is consistent with the loss of the ability of TFDP-3(HCA661)/E2F heterodimers to bind specific E2F DNA-binding sequences. Moreover, the inhibitory effect of TFDP-3(HCA661) also indicated that TFDP-3(HCA661) may play the role of antagonizing TFDP-1 when TFDP-3(HCA661) and TFDP-1 coexist.

Taken together, all the above results suggest that the functional difference between TFDP-3(HCA661) and TFDP-1 may arise from the loss of DNA binding of the E2F/TFDP-3(HCA661) heterodimer and that the molecular basis for the inhibitory effect should be Exist in TFDP-3 (HCA661) molecule, because for a specific E2F protein, its transcriptional activity depends on the strength of the DNA binding ability of the complete DNA binding domain composed of E2F protein and DP protein weak.

Example 10. Exploration of the molecular basis of the unique function of TFDP-3 (HCA661)

In order to explore the molecular basis of the loss of DNA-binding ability of the heterodimer formed by TFDP-3(HCA661) and E2F protein and the inhibition of E2F transcriptional activity by TFDP-3(HCA661), we constructed TFDP-3(HCA661) and For the replacement mutants of TFDP-1, the transcriptional properties of the mutants were analyzed using the 6xE2F-luciferase reporter gene analysis system, and the DNA binding ability of the heterodimer formed by TFDP-3 (HCA661) and E2F protein was indirectly evaluated. The replacement mutants of TFDP-3 (HCA661) and TFDP-1 were constructed by PCR using the corresponding plasmids as templates. First, design forward mutation primers and reverse mutation primers at the replacement site. The primers contain the corresponding bases that need to be replaced. -OA) and the forward primer (DP3CF-OS or DP1CF-OS) were paired, two small fragments were synthesized by PCR, and then the two small fragments were connected into a complete gene by ligation PCR.

The following mutants use pCDNA3-TFDP-3-FLAG and pCDNA3-TFDP-1-FLAG as templates, and the mutation primers used are 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 gcagac 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 ag g 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'.

The domain substitution mutants of TFDP-3ΔH and TFDP-3ΔD, TFDP-1ΔH and TFDP-1ΔD use 3D-1H and 1D-3H as templates, and the mutation primers used in PCR are as follows: TFDP-3ΔH, forward, 5'-acc agcaag aag ACC GTC ATC AAC-3'; reverse, 5'-gtt gat gac ggt CTT CTT GCT GGT G-3'; TFDP-1ΔH, forward, 5'-agt age aag aag ACG GTC ATC GAC-3'; reverse, 5'-gtc gat gaccgt CTT CTT GCT AC-3'; TFDP-3ΔD, forward, 5'-gga gag aag aat GGC AAG GGC CTAC-3'; reverse, 5'-tag gec ctt gcc ATT CTT CTC TCC-3 '; TFDP-1ΔD, forward, 5'-gga gagaag aat GGC ATG GGC CTG-3'; reverse, 5'-cag gcc cat gcc ATT CTT CTC TCC-3'.

TFDP-3ΔDH uses TFDP-3ΔD and TFDP-3ΔH as templates, and the mutation primers are as follows: forward, 5'-ggtctg ccc acc AAC TCG GCT CAG-3'; reverse, 5'-ctg agc cga gtt GGT GGG CAG ACC-3' .TFDP-1ΔDH uses TFDP-1ΔD and TFDP-1ΔH as templates, and the mutation primers are as follows: forward, 5-ggt ctg accacc AAC TCG GCT CAG-3; reverse, 5-ctg agc cga gtt GGT GGT CAG ACC-3'.

TFDP-3MR2～TFDP-3MR5 replacement mutants use the pCDNA3-TFDP-3-FLAG plasmid as a template, and the mutation primers are 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 CACTTC CTG-3'.

The following mutants use the pCDNA3-TFDP-3-FLAG plasmid as a template, and the mutation primers are 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 GGAAGT 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'; TFDP-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 GGCAGC TCT G-3'.

All mutants were sequenced and corrected, digested with Hind III-BamHI restriction endonuclease, and cloned into the pCDNA3-FLAG expression vector. Figure 11 shows the amino acid sequence of the mutants. Figure 12A shows the protein expression of each mutant. The 6xE2F-luciferase reporter gene assay system was used to analyze the effect of its mutants on the transcriptional activity of E2F-4, and indirectly evaluate the DNA-binding ability of the heterodimer formed by TFDP-3 (HCA661) and E2F protein. The specific experimental procedure is the same as the method described in Example 9.

First, we analyzed the differences in the amino acid composition of the DNA-binding domains of TFDP-3 (HCA661) and TFDP-1 based on previous findings, and performed a single amino acid substitution for the third amino acid in the RRXYD DNA recognition motif, yielding Two substitution mutants (TFDP-3M: aa164T→V; TFDP-1M: aa169V→T), to analyze whether the non-conservative substitution of this amino acid is the cause of the unique function of TFDP-3 (HCA661). 6xE2F-firefly luciferase reporter gene analysis showed that the amino acid substitution at this position has nothing to do with the function of TFDP-3(HCA661). Furthermore, within the DNA-binding domain, another 13 amino acids are involved in the heterodimerization of the protein and the interaction with the DNA phosphate backbone, according to the crystal structure. Among them, in TFDP-3 (HCA661), there are three amino acids that are different from TFDP-1 and TFDP-2. Therefore, we designed a 13-amino acid substitution (TFDP-3MR: aa109-121 M GL CRL SMKV W E T → K GL RHF SMKV C E K ; TFDP-1MR: aa114-126 K GL RHF SMKV C E K → M GL CRL SMKV W E T ). However, these substitutions still cannot reverse the function of TFDP-3(HCA661).

Using a similar sequence substitution analysis, we analyzed the full-length amino acid sequences of TFDP-3 (HCA661) and TFDP-1, and divided them into four components according to the composition of the structural domains, namely, the amino-terminal unknown functional region (UNT), DNA-binding domain (DBD), heterodimerization domain (HD) and carboxy-terminal unknown functional domain (UCT), and constructed 3D-1H (containing UNT of TFDP-3 (HCA661) and DBD and TFDP-1 HD and UCT) and 1D-3H (UNT with TFDP-1 and HD and UCT with DBD and TFDP-3 (HCA661)), reporter gene analysis showed that 1D-3H produced levels of E2F-mediated transcriptional activation comparable to wild-type The level produced by type TFDP-1 was not affected by AA192-405 of TFDP-3 (HCA661). In contrast, 3D-1H exhibited a pronounced inhibitory effect on E2F-mediated transcriptional activation. This indicates that the molecular basis for the unique function of TFDP-3(HCA661) should be located in the first half of TFDP-3(HCA661) (AA1-191).

In order to further analyze the molecular basis of function generation, we derived the following substitution mutants from 3D-1H and 1D-3H by PCR method: TFDP-3ΔD (DBD containing TFDP-1), TFDP-1ΔD (containing TFDP-1 3 (DBD of HCA661), TFDP-3ΔH (HD with TFDP-1), TFDP-1ΔH (HD with TFDP-3 (HCA661)), TFDP-3ΔDH (DBD and HD with TFDP-1), TFDP - 1ΔDH (DBD and HD with TFDP-3 (HCA661 )). The 6xE2F-firefly luciferase reporter gene results indicated that TFDP-3ΔH and TFDP-1ΔH produced similar levels of transcriptional repression or activation, respectively, as their wild-type counterparts, whereas, with replacement of the DNA-binding domain, TFDP-3ΔDH and TFDP- 3ΔD produced TFDP-1-like allosteric activation of E2F transcription, while TFDP-1ΔDH and TFDP-1ΔD produced TFDP-3(HCA661)-like repressive effects on E2F transcription. These data indicate that the functional region where TFDP-3(HCA661) efficiently inhibits E2F-mediated transcriptional activation is located in the DNA-binding domain of TFDP-3(HCA661) (see FIG. 12B ).

Next, we used the same strategy as the construction method of TFDP-3MR and TFDP-3M to divide the remaining 13 amino acids in the DNA-binding domain of TFDP-3 (HCA661) that are different from TFDP-1 into three parts, respectively using The corresponding amino acid sequence of TFDP-1 replaces the TFDP-3 (HCA661) sequence to generate TFDP-3MR2 (AA130-145 CQ EV VG ELVA K F R AA S → YN EV AD ELVA E F S AAD ), TFDP-3MR3 (AA148- 161 AS PNESAYD V KNI K → IL PNESAYD Q KNI R ) and TFDP-3MR4 (AA179-190 R EKK K IKWIGL T → K EKK E IKWIGL P ). Reporter gene analysis revealed that the TFDP-3MR3 and TFDP-3MR4 substitution mutants did not increase the transcriptional activity of E2F, producing similar inhibitory effects as wild-type TFDP-3(HCA661). In contrast, TFDP-3MR2 produced a level of transcriptional activation similar to that produced when transfected with E2F alone. This mutant also had the ability to inhibit transcriptional activation of E2F/TFDP-1 (not shown). Another substitution mutant, TFDP-3MR5 (AA134-142 VG ELVA K FR → AD ELVA E FS ) derived within TFDP-3MR2, had a weaker inhibitory effect on E2F-mediated transcriptional activation. Thus, the AA130-145 sequence of the DNA binding domain of TFDP-3 (HCA661 ) is required for the inhibitory effect (see Figure 12C). In addition, in order to find key amino acids, we generated another group of single amino acid substitution mutants TFDP-3CY(AA130C→Y), 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 results suggest that the generation of TFDP-3(HCA661) function may It is the result of the joint action of multiple amino acids.

In summary, the results of the reporter gene analysis indicated that the DNA-binding domain (AA108-AA191) of TFDP-3(HCA661) was the root cause of the loss of DNA-binding ability of the heterodimer formed by TFDP-3(HCA661) and E2F protein, thereby Inhibits the transcriptional activity of E2F independently of the spatial structure of the rest of the protein, including the heterodimerization domain. Further analysis of the DNA-binding domain revealed that AA130-AA145 (CQEVVGELVAKFRAAS) is required for the inhibitory effect of TFDP-3 (HCA661) on E2F DNA binding.

Sequence-listing.txt

SEQUENCE LISTING

<110>Peking University

<120> A liver cancer-testis specific antigenic protein and antigenic peptide

<130>FPI05135

<160>2

<170>PatentIn version 3.1

<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 Ash 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 G1u 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 Ash 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 Ash 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 Set Arg Ser Asn 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 Ash Asp Asp Asp Leu Ser Glu

385 390 395 400

Asn Asp Glu Asp Asp

405

<210>2

<211>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 gaagcccaaa 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 ctccaggggag 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 (8)

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

(a) has aminoacid sequence shown in Figure 1;

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

2, a kind of fusion rotein that comprises the described liver cancer orchis pellet specific antigen protein of claim 1 matter.

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

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

5, a kind of liver cancer orchis pellet specific antigens peptide, it has following (c) or (d) described aminoacid sequence:

(c) it 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 its aminoacid sequence is start-stop AA108-AA191 in sequence shown in Figure 1;

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

6, a kind of fusion rotein that comprises the described liver cancer orchis pellet specific antigens of claim 5 peptide.

7, a kind of claim 5 or the 6 described antigen peptide application in preparation treatment liver-cancer medicine.

8, the gene of the described liver cancer orchis pellet specific antigens of a kind of claim 5 of encoding peptide.

Images