CN101054413A - Tumor correlated albumen, coding gene and application thereof - Google Patents

Abstract

The invention discloses a tumor-associated protein and its coding gene. The tumor-associated protein is a protein of the following (a) or (b): (a) a protein consisting of the amino acid residue sequence of sequence 4 in the sequence listing; (b) the amino acid residue sequence of sequence 4 in the sequence listing is processed A protein derived from (a) with substitution and/or deletion and/or addition of one or several amino acid residues and associated with tumors. The recombinant mammalian gene expression vector containing the above-mentioned tumor-related protein coding gene can be used as an antitumor drug for preventing and/or treating lung cancer, esophageal cancer or laryngeal cancer.

Description

A kind of tumor related protein and its coding gene and application

technical field

The invention relates to a tumor-associated protein and its coding gene and application.

Background technique

Tumor has become one of the major diseases that seriously threaten human survival and health, and its morbidity and mortality are on the rise. The discovery and study of differentially expressed genes in tumors helps to elucidate the molecular mechanism of tumorigenesis, contribute to the diagnosis and prognosis of tumors, and provide new targets for tumor treatment, thus becoming the basis of gene medicine and gene therapy. There are many ways to find differentially expressed genes. The high-throughput screening methods developed in recent years have injected new vitality into this research. In recent years, the research results of disease-related genes reported in the literature, a large part of them are high-throughput screening methods. The method was screened out and validated in the follow-up research. High-throughput methods mainly include serial analysis of gene expression (SAGE), cDNA microarray, oligo-nucleotides microarray, and suppression subtractive hybridization (SSH). ), differential display (differential display, DD), etc. Moreover, in order to improve the treatment effect of lung cancer, many studies are currently devoted to the early diagnosis of lung cancer and the targeted therapy guided by molecular classification. With the emergence and development of systematic genome, transcriptome, and proteome research methods and technology platforms, people's understanding of the occurrence and development of lung cancer and other tumors continues to advance, trying to carry out accurate molecular genetic typing of lung cancer and other tumors, and looking for Molecular markers for early diagnosis, prediction of individual sensitivity to radiotherapy and chemotherapy, accurate judgment and improvement of the prognosis and quality of life of lung cancer patients.

Molecular biology studies of lung cancer have shown that the occurrence and development of lung cancer is a multi-stage, multi-step, multi-gene process involving changes in the structure and expression regulation of a large number of related genes. Therefore, it is of great significance and feasibility to understand the precise molecular mechanism of lung cancer and to find molecular markers closely related to the early detection of lung cancer. Among the many genetic changes in lung cancer, the inactivation of tumor suppressor genes plays an important role. One of the earliest genetic changes in the development of lung cancer is the functional or constitutive loss of chromosome 3p (short arm of chromosome 3). In 30% of lung cancer patients with 3p abnormality, the same change has appeared in the non-malignant bronchial epithelium. Studies have found that chromosome 3p contains multiple tumor suppressor genes, such as RAR (retinoic acid receptor beta) at 3p24, RASSF1A (Ras association domain family I) at 3p21.3, FHIT (fragile histidinetriad) at 3p14.2, and There are CACNA2D2, 101F6, NPRL2, SEMA3B, SEMA3F, FUS1, DLEC1, RBSP3A, RBSP3B, etc. (Zaborovsky, 2005). In addition, chromosomal segments such as 1p, 1q, 5q (APC/MCC cluster), 8p, 9p21 (p16INK4), 11p13 (WT1), 11p15, 13q14 (RB), 17p13 (p53), and 22q are also frequently involved in the process of carcinogenesis , which also contains a number of genes with tumor suppressor functions, which are inactivated through deletion, mutation, methylation, etc., change the characteristics of cell growth and differentiation, and promote the occurrence of cancer.

In the past, it was very difficult to conduct research on a new gene, but now we can flexibly use the laws of bioinformatics to mine as much information as possible to guide further experimental research. DENN domain (differentially expressed in neoplastic versus normal cells domain) exists in a variety of signaling proteins, but the mystery of its function has not been fully solved. Some proteins containing DENN domain, such as Rab3GDP/GTP exchange protein of rat, Rab6IP1 (Rab6 interacting protein 1) of mouse and AEX-3 protein of Caenorhabditis elegans can interact with GTPases of Rab family. Generally speaking, there are uDENN and dDENN domains around the DENN domain, which may be crucial for the function of DENN. In addition to the core hydrophobic amino acid sequence, there are some conserved amino acid sequences in the DENN domain, which may be related to its enzymatic activity. The appearance of the DENN domain in proteins such as Rab3GEP indicates that it may have the activity of guanylate exchange, but this inference has not been confirmed yet.

Contents of the invention

The technical problem to be solved by the present invention is to provide a tumor-associated protein and its coding gene.

The tumor-associated protein provided by the present invention is derived from humans, and the tumor-associated protein is a protein of (a) or (b) as follows:

(a) a protein consisting of the amino acid residue sequence of sequence 4 in the sequence listing;

(b) The amino acid residue sequence of sequence 4 in the sequence listing is subjected to the substitution and/or deletion and/or addition of one or several amino acid residues, and a protein derived from (a) associated with tumors.

The name of the protein in (a) is DENND2D-v2, which consists of 468 amino acid residues, amino acid residues 47 to 145 at the amino terminal of sequence 4 are uDENN domains, and amino acid residues 146 to 330 at the amino terminal of sequence 4 The first amino acid residue is a DENN structural domain, and the 368th to 435th amino acid residues from the amino terminal of sequence 4 are a dDENN structural domain.

The substitution and/or deletion and/or addition of one or several amino acid residues refers to the substitution and/or deletion and/or addition outside the above three domains of Sequence 4.

The amino acid sequence of the protein in (b) may specifically be sequence 3 in the sequence listing. The name of the protein is DENND2D-v1, which consists of 471 amino acid residues, the amino acid residues from the 5th to the 148th amino-terminal of the sequence 3 are uDENN domains, and the amino acid residues from the 149th to the 333rd amino-terminal of the sequence 3 are The DENN structural domain, the 371st to 438th amino acid residues from the amino terminal of sequence 3 are dDENN structural domains.

The genes encoding the above-mentioned tumor-associated proteins also belong to the protection scope of the present invention.

The cDNA gene of the tumor-associated protein is specifically the gene of the following 1) or 2):

1) its nucleotide sequence is sequence 1 or sequence 2 in the sequence listing;

2) A DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the tumor-associated protein.

The stringent conditions may be hybridization at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, and the solution is used to wash the membrane.

Sequence 1 in the sequence listing consists of 2062 deoxyribonucleotides, its coding sequence is from the 201st to 1616th deoxyribonucleotides at the 5' end of sequence 1, and the coding amino acid sequence is DENND2D-v1 of sequence 3.

Sequence 2 in the sequence listing consists of 1905 deoxyribonucleotides, its coding sequence is from the 57th to 1463rd deoxyribonucleotides at the 5' end of sequence 2, and the coding amino acid sequence is DENND2D-v2 of sequence 4.

The genomic gene chromosome location of DENND2D-v1 and DENND2D-v2 is 1p13.3. DENND2D-v1 spans the human genome at about 13.48kb, and DENND2D-v2 spans the human genome at approximately 17.23kb. Both contain 12 exons and share the 2- 12 exons, the first exon is different.

The genes encoding the above tumor-associated proteins can be inserted into existing prokaryotic or eukaryotic expression vectors, and suitable vectors include bacterial plasmids, adenoviruses, adeno-associated viruses, retroviruses, and lentiviruses. The recombinant expression vector containing the gene encoding the above-mentioned tumor-associated protein can be used to transform appropriate host cells, such as Escherichia coli, yeast, insect cells, mammalian cells, etc., so that the host can express the polypeptide.

Recombinant expression vectors, transgenic cell lines and transgenic host bacteria containing the above tumor-related protein coding genes also belong to the protection scope of the present invention.

The above-mentioned tumor-associated proteins, or conservatively mutated polypeptides or fragments, or cells expressing them can be used as antigens to produce antibodies, and are used clinically for clinical diagnosis, treatment, and curative effect evaluation of tumors. The antibody can be a monoclonal antibody or a polyclonal antibody, and also includes chimeric, single-chain and humanized antibodies and Fab fragments, or products of a Fab expression library. Monoclonal or polyclonal antibodies to the above-mentioned tumor-associated proteins can be prepared by existing methods, such as immunizing animals and culturing hybridomas. The antibody can be used to detect the presence or level of the aforementioned tumor-associated protein, or to detect the gene of the aforementioned tumor-associated protein.

The third object of the present invention is to provide an antitumor drug.

The antitumor drug provided by the present invention, its active ingredient is a recombinant mammalian gene expression vector containing the above-mentioned tumor-related protein coding gene.

The starting vector for constructing the recombinant expression vector can be an existing mammalian gene expression vector.

The mammalian gene expression vectors include two categories of plasmid vectors and viral vectors. For example, plasmid expression vectors pTK2, pHyg, and pRSVneo without eukaryotic replication initiation sequences, plasmid expression vectors with eukaryotic regulatory sequence elements, expression vectors pMSG, pSVT7, and pMT2 derived from SV40, bovine papillomavirus ( BPV)-derived vectors, human herpesvirus (EBV)-derived expression vectors, human adenovirus-derived expression vectors, retrovirus-derived expression vectors.

The antineoplastic drug can be used for preventing and/or treating upper aerodigestive tract tumors such as lung cancer, esophageal cancer or laryngeal cancer.

The above-mentioned drugs may also contain adjuvant DDA and/or MPL and/or Quil-A and/or RIBI adjuvant and/or saline (physiological saline) or other adjuvants, such as aluminum adjuvant, Freund's adjuvant and the like.

The medicine of the present invention can be made into various forms such as injection, powder, ointment, nano preparation and the like. The above-mentioned medicines in various dosage forms can be prepared according to the methods in the field of gene medicine/oligonucleotide medicine.

The stable and high expression of the tumor-associated protein of the present invention in mouse fibroblasts can significantly inhibit the malignant transformation of the cells; the results of detecting its expression in cell lines at the mRNA level show that the expression level in lung cancer cell lines is significantly lower than that of the original Subcultured normal bronchial epithelial cells; detection of its expression level in lung cancer clinical tissue specimens showed that the expression level of lung cancer tissue was significantly lower than that of its surrounding normal tissues. The tumor-associated protein of the present invention and its coding gene, the antibody of the present invention and the appropriate carrier expressing the tumor-associated protein of the present invention can be used for in vitro diagnosis and prognosis of tumors. After the recombinant eukaryotic expression vector containing the above-mentioned tumor-related protein coding gene is introduced into the lung cancer cell line, it can independently cause the growth slowdown and tumorigenicity weakening of the lung cancer cell line. The invention provides a new basis for creating new clinical diagnosis, treatment, curative effect evaluation and prognosis index.

Description of drawings

Figure 1a shows the partial forward sequencing results of the pGEM-T Easy-DENND2D-v1 vector

Figure 1b shows the partial forward sequencing results of pcDNA3.1/V5-His TOPO TA-DENND2D-v2 vector

Figure 2 is the physical map of the eukaryotic expression vector pcDNA3.1/mycHis(-)B

Figure 3 is the expression of DENND2D-v2 gene in lung cancer related cell lines

Figure 4 is the RT-PCR analysis of the expression changes of DENND2D-v2 gene in lung cancer tissues

Figure 5 is a schematic diagram of DENND2D-v2 antibody specificity and main nonspecific binding

Figure 6 shows that DENND2D-v2 inhibits NIH/3T3 cells from forming colonies

Figure 7 is the H1299 monoclonal cell line that stably and highly expresses DENND2D-v2 identified by RT-PCR

Figure 8 is the Western Blot identification of H1299 monoclonal cell lines stably and highly expressing DENND2D-v2

Figure 9 is the H1299 stable transfection cell MTT cell proliferation experiment

Figure 10 is a representative diagram of H1299 stably transfected cell colony formation experiment

Figure 11a is the soft agar colony formation experiment of H1299 stably transfected cells (observed with the naked eye)

Figure 11b is the soft agar colony formation experiment of H1299 stably transfected cells (observed under light microscope)

Figure 12a is a photo of tumor formation in nude mice after cell injection

Figure 12b is a schematic diagram of tumor growth curves of nude mice in each group within 3-6 weeks after subcutaneous injection of cells

Figure 13 shows the cell apoptosis detected by flow cytometry 36 hours after transfection of H1299 cells with pcDNA3.1/mycHis(-)B, pcDB3.1-DENND2D-v2, and pcDB3.1-BAX

Figure 14 is the result of flow cytometry detection of the effect of DENND2D-v2 gene on H1299 cell cycle

Figure 15a is the expression of RT-PCR detection of cell cycle-related regulatory factors

Figure 15b is the expression of cell cycle-related regulatory factors detected by Western method

Figure 16 is a scratch test to detect the effect of DENND2D-v2 gene on cell migration

Figure 17 shows the effect of DENND2D-v2 gene on the migration of H1299 cells

Figure 18 is the effect of DENND2D-v2 gene on the invasiveness of H1299 cells

Detailed ways

Cloning of embodiment 1, DENND2D-v2 and DENND2D-v1 and cDNA gene thereof

The specific primer sequences for the full-length reading frames of the DENND2D-v1 and DENND2D-v2 genes were respectively designed for the sequences 1 and 2 in the sequence list as follows:

DENND2D-v1:

Upstream primer 5'CCAGAGATGGAAGGACAAGTG3'

Downstream primer 5'GTCATTCTTATTCACCACAGCTC3'

DENND2D-v2:

Upstream primer 5'CACTCCAGGGGCCATGGATG3'

Downstream primer 5'GTCATTCTTATTCACCACAGCTC3'

Using the above primers, the human normal lung tissue cDNA library (Clontech: K1420-1) was used as a template for PCR amplification reaction, and the reaction conditions were as follows:

The reaction volume is 50 μl, which contains:

Human normal lung tissue cDNA template 5μl (5ng)

Primers The final concentration of forward primer and reverse primer is 0.2 μM each

dNTP final concentration 200μM each

Taq DNA Polymerase 2.5U

10×Taq DNA polymerase buffer 5μl

Make up to 50 μl volume with double distilled water.

Reaction temperature and time: 94°C, denaturation for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 1 minute, and 30 cycles of amplification; finally, extension at 72°C for 10 minutes.

The amplified product is a 3' protruding sticky-end fragment with a base A in the 3', purified with a QIAquick gel recovery kit (Qiagen, 28706) according to the product instructions, and then combined with a linear pGEM-T EASY with a base T in the 3' Carrier (Promega, A1360) was ligated at 16°C for 8 hours, using a 2mm electrode cup, 2500V to transform E. coli DH5α, the transformant was grown on LB plate medium containing ampicillin, clones were selected, plasmids were extracted, and AbI PRISM 3700 DNA was used Analyzer (Perkin-Elmer/Applied Biosystem) sequenced to obtain the full-length cDNA of DENND2D-v1 and DENND2D-v2 genes, the lengths were 2062bp and 1905bp (sequences such as sequence 1, 2), respectively encoding 471 and 468 amino acids, amino acid The sequence is shown in sequence 3,4. The pGEM-T EASY vectors containing the cDNA genes of sequences 1 and 2, namely DENND2D-v1 and DENND2D-v2, were named pGEM-T-DENND2D-v1 and pGEM-T-DENND2D-v2, respectively. The sequencing results of the pGEM-T-DENND2D-v1 vector are shown in Figure 1a.

In order to further test the functions of DENND2D-v1 and DENND2D-v2 genes, a eukaryotic expression vector containing DENND2D-v1 or DENND2D-v2 cDNA was then constructed. The eukaryotic expression vector pcDNA3.1B-DENND2D-v1 (hereinafter abbreviated as pcDB-DENND2D- v1). pcDB is a vector designed for high expression of recombinant proteins in mammalian cell lines. It contains the human cytomegalovirus CMV promoter and can achieve high expression in mammalian cell lines. The cDNA gene fragment of DENND2D-v1 was excised from the pGEM-T-DENND2D-v1 vector with EcoRI (Promega), and the eukaryotic expression vector pcDNA3.1/mycHis(-)B (Invitrogen, V85520) was digested with EcoRI at the same time ( Figure 2), according to J.Sambrook et al., Molecular Cloning Experiment Guide Third Edition, pages 68 to 71, the digested DENND2D-v1 cDNA gene fragment was connected to the vector at 16°C for 8 hours, and transformed into Escherichia coli DH5α, the transformant was grown on the LB plate medium containing ampicillin, the grown colony was selected, the plasmid was extracted, digested with EcoRI, and the digested product was identified by agarose gel electrophoresis, and the positive clone with the inserted 2062bp fragment was selected, passed Sequence determination (using ABI PRISM 3700 DNA analyzer, same as above), select the correct cDNA gene plasmid with forward insertion sequence 1, named pcDB-DENND2D-v1. The cDNA gene fragment of DENND2D-v2 (1905bp) is the sequence 2 obtained by PCR amplification. After recovery, it is directly forward-connected into the pcDNA3.1/V5-His TOPO TA (Invitrogen, K4800) eukaryotic expression vector and bidirectionally sequenced The cDNA gene plasmid with positive insertion sequence 2 was obtained and named pcDNA3.1/V5-His TOPO TA-DENND2D-v2, referred to as pcDB3.1-DENND2D-v2. The sequencing results of the pcDNA3.1/V5-His TOPOTA-DENND2D-v2 vector are shown in Figure 1b.

Example 2, RT-PCR detection of the expression levels of DENND2D-v1 and DENND2D-v2 genes in lung cancer-related cell lines and lung cancer tissues

In order to detect the difference in the expression of DENND2D-v1 and DENND2D-v2 genes between tumor and normal tissue cells, this example extracted the total RNA of lung cancer-related cell lines, and used RT-PCR to detect the changes in mRNA transcription.

1. Expression levels of DENND2D-v1 and DENND2D-v2 in lung cancer-related cell lines

Detect the following lung cancer related cell lines: PAa (human lung adenocarcinoma cell line), A549 (human lung adenocarcinoma cell line, ATCC, CCL-185), GLC-82 (human lung adenocarcinoma cell line), LTEP (human lung adenocarcinoma cell line), cancer cell line), H520 (human lung squamous cell line, ATCC, HTB-182), H2170 (human lung squamous cell line, ATCC, CRL-5928), PG (human lung large cell carcinoma cell line), H460 ( Human lung large cell carcinoma cell line, ATCC, HTB-177), H1299 (human lung large cell carcinoma cell line, ATCC, CRL-5803), Y-BE111 (human immortalized lung bronchial epithelial cell line Y-BE111 generation), M-BE37 (human immortalized lung bronchial epithelial cell line M-BE 37th generation), M-BE206 (human immortalized lung bronchial epithelial cell line M-BE 206th generation), and 15 cases of primary cultured normal bronchial epithelial cells.

(1) The in vitro culture of lung cancer-related cell lines and cells and the extraction of total RNA: the cells of each cell line were spread on a 10 cm plate at a rate of 1.5×10 ⁶ cells/dish, and were mixed with DMEM containing 10% fetal bovine serum (Dulbecco's modified Eagle's medium) medium (Hyclone, SH0022.02) to cultivate PAa, A549, G12-82, LTEP, H520, H2170, pG, H460 and H1299, and cultivate Yp111, Mp37, Mp206 and 15 primary cultures in serum-free conditioned medium normal bronchial epithelial cells. Cells grow to the exponential growth phase, when the confluence rate is about 80-90%, trypsinization, serum terminated. Wash the cells twice with PBS, centrifuge at 1,000 g for 5 minutes, and discard the supernatant. Add 2ml TRIzol reagent to each plate, place at room temperature for 3-5 minutes, blow and mix well, transfer the suspension into a 7ml centrifuge tube, and incubate at room temperature for 5 minutes. Add 400 μl of chloroform (1/5 TRIzol volume) to each tube, invert to mix, centrifuge briefly, and place at room temperature for 5 minutes. Centrifuge at 12,000g at 4°C for 15 minutes, carefully transfer the upper colorless aqueous phase to a new 7ml centrifuge tube (do not touch the middle protein phase), add 1ml isopropanol (1/2 TRIzol volume) to each tube, and place at room temperature for 10 minute. Centrifuge at 12,000g for 10 minutes at 4°C. Remove the supernatant, wash each tube with 2ml of 75% ethanol (prepared with 1 times the volume of TRIzol in DEPC water), and centrifuge at 7000g for 5 minutes at 4°C. repeat. Try to remove ethanol as much as possible, and dry at room temperature for 15-20 minutes on a super-clean bench. Add appropriate amount of pure water to remove RNase and DNase to each tube depending on the amount of RNA precipitation. Leave it for a while to fully dissolve, store at -80°C or directly enter the reverse transcription step.

(2) Quantification of RNA: measure OD260 and OD280 values with a UV spectrophotometer, and calculate the concentration of RNA; take 1 μg of RNA, perform 1% agarose gel electrophoresis, observe the results under ultraviolet light, and identify the quality of RNA.

(3) RT-PCR:

1) Prepare the RNA/primer reaction system and operate on ice:

Oligo dT(12-18)(0.5μg/μl) 1μl

Random Primer(0.5μg/μl) 0.5μl

Total RNA (determine the volume according to the concentration) 5 μg

Nuclease Free _H2O to 10μl

Mix well, bathe in 70°C water for 5 minutes, immediately place on ice for 2 minutes, and collect to the bottom of the tube by instantaneous centrifugation;

2) Prepare the following mixed system and operate on ice:

5×First Strand Buffer 4μl

0.1M DTT 2μl

dNTP mix(2.5mM each) 1μl

RNase inhibitor 1 μl

Nuclease Free H ₂ O 1μl

Add this mixed reaction system into the RNA/primer sample mixed system, mix gently, and collect it to the bottom of the tube by centrifugation, place at 25°C for 10 minutes, and then bathe in 42°C for 2 minutes. Add 1 μl SuperScript II reverse transcriptase to each tube, mix gently, and centrifuge briefly to collect to the bottom of the tube, and react in a water bath at 42°C for 1 hour. The reaction was terminated in a 70°C water bath for 15 minutes. Reverse transcription products were stored at -80°C, or directly into the PCR step.

3) PCR reaction: multiplex PCR was used, and GAPDH was used as the internal control of the PCR reaction system.

① Primer and product length: Table 1 gene name upstream primer downstream primer product length DENND2D-v1 5'CCAGAGATGGAAGGACAAGTG3' 5'CTCCCATCCACATTGGTCAG3' 401bp DENND2D-v2GAPDH 5'TCGAGCCAGCCTGAGACTGAAG3'5'TGAAGGTCGGAGTCAACGGATTTGGT3' 5'CTCCCATCCACATTGGTCAG3'5'CATGTGGGCCATGAGGTCCACCAC3' 363bp983bp

②PCR reaction system

ddH ₂ O 13 μl

10×Ex Taq buffer 2μl

dNTP mix(2.5mM each) 2μl

DENND2D-v1 or DENND2D-v2 primer (10μM) 1μl

GAPDH Primer (10μM) 0.5μl

cDNA template 1 μl

Ex Taq 0.5μl

③PCR reaction conditions: 94°C for 5 minutes; 94°C for 30 seconds, 57°C for 30 seconds, 72°C for 40 seconds (35 cycles); 72°C for 10 minutes; store at 4°C.

The amplification results showed that except for H2170, the expression of DENND2D-v2 mRNA in 8 lung cancer cell lines H520, LTEP, A549, PAa, GLC-82, H1299, PG and H460 were all missing (1/9 11.1%), 15 cases DENND2D-v2 mRNA in primary cultured normal bronchial cells (1N-15N) was still retained (15/15 100%), and in the immortalized bronchial epithelial cell line M-BE37 (early generation ), M-BE206 (late generation), Y-BE111 DENND2D-v2 mRNA expression loss (0/30%), H1299-pcDB (transformed pcDNA3.1/mycHis(-)B prepared according to the method of Example 5 H1299 cells) was a negative control, and SC3-SC5 (the H1299 cell line stably overexpressing DENND2D-v2 prepared according to the method of Example 5) was a positive control after transfection, as shown in Figure 3 shown. The results indicated that the DENND2D-v2 gene was normally expressed in the normal origin tissue of lung cancer, but deexpression occurred in the early stage of lung cancer. In Figure 3, M37 represents M-BE37, M206 represents M-BE206, Y111 represents Y-BE111, L represents LTEP, and DENND2D represents DENND2D-v2.

However, DENND2D-v1 mRNA was not expressed in either normal bronchial epithelial cells or lung cancer-related cell lines. It is speculated that this phenomenon may be determined by tissue-specific expression, so DENND2D-v2 was selected as the research object in subsequent experiments.

2. The expression level of DENND2D-v2 in clinical lung cancer tissues and paired normal tissues:

(1) Source of tissue samples: Tissue samples from 27 patients with lung squamous cell carcinoma admitted to the Department of Thoracic Oncology Surgery, Cancer Hospital, Chinese Academy of Medical Sciences, including tumor tissue and adjacent normal control lung tissue. All patients had not received physical and chemical treatment when the specimens were obtained. All samples were identified with tumor tissue purity greater than 50%. Comprehensively collect clinical data of patients, review all histopathological sections, uniformly evaluate the degree of tissue differentiation (divided into high, medium, and poor differentiation), verify the records of lymph node metastasis, and carry out clinical staging according to the TNM staging standard of the sixth edition of UICC in 2002 (Sobin , 2002). Among the patients, there were 26 males and 1 female, ranging in age from 44 to 76 years, with a median age of 63 years and an average age of 59.7 years.

(2) Extraction of total tissue RNA: Take an appropriate amount of tissue sample (about 0.5mm ³ ), wrap it in aluminum foil and freeze it in liquid nitrogen for more than 5 minutes, knock the sample to crush it, and pour the debris into the RNase removal In a prepared mortar. Add 4ml TRIzol reagent to each sample, continue grinding and digestion on ice for about 5 minutes, pipette the lysate into a new 10ml tube, and react at room temperature for 5 minutes. Subsequent steps are the same as the cellular RNA extraction part in Example 2.

(3) RNA quantification and RT-PCR reaction are the same as before: GAPDH is used as an internal control, and the primer sequences and product lengths are as follows: Table 2 gene name upstream primer upstream primer product length GAPDH (lung cancer tissue experiment) 5'GAAGGTGAAGGTCGGAGTC3' 5'GAAGATGGTGATGGGATTTC3' 226bp

(4) Results: From the RT-PCR results of 27 pairs of paired tissues, it can be seen that the mRNA expression of DENND2D-v2 gene in lung squamous cell carcinoma tissues was significantly reduced or even completely absent compared with adjacent normal lung tissues, and the incidence rate was 48.1% ( 13/27), 8 of the remaining 14 pairs of tissues had no significant difference in expression, accounting for 29.6% (8/27); 2 pairs had no expression, accounting for 7.4% (2/27); The level of DENND2D-v2mRNA was higher than that in adjacent normal lung tissue, accounting for 14.8% (4/27). As shown in Figure 4, the paired tumor and adjacent normal lung tissues are represented by T and N, respectively; DENND2D represents DENND2D-v2.

Example 3. In order to detect the expression of DENND2D-v2 protein, Anti-DENND2D-v2 antibody was prepared

The GPA project team of Beijing Institute of Genomics, Chinese Academy of Sciences was entrusted to prepare and purify DENND2D-v2 polyclonal rabbit antibody, and the antigen used was purified GST-DENND2D fusion protein. Antibody specificity was identified by Western Blot method. The specific operation process is as follows:

1. Expression of DENND2D-v2 protein

Using primers Sense: 5'CCGGAATTCATGGATGGGCTCGGCCGCC3' and Antisense: 5'CCGCTCGAGCGGTTATTCACCACAGCTCTTA3', use pcDB3.1-DENND2D-v2 as a template to PCR amplify the fragment containing sequence 2, respectively digest PCR products with EcoRI and XhoI (Promega) and express in prokaryotic Carrier PGEX-4T-1 (Amersham), recover the digested product, and connect to obtain the pGEX-DENND2D-v2 fusion protein expression vector containing the DENND2D-v2 gene (the N-terminus of the DENND2D-v2 protein is fused with a GST tail), and the vector sequence is sequenced Validation is correct. Concrete digestion, recovery, and connection process are the same as in Example 1. The obtained plasmid was transformed into Escherichia coli strain BL21, cultivated at 37° C. at 220 rpm, induced expression with 0.5 mM IPTG (Takara) for 3.5 hours, lysed the bacteria, and purified the protein with a GST purification column (Amersham) to obtain a purified fusion protein greater than 1 mg. It acts as an antigen.

2. Preparation of DENND2D-v2 rabbit polyclonal antibody

Adult male New Zealand rabbits were selected for immunization. For the first immunization, 200ug antigen (0.1ml) was fully emulsified with an equal volume of Freund's complete adjuvant (FCA), and injected subcutaneously at multiple points on the back. 21, 42, and 63 days after the initial immunization, the antigen protein fully emulsified with Freund's incomplete adjuvant (FIA) was boosted once each, and the dosage was the same as before. 7-10 days after each immunization, the DENND2D-v2 expressed in step 1 was used as the antigen, and the serum titer was detected by ELISA method. When it reached 1×10 ^-4 , the serum was bled to separate. The results of Western blot identification of antibody specificity showed that the polyclonal antibody obtained from DENND2D-v2 had poor specificity, as shown in Figure 5, but it could be used in Western blot experiments. In Figure 5, H1299-empty means the total protein of H1299 cells transfected with pcDNA3.1/mycHis(-)B, and H1299-DENND2D-v2 means the total protein of H1299 cells transfected with pcDB3.1-DENND2D-v2. H1299-DENND2D-v1 represents the total protein of H1299 cells transfected with pcDB-DENND2D-v1.

Example 4. In vitro experiment of DENND2D-v2 inhibiting spontaneous transformation of NIH/3T3 cells

NIH/3T3 cell (ATCC, CRL-1658) is a mouse embryonic fibroblast cell line on the verge of transformation, sensitive to external stimuli, and is an ideal cell model for studying the function of genes promoting or inhibiting transformation. Therefore, this example uses it to prove that the DENND2D-v2 gene can inhibit its ability of spontaneous malignant transformation.

(1) Establishment of NIH/3T3 cell line stably transfected with DENND2D-v2: NIH/3T3 cell line was cultured according to ATCC culture requirements. The day before transfection, 2×10 ⁵ cells/well were spread on a 6-well plate, and the empty vectors pcDNA3.1/mycHis(-)B and pcDB3.1-DENND2D-v2 were respectively transfected the next day, and transfected according to Lipfectamin ^TM 2000 Instructions are carried out. 24 hours after transfection, the cells were digested, and the cells were subcultured according to the ratio of 1 to 5. Each group of cells had 2 wells, and 2ml of medium was mixed and cultured; after 18 hours, fresh medium was replaced, and Geneticin was added to a final concentration of 800μg/ml; Change the fresh medium every day and maintain the Geneticin concentration until the selected colony grows, about 3 weeks; crystal violet staining: take out the culture plate, discard the medium, wash twice with PBS, and fix with ice-cold methanol at -20°C for 30 minutes; Aspirate methanol, add 0.5% crystal violet (prepared with 25% methanol solution, just cover the bottom of the petri dish), let stand at room temperature for 30 minutes, recover crystal violet, carefully rinse with deionized water until the color no longer changes, and leave at room temperature to dry. After three weeks of antibiotic selection, cells transfected with the control empty vector pcDNA3.1/mycHis(-)B formed obvious transformed colonies, while cells transfected with pcDB3.1-DENND2D-v2 had no obvious colony formation, indicating that the gene It has the effect of inhibiting the formation of transformed colonies. The results are shown in Figure 6, NIH3T3-VECTOR represents pcDNA3.1/mycHis(-)B transfected cells, NIH3T3-DENND2D represents pcDB3.1-DENND2D-v2 transfected cells.

Example 5. In vitro experiment of DENND2D-v2 gene's ability to inhibit the growth of lung cancer cell lines

(1) The H1299 cell line stably overexpressing DENND2D-v2 was obtained by the limiting dilution method: the cells were transferred to a 24-well cell culture plate at a ratio of 4×10 ⁴ /well, 5 wells in total. Replace the fresh culture medium one hour before transfection, and transfer the following plasmids according to the instructions of Lipofectamine 2000 transfection reagent: empty vector pcDNA3.1/mycHis(-)B, pcDB3.1-DENND2D-v2, and the remaining one well is untransfected Stained cells control. After 48 hours, the cells were digested, and each group of cells was transferred to four columns in a 96-well plate according to a gradient of 2, 5, and 10 cells/well. Add Geneticin to a screening concentration of 800 μg/ml; at the beginning, observe the cell death every day, replace the fresh medium every three days, and maintain the screening pressure. After about 7-10 days, the screened clones can be seen growing out, and the wells of the single clones are marked , about 3-4 weeks (the cells are nearly full of pores), digest the efferent cells, and maintain the selection pressure.

(2) Identification of H1299-DENND2D stable cell line

1) RT-PCR: extract the total RNA of the pcDNA3.1/mycHis(-)B control cells and the pcDB3.1-DENND2D-v2 candidate clone cells, quantify according to the concentration, and detect the expression of DENND2D-v2 by RT-PCR , the primer sequence is shown in Table 1 in Example 2, and the results are shown in Figure 7. H1299-vector represents the negative control of empty vector pcDNA3.1/mycHis(-)B transfection, and DENND2D-1, -2, -3 represent the selection Three monoclonal cell lines stably expressing DENND2D-v2 mRNA, DENND2D expresses DENND2D-v2.

2) Western Blot: Extract the total protein of the pcDNA3.1/mycHis(-)B control cells and candidate clone cells, quantify the above proteins, and perform Western blot analysis at 60 μg/sample, unpurified DENND2D-v2 polyclonal rabbit antibody Add according to 1:500. The results are shown in Figure 8, DENND2D represents DENND2D-v2, H1299-vector represents the negative control of empty vector pcDNA3.1/mycHis(-)B transfection, DENND2D-1, 2, 3, 4, 5, 6, 7, 8 represents eight H1299 monoclonal cell lines stably and highly expressing DENND2D-v2, and three were selected for further experiments, named H1299-DENND2D-1, H1299-DENND2D-2 and H1299-DENND2D-3.

(3) MTT cell proliferation experiment: H1299 cells (called H1299-vector cells) transfected with pcDNA3.1/mycHis(-)B were used to screen H1299-DENND2D-1 and H1299-vector cells stably expressing DENND2D-v2. Three monoclonal cell lines DENND2D-2 and H1299-DENND2D-3 were tested. MTT operation method is carried out according to CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega, G4000). The results are shown in Figure 9, the growth rate of H1299-DENND2D-1, H1299-DENND2D-2, H1299-DENND2D-3 monoclonal cell lines stably and highly expressing DENND2D-v2 was significantly slower than that of empty vector transfected cells. Each point in the figure is the average number of duplicate wells.

(4) Cell colony formation experiment: Three monoclonal cell lines, H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3 stably expressing DENND2D, were screened for experiments using empty vector H1299-vector cells. The cells were routinely cultured to the logarithmic growth phase, the cells were digested, and 500 cells per well were seeded into 60mm cell culture dishes, and each experiment was repeated for three dishes; the medium was changed twice a week, and the colony formation was observed. After 10-14 days, discard the medium, wash twice with PBS, fix with ice-cold methanol at -20°C for 30 minutes; discard the methanol, add 0.5% crystal violet to stain at room temperature for 30 minutes, wash off the stain and dry at room temperature. Counting standard: colony diameter greater than 1mm. The results are shown in Figure 10 and Table 3. The number of colonies and the size of a single colony formed by H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3 stably and highly expressing DENND2D-v2 were significantly larger than those transfected with the empty vector. Cells are small. Figure 10 is a picture of a representative experiment, and Table 3 is a schematic diagram showing the comparison of the results of three parallel experiments.

Table 3. Number of cell colonies formed Colony formation number (unit/500 cells) H1299-vector H1299-DENND2D-1 H1299-DENND2D-2 H1299-DENND2D-3 first hole 157 45 7 15 second hole 126 61 12 17 third hole 152 48 10 18 average 145 51 10 17

In FIG. 10, Vector represents H1299-vector cells, and DENND2D-1, DENND2D-2, and DENND2D-3 represent H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3, respectively.

(5) Soft agar colony formation experiment: three monoclonal cell lines, H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3 stably expressing DENND2D-v2, were screened using empty vector H1299-vector cells conduct experiment. The experiment was carried out in a six-well plate, and 1.5 ml of DMEM medium containing 10% fetal bovine serum and 0.5% agar was added to each well. Fetal bovine serum, 0.35% agarose in DMEM medium, mix and add to the above bottom medium, culture for 2-3 weeks, add 0.5ml0.2% P-iodonitrontetrazilium chiloride to each dish for staining for more than 1 hour; observe under the microscope Photographs were taken and colonies larger than 200 microns were counted. Three wells were replicated for each group. Figure 11a and 11b are the representative images observed with the naked eye and under a light microscope, and the comparison of the colony formation results of the three parallel experiments is shown in Table 4, indicating that the ability of the cells transferred to DENND2D-v2 to grow independently in soft agar was significantly weakened . In Figure 11b, Vector represents H1299-vector cells, and DENND2D-1, DENND2D-2, and DENND2D-3 represent H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3, respectively.

Table 4. Clones larger than 200 µm Number of clones larger than 200 microns (pieces) H1299-vector H1299-DENND2D-1 H1299-DENND2D-2 H1299-DENND2D-3 first hole 136 1 1 twenty four second hole 165 4 2 50 third hole 110 1 0 25

Example 6. In vivo experiment of DENND2D-v2 inhibiting tumorigenicity of H1299 cells:

Parental cells (untransfected H1299 cells) and established stable transfection cell lines H1299-vector, H1299-DENND2D-1, H1299-DENND2D-2, H1299-DENND2D-3 (representing three stable monoclonals) were respectively According to 5×10 ⁵ cells/mouse, dilute in 200 microliters of 1×PBS, and inject subcutaneously on the back of four-week-old, Balb/c female nude mice. There were 8 rats in the Parental group, 8 rats in the vector group (injected with H1299-vector), and 6 rats in each of the H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3 groups. The animals were observed every three days, and small tumors began to grow at about three weeks. Measure the length and width of the tumor with calipers. After 21 days, the animals were sacrificed by necking, the capsule was carefully peeled off to take out the tumor, and the weight of the tumor was measured. The tumor mass was fixed in 10% formalin, embedded in paraffin, sectioned, and stained with HE for pathological analysis. It was large cell lung cancer. Tumor volume was calculated with the formula: V=(π/6)×((length+width)/2) ³ . The results are shown in Figure 12a and Figure 12b. DENND2D-v2 gene has the ability to inhibit H1299 cells from forming tumors in nude mice subcutaneously. Tumors grew at the cell injection site, and the tumorigenic rate reached 100% (8/8), while only some nude mice in the three monoclonal cell groups had tumors, among which H1299-DENND2D-1 83.3% (5/6), H1299-DENND2D-2 0% (0/6), H1299-DENND2D-3 33.3% (2/6). In addition, the DENND2D-v2 gene also reduced the volume of the tumor. After the nude mice were sacrificed, the tumor was stripped and weighed. It was found that the average weight of the tumor formed by the empty vector cell group was 0.49 g, while that of the three monoclonal cell groups was 0.25 g, 0g, 0.02g. Figure 12b shows the tumor formation of nude mice in each group within 3-6 weeks of subcutaneous injection, and every 3 days is used as an observation time point, and each time point shows the average value of multiple tumor volumes of nude mice in each group.

Example 7. Study on the mechanism of DENND2D-v2's growth inhibitory effect on lung cancer cell line H1299

1. Observation and detection of apoptosis:

(1) Construction of positive control plasmid pcDB3.1-BAX plasmid

pcDB3.1-BAX is the positive control of the apoptosis experiment, which is constructed according to the following method: Using the human normal lung tissue cDNA library (Clontech: K1420-1) as a template, the full-length cDNA gene of BAX is amplified by PCR reaction using Taq enzyme (Genbank No. NM_138761). The PCR product was recovered by agarose electrophoresis, connected to the pGEM-T-easy vector for sequencing, and the sequenced correct fragment was excised from the pGEM-T-Bax vector with the restriction endonuclease EcoRI (Promega), and digested with EcoRI at the same time The eukaryotic expression vector pcDNA3.1/mycHis(-)B (Invitrogen, V85520), ligated the cDNA gene fragment of Bax after digestion with the vector at 16°C for 8 hours, and transformed Escherichia coli DH5α, and the transformant was treated with ampicillin Grow on the LB plate medium, select the growing colonies, extract the plasmid, digest with EcoRI, and identify the digested products by agarose gel electrophoresis, select positive clones with inserts, and sequence (using ABI PRISM 3700 DNA analyzer ), select the correct positive cDNA gene plasmid inserted into Bax, and name it as pcDB3.1-BAX. pcDB3.1-BAX does not have c-myc and his tags.

(2) Transient transfection of H1299 cells: Introduce H1299 cells into a six-well plate at 1×10 ⁵ , and after 24 hours for the cells to adhere to the wall, follow the operation method provided in the Lipofectamine 2000 instruction manual to transfect pcDNA3.1/mycHis(- ) B, pcDB3.1-DENND2D-v2, pcDB3.1-BAX plasmids were transfected into cells in each well, and the amount of plasmid DNA and transfection reagent used was 1/2 of the recommended amount to reduce cytotoxicity. The medium was changed 4 hours after transfection, and the cells were harvested 36 hours after transfection. AnnexinV FITC and PI double staining and flow cytometry detection were performed according to the instructions of Annexin V FITC Apoptosis Detection Kit (CALBIOCHEM ^® , PF032). The results are shown in Figure 13 and Table 5, indicating that the DENND2D-v2 gene only has a slight ability to induce H1299 cell apoptosis.

Table 5. Percentage of apoptotic cells and normal cells Transfection of pcDNA3.1/mycHis(-) B cells Transfection of pcDB3.1-DENND2D-v2 cells Transfection of pcDB3.1-BAX cells apoptotic cell 8.73% 11.74% 23.81% normal cells 91.27% 88.26% 76.19%

2. Observation and detection of cell cycle:

Cell cycle detection by PI flow cytometry: empty vector H1299-vector cells, H1299-DENND2D-1, H1299-DENND2D-2, H1299-DENND2D-3 monoclonal cells stably and highly expressing DENND2D-v2 were mixed with 2×10 ⁵ Into a 60mm cell culture dish, culture at 37°C in 5% CO ₂ until the confluence rate reaches about 80%. Cells were harvested after digestion, and fixed overnight (more than 8 hours) at -20°C with 80% ethanol (prepared with PBS). After staining with PI, it was tested on the machine. The results are shown in Figure 14 and Table 6, the exogenous high expression of DENND2D-v2 can arrest H1299 in G1/S phase.

Table 6. Percentage of cells in each phase installments H1299-vector cells H1299-DENND2D-1 cells H1299-DENND2D-2 cells H1299-DENND2D-3 cells G1 31.82% 42.34% 40.45% 38.00% S 50.11% 41.03% 35.97% 43.73% G2 18.07% 16.63% 23.58% 18.27%

The above examples illustrate that the DENND2D-v2 gene can slightly induce apoptosis and has a clear cell cycle arrest ability in the G1/S phase of the cell cycle, which may be the main reason for its ability to inhibit cell growth and proliferation.

3. RT-PCR and Western Blot to detect the expression of genes related to important functions such as cell cycle arrest:

Design primers or purchase antibodies for genes related to important functions such as cell cycle and metastasis, and perform RT-PCR or Western Blot experiments to detect their expression. The total RNA and protein of cells cultured at the same time were extracted for cell cycle detection, and RT-PCR and Western Blot detection were performed respectively. For RNA quantification, 5 μg of total RNA was used as a template for reverse transcription, and then PCR was performed with different primers (see Table 7 for primer sequences); for protein quantification, Western blot analysis was performed at 20 μg/sample, and the method was as above. p15, CyclinD1 mouse monoclonal antibody (Santa Cruz) was diluted with 5% milk according to 1:200 and added, and β-actin (loaded internal control) mouse monoclonal antibody (Santa Cruz) was diluted with 1:5000 with 5% milk and added . RT-PCR and Western blot results showed that p15, p16 of the INK4 family, p21 and p27 of the CIP/KIP family were down-regulated to a certain extent at the mRNA level, while the expressions of p19 and p57 had no significant changes. In addition, the expressions of p15 and cyclineD1 were increased and decreased at the protein level, respectively, as shown in Figures 15a and 15b. In Fig. 15b, DENND2D-1, DENND2D-2, and DENND2D-3 represent H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3, respectively.

Table 7 Gene upstream primer downstream primer product length p15p16P21P27 5'-CCGTTTCGGGAGGCGC-3'5'-CCCGCTTTCGTAGTTTTCAT-3'5'-CAGGGGACAGCAGAGGAAGA-3'5'-ATGTCAAACGTGCGAGTGTC-3' 5'-ACCCTGCAACGTCGCGGT6-3'5'-TTA TTTGAGCTTTGGTTCTG-3'5'-GGGCGGCCAGGGTATGTAC-3'5'-TCTGTAGTAGAACTCGGGCAA-3' 260bp355bp335bp270bp p57 5'-TCGCTGCCCGCGTTTGCGCA-3' 5'-CCGAGTCGCTGTCCACTTCGG-3' 289bp

Example 8. The effect of DENND2D-v2 on the movement and invasion ability of lung cancer cell line H1299

(1) Scratch healing experiment: H1299-vector cells with empty vector, H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3 monoclonal cells stably and highly expressing DENND2D were propagated at 1×10 ⁶ cells/dish Put into a 60mm cell culture dish, and the fusion rate of the cultured cells reaches 100%; draw a uniform scratch on the bottom of the dish, replace with a new medium, and continue the routine culture; change the medium every 24 hours and take pictures for records. The results are shown in Figure 16, indicating that the scratch healing ability of H1299 cells stably expressing the DENND2D-v2 gene is weakened, indicating that this gene affects the growth and metastasis potential of H1299 cells. In Figure 16, DENND2D-1, DENND2D-2, and DENND2D-3 represent H1299-DENND2D-1, H1299-DENND2D-2, and H1299-DENND2D-3, respectively, and vector represents H1299-vector.

(2) Transwell experiment:

A. Cell migration assay (Migration Assay): Two monoclonal cells H1299-DENND2D-2 and H1299-DENND2D-3 stably and highly expressing the DENND2D-v2 gene were randomly selected from the empty vector H1299-vector cells at 2×10 ⁵ /well, introduced into the Transwell chamber according to the Transwell ^(R) Permeable Supports (Corning) instructions. After incubating at 37°C with 5% CO ₂ for 24 hours, carefully wipe the cells on the upper membrane of each small chamber with a cotton swab, fix with ice methanol for 5 minutes, stain with 0.5% crystal violet for 5 minutes, wash with water, and observe the results with the naked eye and under a microscope. As shown in Figure 17, it shows that the DENND2D-v2 gene can significantly reduce the number of H1299 cells passing through the polycarbonate membrane containing a small hole with a diameter of 8 μm, indicating that this gene can significantly inhibit the migration and movement ability of tumor cells. In Fig. 17, DENND2D-2 and DENND2D-3 respectively represent H1299-DENND2D-2 and H1299-DENND2D-3, and vector represents H1299-vector.

B. Cell invasion assay (Invasion Assay): Matrigel TM Basement Membrane Matrix (BD Biosciences) was plated into the Transwell ^® Permeable Supports (Corning) chamber used in the cell migration assay according to the instructions. In order to detect the effect of DENND2D-v2 gene on the invasiveness of lung cancer cell line H1299 cells, Matrix Gel was added to each chamber at 50 μl/ ^cm2 to form a uniform layer with a thickness of 0.5 mm, which was completely solidified after incubation at 37°C for 30 minutes. The empty vector H1299-vector cells, randomly selected two monoclonal cell lines H1299-DENND2D-2 and H1299-DENND2D-3 stably and highly expressing the DENND2D-v2 gene, were gently dropped into 4×10 ⁵ cells/well. After incubating at 37°C with 5% CO ₂ for 24 hours, carefully wipe the Matrix Gel and cells on the upper layer of the chamber membrane with a cotton swab, fix with ice methanol for 5 minutes, stain with 0.5% crystal violet for 5 minutes, wash with water, and observe with the naked eye and under a microscope The results are shown in Figure 18, DENND2D-v2 gene can significantly reduce the ability of H1299 cells to degrade Matrix Gel and pass through the polycarbonate membrane containing small holes with a diameter of 8 μm, indicating that this gene can significantly inhibit the invasion ability of tumor cells. In Fig. 18, DENND2D-2 and DENND2D-3 represent H1299-DENND2D-2 and H1299-DENND2D-3 respectively, and vector represents H1299-vector.

Sequence Listing

<160>4

<210>1

<211>2062

<212>DNA

<213> Homo sapiens

<400>1

cctgggtccc gtcacatcct tcttgctcaa ccactgggtg cacaggatgg aaacttctat 60

tccctctctg gaagacagcg cgtggcttgg cttcacagag ttgtggctgg agaccgaagc 120

agcccctttc tcaggcttac tgtcaccagt ctgtctgtgt taggggag gggagtccgc 180

tctgtcctga aggcccagag atggaaggac aagtggtagg ccgggtgttc aggctcttcc 240

aacgccgact gcttcaactc cgagcaggac caccccagga caattcaggg gaagctttaa 300

aggaaccaga aagggcccag gagcactctt tgcccaactt tgctgggggg cagcacttct 360

ttgaatacct tcttgtggtt tctctcaaaa agaagcgttc agaggatgat tacgagccta 420

taatcaccta ccaatttccc aagcgggaga acctgcttcg gggtcagcag gaggaggagg 480

agcggctgct caaagctatc cccttgttct gcttcccaga tgggaatgag tgggcatcac 540

tcaccgagta tcccaggggag accttctcct tcgttctgac caatgtggat gggagcagaa 600

agattggata ctgcaggcgc ctcttgcctg ccggccctgg ccctcgcctt cccaaagtgt 660

actgcatcat cagctgcatc ggctgcttcg gcttgttctc caagatcctg gatgaagtgg 720

agaagagaca tcagatctcc atggctgtca tctacccgtt catgcagggc ctccgagagg 780

cagccttccc tgctcctggg aagactgtca ctctcaagag cttcatcccc gactcaggca 840

ctgagttcat ttcactgaca cggcccctgg actcccacct agaacatgtg gattttagtt 900

ctctattgca ctgtctcagt tttgaacaga tacttcagat ctttgcctct gccgtgctgg 960

agagaaaaat catcttcctg gcggaaggtc tcagcacctt gtctcagtgc atccatgctg 1020

ctgccgcact gctctacccc ttcagctggg cgcacaccta catccctgtt gtccctgaga 1080

gccttctggc caccgtctgc tgccccacccc ccttcatggt tggagtacaa atgcgcttcc 1140

agcaggaggt catggacagc cctatggaag aggtcctgct ggtcaatctt tgtgaaggaa 1200

ccttcttaat gtcggttggt gatgaaaaag acatcctgcc accgaagctt caggatgaca 1260

tcttagactc tcttggtcag gggatcaatg agttaaagac tgcagaacaa atcaacgagc 1320

atgtttcagg cccctttgtg cagttctttg tcaagattgt gggccattat gcttcctata 1380

tcaagcggga ggcaaatggg caaggccact tccaagaaag atccttctgt aaggctctga 1440

cctccaagac caaccgccga tttgtgaaga agtttgtgaa gacacagctc ttctcacttt 1500

tcatccagga agccgagaag agcaagaatc ctcctgcagg ctatttccaa cagaaaatac 1560

ttgaatatga ggaacagaag aaacagaaga aaccaaggga aaaaactgtg aaataagagc 1620

tgtggtgaat aagaatgact agagctacac accatttctg gacttcagcc cctgccagtg 1680

tggcaggatc agcaaaactg tcagctccca aaatccatat cctcactctg agtcttggta 1740

tccaggtatt gcttcaaact ggtgtctgag atttggatcc ctggtattga tttctcagga 1800

ctttggaggg ctctgacacc atgctcacag aactgggctc agagctccat tttttgcaga 1860

ggtgacacag gtaggaaaca gtagtacatg tgttgtagac acttggttag aagctgctgc 1920

aactgccctc tcccatcatt ataacatctt caacacagaa cacactttgt ggtcgaaagg 1980

ctcagcctct ctacatgaag tctgtggaca tgtaaggacg agagtaaaga ggaaaatctt 2040

ataaaaaaaa aaaaaaaaa aa 2062

<210>2

<211>1905

<212>DNA

<213> Homo sapiens

<400>2

ataggggggc ggggccagcg cggagctggg agcgggagcc gggcactcca ggggccatgg 60

atgggctcgg ccgccgcctt cgagccagcc tgagactgaa gcgcggccat gggggaccac 120

cccaggacaa ttcaggggaa gctttaaagg aaccagaaag ggcccaggag cactctttgc 180

ccaactttgc tggggggcag cacttctttg aataccttct tgtggtttct ctcaaaaaga 240

agcgttcaga ggatgattac gagcctataa tcacctacca atttcccaag cgggagaacc 300

tgcttcgggg tcagcaggag gaggaggagc ggctgctcaa agctatcccc ttgttctgct 360

tccccagatgg gaatgagtgg gcatcactca ccgagtatcc cagggagacc ttctccttcg 420

ttctgaccaa tgtggatggg agcagaaaga ttggatactg caggcgcctc ttgcctgccg 480

gccctggccc tcgccttccc aaagtgtact gcatcatcag ctgcatcggc tgcttcggct 540

tgttctccaa gatcctggat gaagtggaga agagacatca gatctccatg gctgtcatcc 600

acccgttcat gcagggcctc cgagaggcag ccttccctgc tcctgggaag actgtcactc 660

tcaagagctt catccccgac tcaggcactg agttcatttc actgacacgg cccctggact 720

cccacctaga acatgtggat tttagttctc tattgcactg tctcagtttt gaacagatac 780

ttcagatctt tgcctctgcc gtgctggaga gaaaaatcat cttcctggcg gaaggtctca 840

gcaccttgtc tcagtgcatc catgctgctg ccgcactgct ctaccccttc agctgggcgc 900

acacctacat ccctgttgtc cctgagagcc ttctggccac cgtctgctgc cccaccccct 960

tcatggttgg agtacaaatg cgcttccagc aggaggtcat ggacagccct atggaagagg 1020

tcctgctggt caatctttgt gaaggaacct tcttaatgtc ggttggtgat gaaaaagaca 1080

tcctgccacc gaagcttcag gatgacatct tagactctct tggtcagggg atcaatgagt 1140

taaagactgc agaacaaatc aacgagcatg tttcaggccc ctttgtgcag ttctttgtca 1200

agattgtggg ccattatgct tcctatatca agcgggaggc aaatgggcaa ggccacttcc 1260

aagaaagatc cttctgtaag gctctgacct ccaagaccaa ccgccgattt gtgaagaagt 1320

ttgtgaagac acagctcttc tcacttttca tccaggaagc cgagaagagc aagaatcctc 1380

ctgcaggcta tttccaacag aaaatacttg aatatgagga acagaagaaa cagaagaaac 1440

caagggaaaa aactgtgaaa taagagctgt ggtgaataag aatgactaga gctacacacc 1500

atttctggac ttcagcccct gccagtgtgg caggatcagc aaaactgtca gctcccaaaa 1560

tccatatcct cactctgagt cttggtatcc aggtattgct tcaaactggt gtctgagatt 1620

tggatccctg gtattgattt ctcaggactt tggagggctc tgacaccatg ctcacagaac 1680

tgggctcaga gctccatttt ttgcagaggt gacacaggta ggaaacagta gtacatgtgt 1740

tgtagacact tggttagaag ctgctgcaac tgccctctcc catcattata acatcttcaa 1800

cacagaacac actttgtggt cgaaaggctc agcctctcta catgaagtct gtggacatgt 1860

aaggacgaga gtaaagagga aaatcttaaa aaaaaaaaaa aaaaa 1905

<210>3

<211>471

<212>PRT

<213> Homo sapiens

<400>3

Met Glu Gly Gln Val Val Gly Arg Val Phe Arg Leu Phe Gln Arg Arg

1 5 10 15

Leu Leu Gln Leu Arg Ala Gly Pro Pro Gln Asp Asn Ser Gly Glu Ala

20 25 30

Leu Lys Glu Pro Glu Arg Ala Gln Glu His Ser Leu Pro Asn Phe Ala

35 40 45

Gly Gly Gln His Phe Phe Glu Tyr Leu Leu Val Val Ser Leu Lys Lys

50 55 60

Lys Arg Ser Glu Asp Asp Tyr Glu Pro Ile Ile Thr Tyr Gln Phe Pro

65 70 75 80

Lys Arg Glu Asn Leu Leu Arg Gly Gln Gln Glu Glu Glu Glu Arg Leu

85 90 95

Leu Lys Ala Ile Pro Leu Phe Cys Phe Pro Asp Gly Asn Glu Trp Ala

100 105 110

Ser Leu Thr Glu Tyr Pro Arg Glu Thr Phe Ser Phe Val Leu Thr Asn

115 120 125

Val Asp Gly Ser Arg Lys Ile Gly Tyr Cys Arg Arg Leu Leu Pro Ala

130 135 140

Gly Pro Gly Pro Arg Leu Pro Lys Val Tyr Cys Ile Ile Ser Cys Ile

145 150 155 160

Gly Cys Phe Gly Leu Phe Ser Lys Ile Leu Asp Glu Val Glu Lys Arg

165 170 175

His Gln Ile Ser Met Ala Val Ile Tyr Pro Phe Met Gln Gly Leu Arg

180 185 190

Glu Ala Ala Phe Pro Ala Pro Gly Lys Thr Val Thr Leu Lys Ser Phe

195 200 205

Ile Pro Asp Ser Gly Thr Glu Phe Ile Ser Leu Thr Arg Pro Leu Asp

210 215 220

Ser His Leu Glu His Val Asp Phe Ser Ser Leu Leu His Cys Leu Ser

225 230 235 240

Phe Glu Gln Ile Leu Gln Ile Phe Ala Ser Ala Val Leu Glu Arg Lys

245 250 255

Ile Ile Phe Leu Ala Glu Gly Leu Ser Thr Leu Ser Gln Cys Ile His

260 265 270

Ala Ala Ala Ala Leu Leu Tyr Pro Phe Ser Trp Ala His Thr Tyr Ile

275 280 285

Pro Val Val Pro Glu Ser Leu Leu Ala Thr Val Cys Cys Pro Thr Pro

290 295 300

Phe Met Val Gly Val Gln Met Arg Phe Gln Gln Glu Val Met Asp Ser

305 310 315 320

Pro Met Glu Glu Val Leu Leu Val Asn Leu Cys Glu Gly Thr Phe Leu

325 330 335

Met Ser Val Gly Asp Glu Lys Asp Ile Leu Pro Pro Lys Leu Gln Asp

340 345 350

Asp Ile Leu Asp Ser Leu Gly Gln Gly Ile Asn Glu Leu Lys Thr Ala

355 360 365

Glu Gln Ile Asn Glu His Val Ser Gly Pro Phe Val Gln Phe Phe Val

370 375 380

Lys Ile Val Gly His Tyr Ala Ser Tyr Ile Lys Arg Glu Ala Asn Gly

385 390 395 400

Gln Gly His Phe Gln Glu Arg Ser Phe Cys Lys Ala Leu Thr Ser Lys

405 410 415

Thr Asn Arg Arg Phe Val Lys Lys Phe Val Lys Thr Gln Leu Phe Ser

420 425 430

Leu Phe Ile Gln Glu Ala Glu Lys Ser Lys Asn Pro Pro Ala Gly Tyr

435 440 445

Phe Gln Gln Lys Ile Leu Glu Tyr Glu Glu Gln Lys Lys Gln Lys Lys

450 455 460

Pro Arg Glu Lys Thr Val Lys

465 470

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Met Asp Gly Leu Gly Arg Arg Leu Arg Ala Ser Leu Arg Leu Lys Arg

1 5 10 15

Gly His Gly Gly Pro Pro Gln Asp Asn Ser Gly Glu Ala Leu Lys Glu

20 25 30

Pro Glu Arg Ala Gln Glu His Ser Leu Pro Asn Phe Ala Gly Gly Gln

35 40 45

His Phe Phe Glu Tyr Leu Leu Val Val Ser Leu Lys Lys Lys Arg Ser

50 55 60

Glu Asp Asp Tyr Glu Pro Ile Ile Thr Tyr Gln Phe Pro Lys Arg Glu

65 70 75 80

Asn Leu Leu Arg Gly Gln Gln Glu Glu Glu Glu Arg Leu Leu Lys Ala

85 90 95

Ile Pro Leu Phe Cys Phe Pro Asp Gly Asn Glu Trp Ala Ser Leu Thr

100 105 110

Glu Tyr Pro Arg Glu Thr Phe Ser Phe Val Leu Thr Asn Val Asp Gly

115 120 125

Ser Arg Lys Ile Gly Tyr Cys Arg Arg Leu Leu Pro Ala Gly Pro Gly

130 135 140

Pro Arg Leu Pro Lys Val Tyr Cys Ile Ile Ser Cys Ile Gly Cys Phe

145 150 155 160

Gly Leu Phe Ser Lys Ile Leu Asp Glu Val Glu Lys Arg His Gln Ile

165 170 175

Ser Met Ala Val Ile His Pro Phe Met Gln Gly Leu Arg Glu Ala Ala

180 185 190

Phe Pro Ala Pro Gly Lys Thr Val Thr Leu Lys Ser Phe Ile Pro Asp

195 200 205

Ser Gly Thr Glu Phe Ile Ser Leu Thr Arg Pro Leu Asp Ser His Leu

210 215 220

Glu His Val Asp Phe Ser Ser Leu Leu His Cys Leu Ser Phe Glu Gln

225 230 235 240

Ile Leu Gln Ile Phe Ala Ser Ala Val Leu Glu Arg Lys Ile Ile Phe

245 250 255

Leu Ala Glu Gly Leu Ser Thr Leu Ser Gln Cys Ile His Ala Ala Ala

260 265 270

Ala Leu Leu Tyr Pro Phe Ser Trp Ala His Thr Tyr Ile Pro Val Val

275 280 285

Pro Glu Ser Leu Leu Ala Thr Val Cys Cys Pro Thr Pro Phe Met Val

290 295 300

Gly Val Gln Met Arg Phe Gln Gln Glu Val Met Asp Ser Pro Met Glu

305 310 315 320

Glu Val Leu Leu Val Asn Leu Cys Glu Gly Thr Phe Leu Met Ser Val

325 330 335

Gly Asp Glu Lys Asp Ile Leu Pro Pro Lys Leu Gln Asp Asp Ile Leu

340 345 350

Asp Ser Leu Gly Gln Gly Ile Asn Glu Leu Lys Thr Ala Glu Gln Ile

355 360 365

Asn Glu His Val Ser Gly Pro Phe Val Gln Phe Phe Val Lys Ile Val

370 375 380

Gly His Tyr Ala Ser Tyr Ile Lys Arg Glu Ala Asn Gly Gln Gly His

385 390 395 400

Phe Gln Glu Arg Ser Phe Cys Lys Ala Leu Thr Ser Lys Thr Asn Arg

405 410 415

Arg Phe Val Lys Lys Phe Val Lys Thr Gln Leu Phe Ser Leu Phe Ile

420 425 430

Gln Glu Ala Glu Lys Ser Lys Asn Pro Pro Ala Gly Tyr Phe Gln Gln

435 440 445

Lys Ile Leu Glu Tyr Glu Glu Gln Lys Lys Gln Lys Lys Pro Arg Glu

450 455 460

Lys Thr Val Lys

465

Claims (10)

1, tumor correlated albumen is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 4;

(b) with the amino acid residue sequence of sequence in the sequence table 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with tumour by (a) deutero-protein.

2, tumor correlated albumen according to claim 1 is characterized in that: the proteinic aminoacid sequence of described (b) is the sequence 3 in the sequence table.

3, the encoding gene of claim 1 or 2 described tumor correlated albumens.

4, gene according to claim 3 is characterized in that: the cDNA gene of described tumor correlated albumen is following 1) or 2) gene:

1) its nucleotide sequence is sequence 1 or the sequence 2 in the sequence table;

2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular of the described tumor correlated albumen of encoding.

5, the recombinant expression vector, transgenic cell line and the transformed host bacterium that contain claim 3 or 4 described tumor correlated albumen encoding genes.

6, with the described tumor correlated albumen of claim 1 be the polyclonal antibody or the monoclonal antibody of antigen prepd.

7, a kind of antitumor drug, its activeconstituents are the recombinant mammalian expression vectors that contains claim 3 or 4 described tumor correlated albumen encoding genes.

8, medicine according to claim 7 is characterized in that: described tumour is lung cancer, the esophageal carcinoma or laryngocarcinoma.

9, described tumor correlated albumen of claim 1 and encoding gene thereof preparation prevent and/or treat tumour and with the medicine of metastases relative disease in application.

10, application according to claim 9 is characterized in that: described tumour is lung cancer, the esophageal carcinoma or laryngocarcinoma.

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