CN101168566A - Antibody preparation and application of anti-tumor specific marker protein TS/MEDP - Google Patents

Abstract

The invention discloses the preparation and application of an antibody against tumor-specific marker protein TS/MEDP. Using molecular cell biology techniques, a new gene was cloned in gastric cancer cells, named TS/MDEP. A polyclonal antibody against TS/MDEP was prepared, and a highly specific antibody was obtained through antibody purification technology. The expression characteristics of TS/MDEP gene in tumor cell lines and tissues were verified from the mRNA and protein levels, and it was confirmed that it was expressed in 14 tumor cell lines including gastric cancer cell lines, and showed cell cycle dependence. It is overexpressed in breast cancer and cervical cancer tissues, but not in corresponding normal tissues, and expressed in various embryonic tissues. It has been confirmed that TS/MDEP is highly expressed in embryonic tissues with vigorous cell proliferation, and this gene is turned off in adulthood, and it is highly expressed in tumor cells again. TS/MDEP is cycle-specific expression, suggesting that high expression of TS/MDEP leads to abnormal regulation of cell cycle and participates in the occurrence and development of tumors. The first anti-TS/MDEP antibody can be used to monitor cell cycle progression and clinical biological behavior of gastrointestinal tumors.

Description

Antibody preparation and application of anti-tumor specific marker protein TS/MEDP

technical field

The present invention involves a variety of cell and molecular biology techniques, including cell synchronization technology, mRNA differential display technology, RACE technology, prokaryotic expression of protein, RT-PCR, mRNA in situ hybridization, polyclonal antibody preparation, immunochemistry, Western blot, Flow cytometry, RNAi and animal experiments. .

Background technique

Gastric cancer ranks second among tumor-related diseases in the world, and is the most common malignant tumor in Asian populations, seriously endangering human health. People expect to clarify the mechanism of occurrence and development of gastric cancer, so as to effectively prevent and treat this malignant tumor. Although a large number of studies have confirmed that the occurrence of gastric cancer is related to many factors including diet, environment, and bacterial or viral infection, such as Helicobacter pylori infection, in recent years, molecular mechanism studies have found that many genes are involved in the occurrence and development of gastric cancer, such as the expression of E-cadherin Deletion, p53 gene, TGF-β, c-met, erbB-2 and RUNX3, etc. These studies have explained part of the pathogenesis of gastric cancer, but we still know little about the detailed molecular pathogenesis of gastric cancer.

Compared with normal cells, one of the most fundamental characteristics of tumor cells is the abnormal regulation of the cell cycle. During the transformation of normal cells into malignant cells, the accumulation of polygenic mutations endows tumor cells with unique functions, such as tumor cells producing many of their own growth Factors are insensitive to signals that inhibit cell growth, escape apoptosis, and have unlimited replication potential, all of which can be attributed to mutated genes that lead to abnormal cell cycle regulation, especially cell cycle checkpoint abnormalities, which endow tumor cells with uncontrolled growth characteristics . Although the discovery of Cyclin and CDK has clarified the basic regulatory mechanism of the cell cycle, there is also a lot of understanding of the G1-S phase checkpoint, such as P53, P21, GADD45, TGF-β, P27, P16, Rb, and E2F participate in the G1-S phase checkpoint. S-phase checkpoint regulation, but we are still far from understanding the detailed molecular regulation mechanism of cell cycle regulation.

Contents of the invention

The purpose of the present invention is to comprehensively utilize the above-mentioned technologies, and it is necessary to find key genes involved in the occurrence and development of gastric cancer and cell cycle regulation, so as to clarify the molecular mechanism of the occurrence and development of gastric cancer, and further screen for molecular markers of gastric cancer and molecular targets for the treatment of gastric cancer . This study combined mRNA differential display technology and cell synchronization technology. Taking gastric cancer cell line BGC823 as the research object, a cycle differentially expressed gene was obtained. The full-length mRNA was cloned and named TS/MDEP, and submitted to Genbank. The gene number is DQ150361. The TS/MDEP gene encodes a 389aa protein with a molecular weight of 42.3kd. Biological software predicts that it contains multiple phosphorylation sites, one proline-rich region that can bind to proteins with SH3 Domain, one with CC domain, Therefore, it is speculated that the TS/MDEP gene has important biological functions.

The differential expression of the gene suggests that it has a specific biological function, so we used different techniques to verify the expression characteristics of the TS/MEDP gene at the cell and tissue levels. This gene was expressed in 14/17 tumor cell lines and showed a cycle Specifically expressed, the expression of TS/MEDP protein was the highest in BGC823 in the M phase, and its expression level gradually decreased with the completion of cell division. More importantly, it is expressed in paired gastric cancer tissues, but not in normal gastric mucosa. It has also been confirmed that the TS/MEDP gene is expressed in a variety of embryonic tissues with vigorous cell proliferation, so the TS/MEDP gene has the following expression in the tissue Features: TS/MDEP is highly expressed in embryonic tissues with vigorous cell proliferation. After adulthood, this gene is turned off in tissue cells, and it is highly expressed in tumor cells, which is in line with the expression law of oncogenes. Therefore, TS/MEDP gene is a new candidate oncogenes.

Especially in BGC823, after RNAi interfered with TS/MEDP gene, cell morphology changed significantly, cell proliferation was significantly inhibited, cell colony formation ability and tumorigenicity were significantly reduced, and cells appeared G2/M phase arrest after interference. This result indicates that TS/MEDP gene has important biological functions in the regulation of cell proliferation and the occurrence and development of tumors. And it may become a molecular marker to predict the biological behavior of gastric cancer.

The technical content of the present invention is: cloned the new gene TS/MEDP (TumorSpecificity and Mitosis Phase-dependent Expression protein) with cycle-dependent expression, and submitted it to Genebank with the acceptance number DQ150361. After synthesizing the polypeptide and linking it with the macromolecular protein KLH, immunize New Zealand white rabbits, and after measuring the antibody titer by ELISA, take the rabbit serum, and perform affinity chromatography with prokaryotically expressed TS/MDEP protein, cell cycle and tumor-specific expression, The specific polyclonal antibody against TS/MEDP was prepared after purification.

This antibody can detect the expression of TS/MEDP protein by Western blot and immunohistochemical techniques.

By detecting gene expression, we found its expression regularity in cells and tissues, and showed cycle-specific expression in cells. The expression of TS/MEDP protein was the highest in BGC823 in the M phase, and its expression level gradually increased with the completion of cell division. In tissues, TS/MDEP is highly expressed in embryonic tissues with vigorous cell proliferation. More importantly, TS/MEDP genes are expressed in gastric cancer tissues, but not in normal gastric mucosa.

Further functional research shows that TS/MEDP gene plays an important role in cell proliferation and is closely related to cell division, and has important biological functions in the occurrence and development of tumors. Because dysregulation of the cell cycle is a fundamental feature of tumor cells, TS/MEDP is a candidate oncogene. The anti-TS/MDEP antibody we prepared can be used to diagnose gastric cancer, and based on the function of this gene that we have elucidated, the TS/MEDP gene may become a molecular marker for the diagnosis of gastric cancer or a molecular target for the treatment of gastric cancer.

Using the technical solution provided by the present invention, a new gene was cloned in gastric cancer cells by using molecular cell biology techniques, named TS/MDEP (Tumor Specificity and Mitosis Phase-dependent Expression protein, DQ150361). A polyclonal antibody against TS/MDEP was prepared, and a highly specific antibody was obtained through antibody purification technology. The expression characteristics of TS/MDEP gene in tumor cell lines and tissues were verified from the mRNA and protein levels, and it was confirmed that it was expressed in 14 tumor cell lines including gastric cancer cell lines, and showed cell cycle dependence. It is overexpressed in breast cancer and cervical cancer tissues, but not in corresponding normal tissues, and expressed in various embryonic tissues. Therefore, we confirmed that TS/MDEP is highly expressed in embryonic tissue with vigorous cell proliferation, and this gene is turned off in adulthood, and it is highly expressed in tumor cells again. Further research found that TS/MDEP is cycle-specific expression, suggesting that high expression of TS/MDEP leads to abnormal cell cycle regulation and participates in the occurrence and development of tumors. Our first anti-TS/MDEP antibody can be used to monitor cell cycle progression and clinical biological behavior of gastrointestinal tumors.

Description of drawings

Figure 1 shows the differentially expressed cDNA fragment A54-3, whose full-length mRNA was predicted by Northern blot, and the full-length mRNA of TS/MDEP gene was obtained by RACE technology.

Figure 2 shows that the prepared polyclonal antibody can specifically bind to the prokaryotic expressed TS/MDEP protein, indicating that it has high specificity.

Figure 3 shows that the TS/MDEP gene is expressed in various tumor cell lines and presents cycle-specific expression. The expression level of mRNA in G1/M phase is higher than that in G2/S phase; while the protein level is highest in M phase, and the expression gradually decreases with the completion of cell division.

Figure 4 shows the expression characteristics of TS/MDEP gene in tissues: TS/MDEP gene showed significant differential expression at the mRNA and protein levels, expressed in gastric tumor tissue but not in normal gastric mucosa; TS in various embryonic tissues /MDEP gene expression.

Figure 5 shows the effect of inhibiting TS/MDEP gene expression on cell morphology and cycle. After RNAi inhibited the expression of TS/MDEP gene, the cell morphology changed significantly, the cells were flattened, enlarged, giant cells appeared, synapse-like pseudopodia appeared, and the cells appeared G2/M phase arrest.

Figure 6 shows that the TS/MDEP gene interference inhibits the proliferation and tumorigenicity of cells. The proliferation of cells after interference was significantly lower than that of the control, and the soft agar and nude mouse tumor formation experiments confirmed that the tumorigenic ability of cells after interference was significantly reduced in vivo and in vitro.

Detailed ways

The specific implementation manner of the present invention will be described below in conjunction with the accompanying drawings.

material method

1. Cell culture

Gastric cancer cell lines BGC823, MGC803, SGC7901, PAMC82, MKN45, SNU1, SNU5, SNU16, RF1, RF48 were cultured in 5% fetal bovine serum DMEM medium, AGS, N87 and colon cancer cell lines LOVO were cultured in 10% fetal bovine serum DMEM Cultured in culture medium, prostate cancer cell line PC-3, liver cancer cell line BEL7421, breast cancer cell line MCF7, esophageal cancer cell line EC9706 were cultured in 10% fetal bovine serum 1640 medium, and there were final concentrations in the medium 100,000units/L penicillin and 100mg/L streptomycin, cultured at 37°C in 5% CO ₂ .

Second, cell synchronization

The gastric cancer cell line BGC823 was seeded in several 100mm diameter petri dishes at a density of 20%, cultured in a CO ₂ incubator at 37°C for 24 hours to reach the logarithmic growth phase, and replaced with 2.5mMTdR/DMEM when the cell density was 40%-80% The conditioned medium was continued to be cultivated in a 5% CO ₂ incubator at 37°C for 18 hours, then the TdR conditioned medium was discarded, washed once with the conventional medium, and the conventional medium was added to cultivate at 37°C for 6 hours. Afterwards, the culture dish was put into the high-pressure N ₂ O tank developed by the cell laboratory of our institute, 400ml of CO ₂ was injected into the tank, and the lid was sealed. Turn on the N ₂ O inlet switch, release the upper layer of pseudo gas, slowly inflate the tube, and make the pressure reach 0.55MP within 30 minutes, and then put the N ₂ O tank in a 37°C incubator for 18 hours, and the full 18 After 1 hour the _N2O tank was removed and the _N2O was vented. A large number (90%) of these cells are rounded under an inverted microscope. These cells are cells in the M phase, and some of them have been floating in the culture medium. Gently shake the culture bottle to suspend the cells in the M phase in the culture medium, suck out the cell culture medium, and centrifuge Discard the supernatant, and the collected cells are the M phase cells. Cells in G1 phase can be collected after the remaining M phase cells continue to culture for 5 hours. Part of the G1 phase cells were replaced with TdR conditioned medium and continued to culture for 18 hours. After 18 hours, replace the conventional medium and continue to culture for 5 hours to harvest the S phase cells, and continue to culture the S phase cells for 7 hours to harvest the G2 phase cells. Harvested cells of different cycles were detected by PROFILE II flow cytometer (COULTER).

Three, mRNA differential display

The total RNA of the gastric cancer cell line BGC823 in the G1 and S phases was extracted by the acidic phenol-isocyanuridine-isosulfate-step method, and the operation was performed according to the instructions of the HIEROGLYPH ^TM mRNA Profile Kit (Genomyx Corporation) for mRNA differential display, as follows, reverse transcription reaction system 20 μl : DEPC-H ₂ O 8.8 μl, 5×buffer 4.0 μl, dNTP (250 μM) 2.0 μl, DTT (100 mM) 2.0 μl, DNase-treated total RNA (0.5 μg/μl) 1.0 μl, 3' anchor primer AP 2.0 μl, MMLV (200u/μl) 0.2 μl; the reaction is as follows: add 3' anchor primer AP to RNA and incubate at 70°C for 5 minutes, immediately put it on ice and cool it, then add other components (DEPC-H ₂ O, dNTP, DTT, MMLV , buffer), 42°C for 5 minutes, 50°C for 50 minutes, 70°C for 15 minutes, and the reverse transcription product was stored at -20°C. DD-PCR reaction system 20μl is as follows: dH ₂ O 8.2μl, 10×buffer 2.0μl, MgCl ₂ (25Mm) 1.2μl, dNTP (250μM) 1.6μl, 3' anchor primer AP (2.5μM) 2.0μl, 5' Anchor primer ARP (2.5 μM) 2.0 μl, TaqE (5u/μl) 0.2 μl, RT-Mix 2.0 μl, [α- ³⁵ S]Datp (12 μci/μl) 0.8 μl, PCR program: 95°C for 2 minutes, 92 15 seconds at ℃, 30 seconds at 50℃, 2 minutes at 72℃, 4 cycles, 15 seconds at 92℃, 30 seconds at 60℃, 2 minutes at 72℃, 25 cycles, 7 minutes at 72℃, and the product was stored at -20℃. 6% polyacrylamide gel electrophoresis of DD-PCR products: Prepare 6% polyacrylamide gel, polymerize overnight, pre-electrophoresis for 30 minutes before adding samples, temperature 55°C, voltage 1760v, current 28mA, constant power 50W; sample 6μl denatured Then load the sample, remove the gel after electrophoresis for 4 hours, dry in vacuum at 80°C for 2 hours, and perform autoradiography. The cDNA fragments differentially expressed in G1 and S phases were found on the X-ray film, and the gel was cut and recovered for PCR re-amplification, and then the products were sequenced and identified. As shown in Fig. 1A, mRNA difference shows that the differentially expressed cDNA fragment A54-3 in G1/S phase was obtained, and this fragment is highly expressed in G1 phase.

4. Northern Blot

The total RNA extracted from the cell line was calculated based on the OD value. Take 20 μg of total RNA and separate it with RNA denaturing gel. Rinse the electrophoresis RNA gel several times with distilled water to remove formaldehyde, shake and wash it with 100ml of 10×SSC for 1 hour to melt, and use Use 150ml 20×SSC as the transfer solution, transfer the RNA to the nitrocellulose membrane by capillary macrosuction, and dry it in an oven at 80°C for 2 hours; pre-hybridize in the hybridization solution at 65°C overnight, and label the probe as follows before hybridization: Add water (provided by the kit) to 150ng needle to 30μl, denature in boiling water for 5 minutes, quickly insert ice to cool, after about three minutes, add the following reagents in sequence, Labeling 5×buffer 10μl, Mix dA(G, T)TP 2μl, 10mg/ml BSA 2μl, α- ³² P dCTP (50μci) 5μl, Klenow enzyme (5u/μl) 1μl, mix well and leave at room temperature for 2 hours, then purify the probe according to the purification column instructions. The labeled probe was denatured at 70°C, and 10ml of new hybridization solution was added, and the denatured probe was added, and hybridized overnight at 65°C. After the eluate was eluted, it was exposed to light at -70°C for 2 weeks for development. As shown in Fig. 1B, in five tumor cell lines, the expression of TS/MDEP gene was confirmed, and the full length of its mRNA was about 4.0 kb.

Five, RT-PCR

According to the instructions, TRIZOL extracted total RNA from tumor cell lines and tissues, quantified it with a spectrophotometer, took 5 μg total RNA for reverse transcription, added 0.4 μl oligodT to each, added DEPC-treated H ₂ O to 20 μl, denatured at 70°C for 10 minutes, and rapidly Insert ice to cool, add 5×RT buffer 6μl, dNTP 2μl (2.5mM), RNase inhibitor 0.5μl (40u/μl), mix thoroughly, add M-MLV reverse transcriptase 1μl each, mix well, and incubate at 37°C for 1 Hours, inactivated at 70°C for 15 minutes, and stored at -20°C after aliquoting. Then PCR amplification, primers used, upstream primer sequence: 5'-TGGCATCTTTACTGGACTGG-3, downstream primer sequence: 5'-TGGCACCTCGTGGATAGAGC-3', the length of the amplified fragment is 385bp, including SAGE tag. PCR reaction system 20 μl: dH ₂ O 15.0 μl, 10×Buffer (containing MgCl ₂ ) 2.0 μl, dNTP (2.5mM) 0.5 μl, upstream primer (5 μM) 0.5 μl, downstream primer (5 μM) 0.5 μl, Taq DNA polymerase (5U/μL) 0.5μl, DNA template (100ng/μL) 0.5μl. PCR amplification conditions, the parameters are as follows: pre-denaturation at 95°C for 2min30sec, the cycle reaction program is denaturation at 94°C for 500sec, annealing for 50sec; extension at 72°C for 50sec, 30cycles, after the cycle reaction is completed, fully extend at 72°C for 10min, store at 4°C, and perform PCR The product was identified on 1.2% agarose gel. β-actin was used as an internal control, and Mock was used as a negative control. As shown in Figure 3A and Figure 4A, respectively, it was confirmed that the TS/MDEP gene was expressed in a variety of tumor cell lines, and showed a cell cycle-dependent expression, and RT-PCR confirmed that its mRNA expression was higher than that of Argo and Q /S phase; it was confirmed in tissues that this gene is expressed in various embryonic tissues, such as stomach, small intestine, large intestine, etc., and the most important thing is to confirm that TS/MDEP gene is expressed in gastric cancer tissues in paired gastric cancer tissues while paired normal Stomach tissue was negative.

6. Antibody preparation

Analyze the hydrophilic region and epitope region of T/MDEP by software, refer to the general principle of using peptides to prepare antibodies, and select three peptides, which are located at the N-terminal, middle segment and C-terminal of the protein, respectively. Because the molecular weight of the synthetic peptide is small, it cannot fully trigger the immune response, so it is necessary to select a suitable carrier to enhance its immunogenicity. We linked the synthetic peptide to KLH and injected it at multiple points on both sides of the New Zealand white rabbit's spine. For the first immunization, 500ug of the peptide was injected, and then the dose was halved, injected once a week, and immunized four times in total. Finally, the whole blood of the rabbit was taken, the serum was separated, and stored in aliquots. After the polyclonal antibody was purified by antibody purification technology (CNBr-activated Sepharose 4B), the specificity of the polyclonal antibody was verified by immunohistochemistry and Western blot, and the expression characteristics of TS/MDEP protein were detected. As shown in Figure 2, the TS/MDEP protein was obtained after prokaryotic expression and purification, and was identified by mass spectrometry. After hybridization with serum containing anti-TS/MDEP antibody, it was confirmed that the antibody prepared by our polypeptide can specifically bind to the expressed whole protein. Therefore, we Further adopting the method of affinity chromatography, using the prokaryotic expression antibody to purify the antibody in the serum, significantly improving the specificity of the antibody.

7. Immunohistochemistry

Using the ABC method, the cell lines were fixed with 2% formaldehyde after dropping, 0.3% H ₂ O ₂ methanol at room temperature for 10 minutes, and washed 3 times with PBS for 5 minutes. Add primary antibody (1:50) dropwise, overnight at 4°C in a wet box, wash 3 times with PBS for 5 minutes, add secondary antibody 1:200 dilution, incubate at room temperature for 30 minutes, wash 3 times with PBS for 5 minutes, add third antibody dropwise Dilute 1:400, incubate at room temperature for 30 minutes, wash with PBS 3 times×5 minutes, develop color with DAB-H ₂ O ₂ , control under microscope for 3-5 minutes, rinse with PBS, stop color development, counterstain with hematoxylin for 45 seconds, 1% Separation with hydrochloric acid and alcohol, rinse with warm tap water to turn blue, dehydrate with 95% and 100% ethanol for 5 minutes each, transparent with xylene, and seal with neutral gum. As shown in FIG. 4C , the purified antibody was used to stain tissue arrays prepared from gastric cancer and normal tissues, and it was confirmed that TS/MDEP protein was expressed in gastric tumor tissues but not in normal gastric tissues. See Table 2 for statistical data.

Eight, mRNA in situ hybridization

Using the same primers as above, a large amount of TS/MEDP gene fragments were amplified in BGC823 by RT-PCR. After recovery and purification from rubber tapping, the cDNA fragments were inserted into pGEM-T Easy vector T/A clone (Promega), and transformed into competent DH5α, small Quantitative extraction of plasmids and identification by sequencing. Then label the probe: use Digoxigenin Probe Labeling Kit (Boehringer Mannheim company product), operate according to the instructions, label Digoxigenin on the probe, use RNA polymerase Sp6 and RNA polymerase T7 to label the sense probe and RNA polymerase T7 respectively. Antisense probes, labeled probes should be stored at -20°C. Carry out mRNA in situ hybridization according to the following procedure: deparaffinize the sections, return to water with ethanol step by step, equilibrate the sections with 0.2N HCl, incubate the sections in 2×SSC (preheated) at 70°C for 15 minutes, wash the sections with DEPC-treated PBS for 2 minutes , the sections were equilibrated in 4% PFA (pre-cooled) at 4°C for 5 minutes, and the sections were washed twice with DEPC-treated PBS, 2 minutes each time. The slices were equilibrated in 2×SSC for 5 minutes, and an appropriate amount of pre-hybridization solution was added to each slice, incubated in a wet box at 70°C for 8 minutes and then 37°C for 1 hour, and the slices were washed twice with 2×SSC, 2 minutes each time. Dehydrate with ethanol step by step, heat the probe to 70°C and add it to the ice-cold prehybridization solution (according to 10ng/μl) to prepare a single-stranded probe, add 200μl hybridization solution on the slice, cover with a cover glass, and wet box 43 Incubate overnight at ℃, add 2×SSC dropwise to the slices to remove the coverslip, wash the slices with 2×SSC at room temperature for 20 minutes, 1×SSC for 20 minutes, 0.5×SSC at 43°C for 20 minutes, and room temperature 0.5×SSC for 20 minutes. The slices were equilibrated in the washing solution for 5 minutes, and sheep serum diluted with blocking solution (1:20 with blocking solution) was added to each slice, incubated at room temperature in a wet box for 20 minutes, the blocking solution was discarded, and the blocking solution was added to each slice Diluted alkaline phosphatase anti-digoxigenin antibody (1:500), incubate at room temperature for 1 hour or overnight at 4°C in a wet box, discard the antibody solution, wash the slices twice in washing solution, 15 minutes each time, slice in Equilibrate in the substrate solution for 5 minutes, add freshly prepared color solution to each section, incubate at room temperature in a wet box, and keep away from light for half an hour. Dehydration, sealing (digoxigenin nucleic acid detection reagent and also a product of Boehringer Mannheim). As shown in Figure 4B, in the tissue arrays containing large samples, different methods were used to confirm again that the mRNA of TS/MDEP gene was expressed in gastric cancer tissues, but negatively expressed in normal gastric mucosa. Using the sense probe as a control, it was confirmed that the probe we selected had good specificity. See Table 1 for statistical data.

Ten races

Use SMART ^TM RACE cDNA Amplification Kit (CLONTECH) for 5′-end and 3′-end RACE. The specific process is according to the instructions: extract the total RNA of gastric cancer cell line BGC823, identify it by denaturing gel electrophoresis, and reverse-transcribe the mRNA into 5′- Both RACE-Ready cDNA and 3′-RACE-Ready cDNA were nested PCR, primers used in 5′RACE: GSP1: GGATCGGTCCCCTCGTCCACCCG, NGSP1: CCCACGGCGACAATAGCGACTACTT, PCR reaction system 50μl: 5′-RACE-ReadycDNA 2.5μl, UPM ( 10×) 5 μl, GSP1 (or NGSP1) 1 μl, Master Mix 41.5 μl, PCR reaction program: 95°C for 3 minutes for full denaturation, 95°C for 2 minutes, annealing temperature gradient of 62°C, 63.4°C, 64.4°C for 50 seconds, extension at 72°C 50 seconds, a total of 30 cycles of amplification, fully extended at 72°C for 10 minutes; primers used for 3′RACE: GSP2: TCCCAGCACTTGAGGCCAGGAGT, PCR reaction system 50 μl: 3′-RACE-Ready cDNA 2.5 μl, UPM (10×) 5 μl, GSP2 1μl, Master Mix 41.5μl, PCR reaction program: fully denatured at 95°C for 2 minutes, 1 minute and 30 seconds at 95°C, annealing temperature gradient of 56°C, 58.4°C, 60°C for 50 seconds, extension at 72°C for 50 seconds, a total of 30 cycle, fully extended at 72°C for 10 minutes. The amplified fragments were gel-cut, recovered and purified, and verified by sequencing after T/A cloning. See Figure 1C, D, through RACE technology, the full length (3877bp) of TS/MDEP mRNA is obtained, which contains the G-Caping structure at the 5' end and the tailing signal at the 3' end, and this mRNA contains a complete CDS ( coding sequence) and contains a Kozak sequence at the translation initiation site.

Eleven, RNA interference

According to the common standard of siRNA target sequence design and psiRNA-hHlneo plasmid characteristic application software siRNA Wizard ^TM v2.4, design the targeting sequence of TS/MDEP gene, select 3 segments of TS/MDEP gene cDNA that meet the conditions and exclude them by BLAST analysis The homologous sequence, according to the plasmid instructions, designed and synthesized a hairpin structure, annealed to form a double-stranded structure, with a Bbs I enzyme-cut restriction site at the end, and then ligated with the psiRNA-hHlneo plasmid after Bbs I digestion, and transformed bacteria. After antibiotic screening, clones were picked, sequenced and identified, and the large-scale plasmids were subpackaged and stored at -70°C. Transfect the gastric cancer cell line BGC823, add selective medium (400μg/ml G418) for 48 hours, change the medium every 3 days, pick single clones after 69 days of screening, pick 6 for each fragment and expand culture, and then extract RNA and protein, RT-PCR and Western blot verification were performed to confirm that the TS/MDEP gene was disturbed in the 3c clone, and the cell morphology was observed. MTT confirmed that the TS/MDEP gene was disturbed after birth. Cell growth status, and cell cycle changes detected by cell flow cytometry , soft agar colony formation experiment to observe the cell colony formation ability, and nude mouse tumorigenicity experiment to observe the changes of cell tumorigenicity after TS/MDEP gene was disturbed. As shown in Figures 5 and 6, after RNAi inhibited the expression of TS/MDEP gene, the cell morphology changed significantly, the cells were flattened, enlarged, giant cells appeared, synapse-like pseudopodia appeared, and the cells appeared G2/M phase arrest; After the TS/MDEP gene was disturbed, the proliferation and tumorigenicity of the cells were inhibited. reduce.

Attached:

Table 1. In situ hybridization confirmed the differential expression of TS/MDEP gene in normal gastric mucosa and gastric cancer.

× ² tests, p<0.01

Table 2. Immunohistochemistry confirmed the differential expression of TS/MDEP gene in normal gastric mucosa and gastric cancer.

× ² test, p=0.01

Claims (4)

1. the Antibody Preparation of antineoplastic specificity marker protein TS/MEDP, it is characterized in that: cloned a new tumor-related gene TS/MDEP and be submitted to Genebank, accept number is (DQ150361), turn out to be cell cycle and tumor specific expression, and prepared antibody with high degree of specificity.

2. the purposes of the antibody of anti-TS/MDEP is characterized in that: the proteic expression level of TS/MDEP during this antibody can detect cell and organize with Western blot and immunohistochemistry.

3. the purposes of the antibody of anti-TS/MDEP according to claim 2 is characterized in that: in various kinds of cell is that TS/MDEP protein presents cyclin dependent and expresses, and promptly expresses the highlyest in the M phase, finishes along with fissional, and its expression level reduces gradually.

4. according to the purposes of the antibody of the described anti-TS/MDEP of claim 2, it is characterized in that: TS/MDEP albumen may become the molecular target of tumor diagnosis and treatment.

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