CN1330759C - Nucleusase gene hTERT-5'R2 cDNA of inhibiting endgranulase activity and its recombined carrier and nucleusase - Google Patents
Nucleusase gene hTERT-5'R2 cDNA of inhibiting endgranulase activity and its recombined carrier and nucleusase Download PDFInfo
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- CN1330759C CN1330759C CNB2004100405201A CN200410040520A CN1330759C CN 1330759 C CN1330759 C CN 1330759C CN B2004100405201 A CNB2004100405201 A CN B2004100405201A CN 200410040520 A CN200410040520 A CN 200410040520A CN 1330759 C CN1330759 C CN 1330759C
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Abstract
The present invention discloses a ribozyme gene hTERT-5' RZ cDNA for inhibiting telomerase activity, a recombinant carrier containing the gene and ribozyme hTERT-5' RZ for transcribing the recombinant carrier. The ribozyme gene is composed of two 40 nt complementary strands which are annealed to form the ribozyme gene; the nucleotide sequence of the gene is disclosed in the figure 1. The ribozyme gene hTERT-5' RZ cDNA is inserted into the EcoRI-XhoI site of the carries pcDNA3.1(+), and then, the recombinant plasmid pcDNA3.1hTERT-5' RZ is obtained. The recombinant plasmid pcDNA3.1hTERT-5' RZ is cut by XhoI and EcoRI enzymes to be linearized. The ribozyme hTERT-5' RZ can be transcribed via T7RNA pclymerase catalysis by a T7/SP6 in-vitro transcription reagent kit; the secondary structure of the ribozyme is disclosed in the figure 3. The ribozyme hTERT-5' RZ and the ribozyme teloRZ can also form into a dicaryon enzyme. Detection results indicate that the ribozyme hTERT-5' RZ and the dicaryon enzyme can both effectively inhibit tumor growth.
Description
Technical field
The invention belongs to gene engineering technology field, relate in particular to the pharmaceutical use of the ribozyme gene, the recombinant vectors that contains this gene and transcription product ribozyme and the ribozyme that suppress telomerase activation.
Background technology
Telomere is a kind of protective structures of eukaryote end of chromosome, and normal somatic cell is because end duplicates problem, and telomere is with the shortening of carrying out property of cell fission.Telomerase (telomerase) is a kind of special ribonucleoprotein matter polymkeric substance, and it can synthesize telomere as template with self RNA, duplicates the telomere shortening that causes to remedy, and makes cell can apoptosis not occur because of telomere exhausts.On the other hand, Telomerase is one of important regulation and control factor of cell cycle, affects the process of cell cycle.Tumour cell has been kept the length of telomere on the one hand owing to the activation of Telomerase, and cell is immortalized; Cell cycle is shortened, and growth accelerates.A large amount of researchs show that the generation of activation of telomerase and cancer is closely related, and Telomerase has become a novel targets of antineoplaston.
Ribozyme is the microRNA with a fixed structure, the cutting said target mrna of fixing a point, thereby inhibition of gene expression.Liu Bailin, Qu Yi etc. (see " Chinese science (C collects) " the 32nd the 2nd phase of volume in " Ribozyme suppresses the therapy of tumor of telomerase activation ", P159-164, in April, 2002) a kind of teloRZ ribozyme that suppresses telomerase activation is disclosed in the literary composition, this ribozyme is imported cultured tumor cells in vitro and transplanted tumor in nude mice, adjustable point cutting hTR (people's telomerase RNA, be human telomerase RNA), make it to lose the template function of synthetic telomeric dna, thereby suppress the growth of tumour cell and tumor tissues.Therefore, developing the ribozyme of efficient, special inhibition telomerase activation, is new strategy and important measures that the mankind capture Cancerous disease.
Qu Yi etc. (see the 19th the 5th phase of volume of " Chinese Journal of Medical Genetics " October in 2002 in " reverse transcriptase of telomere ribozyme hTERT-5 ' RZ suppresses the experimental study of Hela cell telomerase activation ", P389-392) disclose reverse transcriptase of telomere ribozyme hTERT-5 ' RZ in the literary composition and had the effectiveness that suppresses Hela cell telomerase activation, and its telomerase activation suppresses effectiveness and is better than the teloRZ ribozyme, but the gordian technique contents such as structure of unexposed reverse transcriptase of telomere ribozyme gene hTERT-5 ' RZ cDNA and reverse transcriptase of telomere ribozyme hTERT-5 ' RZ cause the person of ordinary skill in the field to implement.
Summary of the invention
The object of the present invention is to provide a kind of ribozyme gene that suppresses telomerase activation---the reverse transcriptase of telomere ribozyme hTERT-5 ' RZ that reverse transcriptase of telomere ribozyme gene hTERT-5 ' RZ cDNA, the recombinant vectors that contains this gene and recombinant vectors are transcribed, to realize the application of ribozyme as the therapy of tumor medicine.
Existing achievement in research shows: human telomerase reverse is made up of RNA and protein, its 3 main components comprise human telomerase RNA (human Telomerase RNA, hTR), Telomerase associated protein (Telomerase Protein 1, TPI) and reverse transcriptase of telomere (human Telomerase Reverse Transcriptase, hTERT).HTR and hTERT and telomerase activation have closely related property, hTERT is the catalytic subunit and the active centre of Telomerase, the key factor that is the decision telomerase activation (is seen Kim NW, Piatyszek MA, Prowse KR, et al.Specific association of humantelomerase activity with immortal cells and cancer.Science, 1994,266:2011-2015 and Nakamura TM, Morin GB, Chapman KB, et al.Telomerase catalytic subunit homologs fromfission yeast and human.Science, 1997,277:955-959.).
Technical scheme of the present invention: at the synthetic Telomerase reverse transcription ribozyme gene of reverse transcriptase of telomere hTERT mRNA 5 ' petiolarea GUC point of contact design, ribozyme gene is made up of the complementary strand of two 40nt.For ease of making up recombinant vectors, the complementary strand two ends are respectively equipped with restriction enzyme site sequence and 2～3 protection bases, long altogether 57bp.Two complementary strands annealing are formed ribozyme genes, this ribozyme gene called after hTERT-5 ' RZ cDNA, its nucleotide sequence is as described in the accompanying drawing 1.With the EcoRI-XhoI site that synthetic ribozyme gene hTERT-5 ' RZ cDNA inserts the carrier for expression of eukaryon pcDNA3.1 (+) that has the in-vitro transcription function concurrently, promptly obtain recombinant vectors, this recombinant vectors called after pcDNA3.1 hTERT-5 ' RZ.Recombinant plasmid pcDNA3.1 hTERT-5 ' RZ cut with XhoI and EcoRI enzyme make it linearizing, adopt T
7/ SP
6The in-vitro transcription test kit is through T
7RNA polymerase catalysis can be transcribed out reverse transcriptase of telomere ribozyme of the present invention, this ribozyme called after hTERT-5 ' RZ, and its secondary structure is as described in Figure 3.Ribozyme hTERT-5 ' RZ and ribozyme teloRZ combination can form binuclear enzymes, and the mass ratio of ribozyme hTERT-5 ' RZ and ribozyme teloRZ is 1: 1.
With recombinant vectors pcDNA3.1 hTERT-5 ' RZ of the present invention transfection to human cervical carcinoma Hela cell's strain and carry out telomerase activation and detect.Detected result shows that Hela cell telomerase activation significantly descends behind the importing ribozyme hTERT-5 ' RZ, and its telomerase activation suppresses effectiveness and is better than the TeloRZ ribozyme, is approximately about the twice of TeloRZ ribozyme.
Ribozyme hTERT-5 ' RZ and binuclear enzymes are used for the treatment of the experiment of people's liver cancer transplanted tumor in nude mice, and experimental result shows that ribozyme hTERT-5 ' RZ and binuclear enzymes all can suppress tumor growth effectively.
Description of drawings
Fig. 1 is the nucleotide sequence of ribozyme gene hTERT-5 ' RZ cDNA of the present invention;
Fig. 2 inserts the process synoptic diagram that carrier for expression of eukaryon pcDNA3.1 (+) makes up recombinant vectors with synthetic ribozyme gene hTERT-5 ' RZ cDNA;
Fig. 3 is the secondary structure figure of ribozyme hTERT-5 ' RZ;
Fig. 4 is the electrophoresis evaluation figure of recombinant vectors;
Fig. 5 is the polyacrylamide gel electrophoresis figure that telomerase activation detects, among the figure, the 1st group of 1 expression (hTERT-5 ' RZ+teloRZ), 2 the 2nd group of expression (hTERT-5 ' RZ), the 3rd group (teloRZ) of 3 expressions, the 4th group (control RNA) of 4 expressions, 5 expression negative controls (lysis-buffer).
Embodiment
Embodiment 1: the design of ribozyme gene hTERT-5 ' RZ cDNA, synthetic
At reverse transcriptase of telomere hTERT mRNA 5 ' petiolarea GUC point of contact, become the ribozyme gene order by computer aided design (CAD), ribozyme gene is formed (as shown in Figure 1) by the complementary strand of two 40nt.For the ease of the clone, be respectively equipped with the sequence at EcoRI and XhoI enzyme point of contact at 5 ' and 3 ' end of ribozyme gene, and add 2～3 protection bases.Adopt solid phase phosphoramidite triester method (Pon R.T., Buck G.A., Hager K.M.et al.Deoxynucleosidephosphoramidates:A new class of key intermediates for deoxypolynucleotide synthesis.Tetrahedron Lett.22:1859-1862,1981), synthetic with ABI 3900 high-throughput dna synthesizers (PE company, BIO-RAD company on sale).The solid support that uses is controlled micropore glass pearl (available from a NEW Life Science company), and monomer is nucleoside phosphoramidites (available from a PE Applied Biosystems company).From the nineties, synthetic all specialized, the commercialization of relevant nucleotide sequence both at home and abroad.Therefore, also designed ribozyme gene sequence can be transferred to relevant specialized company synthetic (it is synthetic that ribozyme gene hTERT-5 ' the RZ cDNA among the present invention once transferred to GIBCO-BRL Life Technologies, Inc.).
Embodiment 2: the structure of recombinant vectors
The process of structure recombinant vectors as shown in Figure 2.Get each 33 μ g of strand ribozyme gene hTERT-5 ' RZ cDNA fragment of embodiment 1 synthetic also purifying, be annealed into complementary double-stranded.Reclaim big fragment with EcoRI and XhoI (all available from TakaRa company) double digestion, gel electrophoresis.Carrier pcDNA3.1 (+) (can from Invitrogen company buy), after cutting with EcoRI and XhoI enzyme, electrophoresis reclaims big fragment.1: 4 in molar ratio usefulness DNA of carrier and ribozyme gene Ligationkit ver.2 (available from TakaRa company) method is connected, and transformed competence colibacillus bacterium JM109 (available from TakaRa company) filters out transformant with penbritin.Extract plasmid with pillar extraction agent box (available from TakaRa company), enzyme is cut and is identified recon and order-checking, obtains recombinant plasmid pcDNA3.1 hTERT-5 ' RZ.6 pcDNA3.1 hTERT-5 ' of random choose RZ bacterium colony amplification cultivation and extracting plasmid, after EcoRI and XhoI enzyme are cut, 10% polyacrylamide (PAGE, being Fluka company product) gel electrophoresis all presents 46bp band (see figure 4), and is consistent with ribozyme gene hTERT-5 ' the RZcDNA clip size of expection.Selected clone 1 checks order, and the result is in full accord with design synthetic ribozyme gene hTERT-5 ' RZcDNA sequence.
Embodiment 3: ribozyme hTERT-5 ' RZ is obtained in the in-vitro transcription reaction
Recombinant plasmid pcDNA3.1 hTERT-5 ' RZ cuts with above-mentioned XhoI and EcoRI enzyme and makes it linearizing, adopts T
7/ SP
6In-vitro transcription test kit (available from Bao Ling Man) is through T
7RNA polymerase (available from GIBCO-BRL company) catalysis is carried out quantitatively with uv detection method to transcribe out corresponding small segment ribozyme hTERT-5 ' RZ, and yield is 3.5 μ g RNA/ μ g plasmids.The secondary structure of ribozyme hTERT-5 ' RZ as shown in Figure 3.
Embodiment 4: the preparation of binuclear enzymes
Ribozyme hTERT-5 ' RZ that embodiment 3 is obtained and ribozyme teloRZ press 1: 1 batching of mass ratio, and promptly ribozyme hTERT-5 ' RZ10 μ g, ribozyme teloRZ10 μ g mix with 40 μ l liposome Lipofectamine (available from Roche company) and promptly can be made into binuclear enzymes hTERT-5 ' RZ-teloRZ.
Ribozyme teloRZ is by disclosed method preparation in " Ribozyme suppresses the therapy of tumor of telomerase activation " (seeing " Chinese science (C collects) " the 32nd the 2nd phase of volume, P159-164, in April, 2004) literary composition such as Liu Bailin, Qu Yi.
Embodiment 5: cell transfecting
In 6 orifice plates, inoculate 5 * 10
4Individual human cervical carcinoma Hela cell/hole (human cervical carcinoma Hela cell can buy from ATCC company), is used OPTi-MEM substratum (available from Roche company) 2ml instead and is cultivated after 2 days with the conventional cultivation of 1640 substratum (available from GIBCO-BRL company).Experiment is divided into 4 groups, and the 1st group is 10 μ g hTERT-5 ' RZ+10 μ gteloRZ+40 μ l Lipofectamine; The 2nd group is 15 μ g hTERT-5 ' RZ+40 μ l Lipofectamine; The 3rd group is 15 μ g teloRZ+40 μ l Lipofectamine; The 4th group is contrast, adds empty carrier pcDNA-3.1 (can buy from Invitrogen company)/XhoI and transcribes synthetic small fragment RNA and 40 μ l Lipofectamine.Liposome Lipofectamine in above-mentioned each experimental group (available from Roche company) is with after small segment ribozyme rna (being dissolved among the 160 μ l OPTi-MEM) mixes, room temperature leaves standstill 20min, splash in the 6 orifice plate nutrient solutions, use 1640 culture medium culturing that contain 10% foetal calf serum after 6 hours instead.Every 24h repeats transfection once, and collecting cell behind the 72h is used for telomerase activation and detects.
Embodiment 6: telomerase activation detects
Centrifugal collection HeLa cell, and with no K
+, Ca
2+PBS (available from GBC-BRL company) wash secondary, add 100 μ l TRAP-lysis buffer[10mmol/L Tris-HCl (pH7.5), 1mmol/L MgCl
2, 1mmol/L EGTA, 0.1mmol/L PMSF, 5mmol/L β-thioglycol, 0.5%CHAPs, 10% glycerine], fully place 30min on ice, 4 ℃ of centrifugal 20min of 12000r/min behind the mixing, collect supernatant liquor, the by specification method is carried out Coomassie brilliant blue dyeing, in ultraviolet spectrophotometer (751-GW spectrophotometer, Shanghai Analytical Instrument Co., Ltd of Hewlett-Packard, production code member 910389) wavelength 595nm place mensuration protein concn and dilution are 2 μ g/ μ l, and-70 ℃ frozen standby.Adopt the TRAP method of kim report (to see Kim NW, Piatyszek MA, Prowse KR, et al.Specific association of human telomeraseactivity with immortal cells and cancer.Science, 1994,266:2011-2015), carry out telomerase activity with Telomerase activity test kit (available from Oncogene company).Each experimental group is got 2 μ g protein extracts respectively, adds reaction solution [5 μ l TRAP-buffer, 50umol/L dNTP, 1 μ l TS primer, PCR-Grade Water] to final volume 50 μ l.Incubated at room 30min is to finish the telomere extension that carries out at TS primer 3 ' end of Telomerase mediation.97 ℃ of 10min deactivation Telomerases add 1 μ l Primer Mix and 2u Taq enzyme on ice to reaction system.In pcr amplification instrument (PERKINELMER CETUS company), carry out cyclic amplification 35 times, 94 ℃ of loop parameters, 30s; 50 ℃, 30s; 72 ℃, 90s, last circulation prolongs 10min at 72 ℃ again.And substitute cell pyrolysis liquid as negative control with lysis buffer, getting capable 12% native polyacrylamide gel electrophoresis of 40 μ l PCR products separates, SYBR Green dyestuff (available from FMCBioproducts company) dyeing, mark in the 36bp band shows quantitatively, gradient zone shows telomerase activation.With gel scanning analysis instrument (the digital gel images analytical system of GIS-1000) running gel band gray scale is carried out scanning and counting, 36bp band gray scale is counted A1, and Telomerase gradient zone gray scale adds up to A2, and then A2/A1 is a cell Telomerase relative quantity.
The polyacrylamide gel electrophoresis result that telomerase activation detects as shown in Figure 5, each experimental group sees Table 1 through the resulting HeLa cell of scanning quantitative analysis Telomerase relative quantity.
Table 1
| Group | HeLa cell Telomerase relative quantity |
| The 1st group of 10 μ g hTERT-5 ' RZ+10 μ gteloRZ | 1.1 |
| The 2nd group of 15 μ g hTERT-5 ' RZ | 1.3 |
| The 3rd group of 15 μ g teloRZ | 4.7 |
| The 4th group of control RNA | 43 |
From Fig. 5 and table 1 as can be seen, import ribozyme hTERT-5 ' RZ after HeLa cell telomerase activation significantly descend, its Telomerase suppresses to render a service and obviously is better than ribozyme teloRZ.The mixture of two kinds of ribozymes is better than single ribozyme to the active inhibition effect of Telomerase.
Embodiment 7: ribozyme treatment people liver cancer transplanted tumor in nude mice
1. materials and methods
1.1 reagent and material
Human hepatoma cell strain SMMC-7721 (can from ATCC company buy) is the cultivation of going down to posterity of this chamber.BALB/C nude mice (heavily about 20g/ only) is purchased the institute of Antibiotics in Sichuan Province.
RNA transfection reagent TransMessanger
TMTransfection Reagent is a Qiagen company product.
1.2 the foundation of SMMC-7721 transplanted tumor in nude mice and program control illumination are raised
Make up the liver cancer transplanted tumor in nude mice, the knurl piece is to 55mm
3Be used for experiment during size.
1.3 medication experiment
Nude mice is divided into 4 groups, 6 every group (male and female half and half).The 1st group is control group, the interior multi-point injection 5 μ g/ of knurl body every day only contrast RNA (5 μ g Control RNA+10 μ l Enhancer R+TransMessanger Reagent, 25 μ l), the 2nd group is the teloRZ group, the interior multi-point injection 5 μ g/ teloRZ (5 μ g teloRZ+10 μ l EnhancerR+TransMessanger Reagent, 25 μ l) of knurl body every day, the 3rd group is hTERT-5 ' RZ group, the interior multi-point injection 5 μ g/ hTERT-5 ' RZ (5 μ g hTERT-5 ' RZ+10 μ l Enhancer R+TransMessanger Reagent, 25 μ l) of knurl body every day, the 4th group is the binuclear enzymes group, and (5 μ g binuclear enzymes: the mass ratio of hTERT-5 ' RZ and teloRZ is 1 to the interior multi-point injection 5 μ g/ binuclear enzymes of knurl body every day: 1+10 μ lEnhancer R+TransMessanger Reagent 25 μ l).Injection is 14 days continuously, and the unified neck method of breaking of using is put to death animal after 14 days, measures the tumour weight in wet base.
2. result
Medication after 14 days ribozyme growth of nude mice transplantation tumor is seen Table 2.
Table 2
| Croups (grouping) | Mean tumor weight on 14 day (14 days time tumour average weight in wet base) | PValue (P value) | Tumor inhibition ratio (tumour inhibiting rate %) |
| Small fragment RNA control | 0.694±0.121 | - | - |
| teloRZ group | 0.3434±0.024 | ＜0.005 | 51％ |
| hTERT-5’RZ group | 0.271±0.030 | ＜0.005 | 61％ |
| Binuclear enzymes group | 0.252±0.031 | ＜0.005 | 63％ |
As can be seen from Table 2, its knurl piece speed of growth of three groups of nude mices of injection ribozyme is considerably slower than the knurl piece speed of growth of nude mice of control group.Wherein, the knurl piece of hTERT-5 ' RZ group, binuclear enzymes group is organized less than teloRZ.The tumour inhibiting rate of hTERT-5 ' RZ group is 61%, the tumour inhibiting rate of binuclear enzymes group is 63%, the tumour inhibiting rate 51% of teloRZ group.Show that ribozyme hTERT-5 ' RZ and binuclear enzymes can suppress tumor growth better than ribozyme teloRZ.
Claims (4)
1, a kind of ribozyme gene hTERT-5 ' RZ cDNA that suppresses telomerase activation is characterized in that it has accompanying drawing 1 described nucleotide sequence.
2, a kind of recombinant vectors is characterized in that it contains the insertion sequence of the described ribozyme gene hTERT-5 ' of claim 1 RZ cDNA.
3, a kind of ribozyme hTERT-5 ' RZ that suppresses telomerase activation is characterized in that it has accompanying drawing 3 described structures.
4, a kind of mixture that suppresses two kinds of ribozymes of telomerase activation is characterized in that mainly being made up of claim 3 described ribozyme hTERT-5 ' RZ and ribozyme teloRZ, and the mass ratio of ribozyme hTERT-5 ' RZ and ribozyme teloRZ is 1: 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000043501A2 (en) * | 1999-01-22 | 2000-07-27 | Humboldt-Universität Zu Berlin Universitätsklinikum Charite | Ribozymes directed against the catalytic subunit of the human telomerase (htert) |
| CN1450170A (en) * | 2002-04-05 | 2003-10-22 | 北京科宇联合干细胞生物技术有限公司 | Method for detecting telomerase activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000043501A2 (en) * | 1999-01-22 | 2000-07-27 | Humboldt-Universität Zu Berlin Universitätsklinikum Charite | Ribozymes directed against the catalytic subunit of the human telomerase (htert) |
| CN1450170A (en) * | 2002-04-05 | 2003-10-22 | 北京科宇联合干细胞生物技术有限公司 | Method for detecting telomerase activity |
Non-Patent Citations (4)
| Title |
|---|
| 人端粒酶逆转录酶核酶抑制端粒酶活性的实验研究 屈艺 刘菽秋 彭文珍 刘柏林，四川大学学报(自然科学版)，第39卷第0期 2002 * |
| 端粒酶核酶hTERT5'RZ时辰治疗人肝癌裸鼠移植瘤 屈艺 黄翔 杨春蕾 刘菽秋 刘柏林 王正荣，四川生理科学杂志，第25卷第4期 2003 * |
| 端粒酶核酶hTERT5'RZ时辰治疗人肝癌裸鼠移植瘤 屈艺 黄翔 杨春蕾 刘菽秋 刘柏林 王正荣，四川生理科学杂志，第25卷第4期 2003;端粒酶逆转录酶核酶hTERT-5′RZ抑制HeLa细胞端粒酶活性的实验研究 屈艺 刘菽秋 刘柏林，中华医学遗传学杂志，第19卷第5期 2002;人端粒酶逆转录酶核酶抑制端粒酶活性的实验研究 屈艺 刘菽秋 彭文珍 刘柏林，四川大学学报(自然科学版)，第39卷第0期 2002 * |
| 端粒酶逆转录酶核酶hTERT-5′RZ抑制HeLa细胞端粒酶活性的实验研究 屈艺 刘菽秋 刘柏林，中华医学遗传学杂志，第19卷第5期 2002 * |
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