WO2018170759A1 - Adénovirus recombinant ad-140-148a-185-tud, construction et application de celui-ci - Google Patents
Adénovirus recombinant ad-140-148a-185-tud, construction et application de celui-ci Download PDFInfo
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- WO2018170759A1 WO2018170759A1 PCT/CN2017/077601 CN2017077601W WO2018170759A1 WO 2018170759 A1 WO2018170759 A1 WO 2018170759A1 CN 2017077601 W CN2017077601 W CN 2017077601W WO 2018170759 A1 WO2018170759 A1 WO 2018170759A1
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Definitions
- the present invention belongs to the field of genetic engineering, and relates to the construction of an adenovirus, and more particularly to a recombinant adenovirus which interferes with the expression of miR-140, miR-148a and miR-185.
- MicroRNAs are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.
- miR-140 is closely related to the development of various diseases, such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases. miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TG FBR1 and other gene expression. miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis.
- miR-148a is a study in recent years More micr 0 RNA is obtained. It is reported that miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers; miR-185 is a 22nt miRNA, located on human chromosome 22ql 1.21 It plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, and liver cancer.
- DNMT1 methylation-related tumor suppressor miRNA
- the expression affects the methylation level of the whole genome, which in turn regulates the methylation status of certain genes and affects gene expression.
- miR-140, miR-148a and miR-185 By controlling the expression of miR-140, miR-148a and miR-185, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.
- Tough Decoy RNA is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.
- the present invention provides a recombinant Ad-140-148a-185-Tud adenovirus containing a Tud RNA which interferes with the expression of iR-140, miR-148a and miR-185, the Tud RNA has SEQ
- the shuttle plasmid used to construct the recombinant Ad-140-148a-185-Tud adenovirus is pHBAd-U6-
- the Tud RNA that interferes with the expression of miR-140, miR-148a and miR-185 is ligated under the U6 promoter of the shuttle plasmid.
- framework plasmid used in the construction of the recombinant adenovirus of the present invention is pHBAd-BHG, and the packaging cell is 293 cells.
- Ad-140-148a-185-Tud adenovirus of the present invention is constructed by designing a Tud RNA of the nucleotide sequence shown in SEQ ID No. 1, and chemically synthesizing the corresponding SEQ ID No 2 and SEQ ID No.
- the Tud RNA complementary single-stranded target gene fragment shown in 3 was annealed to obtain a double-stranded expression template of the target gene fragment, and ligated with BamHI and EcoRI double-digested vector pHBAd-U6-GFP to construct an adenoviral shuttle plasmid, and then The recombinant adenovirus Ad-140-148a-185-Tud was obtained by co-transfecting the adenovirus shuttle plasmid with the backbone plasmid pHBAd-BHG into 293 cells.
- Ad-140-148a-185-Tud adenovirus constructed by the present invention can be used as a tool for blocking/reducing the expression of miR-140, miR-148a and miR-185.
- 1 is the expression level of miRNA, wherein a. miR-140 expression, b. miR-148a expression, c. miR-185 expression.
- Tud DNA-F/Tud DNA-R 1/1 ⁇ l
- Procedure 95 ° C for 5 min, cooled to 25 ° C at a rate of 0.1 ° C / s, and then stored at 4 ° C.
- the linearized pHBAd-U6-GFP vector was recovered using a PCR reaction recovery kit (Axygen).
- the annealing product of the above Tud RNA expression sequence was ligated to the BamHI and EcoRI sites of the vector pHBAd-U6-GFP with T4 ligase to construct an adenoviral shuttle plasmid, which was regulated by the U6 promoter.
- Tud RNA and carrier-coupled reaction system 1 ⁇ annealing product; 100-200
- Ngx linearized pHBAd-U6-GFP vector 2 ⁇ 1 T4 DNA Ligase Buffer; ⁇ ⁇ 4 DNA Ligase; diluted to ⁇ 20 to a total volume of 20 ⁇ 1.
- the ligation reaction system was allowed to stand at 4 ° C overnight.
- the constructed adenoviral shuttle plasmid was transformed into competent cells Jml07, resistant: ampicillin; cultured overnight at 37 °C.
- the transformed Tud RNA was picked by a plate, and the bacteria were shaken at 37 ° C for 200 hours at 200 rpm.
- the bacterial solution was sequenced, and the sequencing result was consistent with the target sequence, which was obtained to contain 13 ⁇ 4 ⁇ (1-1 1(1-140-148&- 185 plasmid of E. coli.
- the recombinant adenoviral vector plasmid pHBAd-Tud-14 is taken.
- pHBAd-Tud-140- 148a- 185 pHBAd-Tud-140- 148a- 185 , lO g backbone plasmid pHBAd-BHG, diluted with 300 ⁇ DMEM medium and allowed to stand at room temperature for 5 min.
- the 50 ml centrifuge tube was repeatedly freeze-thawed three times in a liquid nitrogen and a 37 ° C constant temperature water bath, centrifuged at 3000 rpm for 5 min, and the virus-containing supernatant was collected, and the precipitate was discarded.
- the supernatant is the first generation of Ad-140-148a-185-T U d (P1), which is used as a virus for subsequent amplification of a large number of viruses.
- Min take the supernatant as a third generation virus (P3).
- the WRL-68 cells were seeded into a six-well plate at a density of 20,000 cells/well, and the Ad-140-148a-185-Tud adenovirus was infected at 37° after the cells were grown to 60% confluence. Incubate for 2 h in a C 5% C02 incubator, change the medium, and continue to culture for 36 hours.
- the miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, followed by S-PolyC hsa-miR-140 qPCR-assay primer set, S-Poly(T) hsa-miR- 148a qPCR-assay primer set and S -Poly(T) hsa-miR- 185 qPCR-assay primer seti box to reverse transcribe and tail the miRNA to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
- the expression levels of miR-140, miR-148a and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times, and 3 parallel samples were set per well, and snord 44 was used as an internal reference.
- the results are shown in Figure 2. It can be seen that the expression level of miR-140 in TuD-140-148a-185 cells is 43 ⁇ 3 ⁇ 4 lower than that of WRL-68 cells, and the expression level of miR-148a is 62% lower than that of WRL-68 cells. The expression level of miR-185 was 76% lower than that of WRL-68 cells. The difference was statistically significant (p ⁇ 0.01), indicating that the TuD-140-148a-185 cell line was successfully constructed.
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L'invention concerne un TudARN servant à réduire les expressions de miARN-140, miARN-148a et miARN-185 ; une séquence nucléotidique de celui-ci est représentée par SEQ ID NO.1. L'invention concerne également un vecteur d'adénovirus recombinant comprenant le TudARN.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077601 WO2018170759A1 (fr) | 2017-03-22 | 2017-03-22 | Adénovirus recombinant ad-140-148a-185-tud, construction et application de celui-ci |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/077601 WO2018170759A1 (fr) | 2017-03-22 | 2017-03-22 | Adénovirus recombinant ad-140-148a-185-tud, construction et application de celui-ci |
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| WO2018170759A1 true WO2018170759A1 (fr) | 2018-09-27 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010138263A2 (fr) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Nouveaux virus adéno-associés (aav) et leurs utilisations |
| WO2011127625A1 (fr) * | 2010-04-13 | 2011-10-20 | 江苏命码生物科技有限公司 | Procédé pour réguler la teneur en microarn dans des organismes et utilisations de celui-ci |
| CN102851291A (zh) * | 2006-04-03 | 2013-01-02 | 桑塔里斯制药公司 | 包含抗微小rna 反义寡核苷酸的药物组合物 |
| CN103623425A (zh) * | 2012-08-27 | 2014-03-12 | 苏州圣诺生物医药技术有限公司 | 应用双靶标拮抗寡核酸抑制新血管增生疾病的药物 |
| WO2015164786A1 (fr) * | 2014-04-25 | 2015-10-29 | University Of Massachusetts | Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques |
| CN105457042A (zh) * | 2015-12-23 | 2016-04-06 | 深圳市疾病预防控制中心 | miR-34a沉默表达重组载体及其应用 |
-
2017
- 2017-03-22 WO PCT/CN2017/077601 patent/WO2018170759A1/fr active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851291A (zh) * | 2006-04-03 | 2013-01-02 | 桑塔里斯制药公司 | 包含抗微小rna 反义寡核苷酸的药物组合物 |
| WO2010138263A2 (fr) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Nouveaux virus adéno-associés (aav) et leurs utilisations |
| WO2011127625A1 (fr) * | 2010-04-13 | 2011-10-20 | 江苏命码生物科技有限公司 | Procédé pour réguler la teneur en microarn dans des organismes et utilisations de celui-ci |
| CN103623425A (zh) * | 2012-08-27 | 2014-03-12 | 苏州圣诺生物医药技术有限公司 | 应用双靶标拮抗寡核酸抑制新血管增生疾病的药物 |
| WO2015164786A1 (fr) * | 2014-04-25 | 2015-10-29 | University Of Massachusetts | Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques |
| CN105457042A (zh) * | 2015-12-23 | 2016-04-06 | 深圳市疾病预防控制中心 | miR-34a沉默表达重组载体及其应用 |
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