WO2018170757A1

WO2018170757A1 - Recombinant ad-29a-185-424-tud adenovirus, and construction and application thereof

Info

Publication number: WO2018170757A1
Application number: PCT/CN2017/077599
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-03-22
Filing date: 2017-03-22
Publication date: 2018-09-27

Abstract

Provided are a recombinant Ad-29a-185-424-Tud adenovirus and a method for preparing same. The adenovirus contains a TudRNA, inhibiting the expressions of human miR-29a, miR-185 and miR-424, of a nucleotide sequence as represented by SEQ ID NO.1. The method for preparing the adenovirus comprises: connecting the TudRNA to a U6 promoter of a shuttle plasmid, pHBAd-U6-GFP, to prepare an adenovirus shuttle plasmid, and transfecting 293 cells with the adenovirus shuttle plasmid and a skeleton plasmid, pHBAd-BHG, to obtain the recombinant Ad-29a-185-424-Tud adenovirus.

Description

Inventive name: Recombinant Ad-29a-185-424-Tud adenovirus and its construction and application

[0001] 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-29a, miR-185 and miR-424.

Background technique

[0002] MicroRNAs (miRNAs) 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.

[0003] miR-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer. Correlation, its expression level is an important reference for evaluating the benign and malignant gliomas. It is also associated with diseases such as atherosclerosis and liver fibrosis. It has important potential application value for the treatment of various tumors; miR-185 is a length 22nt miRNA, located in human chromosome 22ql l.21, 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, liver cancer, etc. The tumor suppressor miRNA can directly affect the methylation level of the whole genome by directly targeting the expression of DNMT1, thereby regulating the methylation status of some genes and affecting gene expression; miR-424 is a miRNA discovered in recent years. It affects the biological effects and occurrence of tumor cells by acting on target genes in various tumors and participating in the signal pathway of target gene regulation. Exhibition, play similar oncogenes, tumor suppressor gene, or promoting, inhibition of tumor invasion and metastasis. Studies have shown that miR-424 is a multifunctional miRNA, which is involved in cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and T NF-ot; since the miR-424 promoter region has CpG Island, which is also associated with methylation-induced gene silencing. By controlling the expression of miR-29a, miR-185 and miR-424, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.

technical problem [0004] Functional studies of MiRNAs are primarily accomplished through miRNA interference and overexpression techniques. Among the existing miRNA interference technologies, anti-miR and antagomiR are transient transfection techniques, and the interference effect cannot be stably maintained, while the mi RNA sponge effect is far from optimal. The prior art lacks a good interference effect and can achieve long-term effects. Stable interference technology.

[0005] Tough Decoy RNA (Tud 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.

Problem solution

Technical solution

[0006] It is an object of the present invention to provide a recombinant Ad-29a-185-424-Tud adenovirus, and a method for constructing the recombinant adenovirus for blocking/reducing miR-29a, miR-185 and miR- Expression of 424.

To achieve the above object, the present invention provides a recombinant Ad-29a-185-424-Tud adenovirus containing a Tud RNA which interferes with the expression of iR-29a, miR-185 and miR-424, the Tud RNA has SEQ

The nucleotide sequence shown by ID No. 1.

The shuttle plasmid used to construct the recombinant Ad-29a-185-424-Tud adenovirus is pHBAd-U6-

GFP, the Tud RNAs that interfere with the expression of miR-29a, miR-185 and miR-424 are ligated under the U6 promoter of the shuttle plasmid.

Further, the framework plasmid used in the construction of the recombinant adenovirus of the present invention is pHBAd-BHG, and the packaging cell is 293 cells.

[0010] The recombinant Ad-29a-185-424-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 is annealed to obtain a double-stranded expression template of the target gene fragment, and ligated with the BAHHI and EcoRI double-digested vector pHBAd-U6-GFP to construct an adenoviral shuttle plasmid, and then The adenovirus shuttle plasmid was co-transfected with the backbone plasmid pHBAd-BHG into 293 cells to obtain recombinant adenovirus A d-29a-185-424-Tud.

The recombinant Ad-29a-185-424-Tud adenovirus constructed by the present invention can be used as a tool for blocking/reducing the expression of miR-29a, miR-185 and miR-424. Advantageous effects of the invention

Beneficial effect

[0012] Through the design of the above-mentioned tool medicine of the present invention, the interaction of miR-29a, miR-185 and miR-424 in the process of tumor development and development can be further explored, thereby contributing to the research of tumor treatment.

Brief description of the drawing

DRAWINGS

1 is the expression level of miRNA, wherein a. miR-29a expression, b. miR-185 expression, c. miR-424 expression.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Embodiment 1

[0015] A Tud RNA sequence designed to interfere with the expression of miR-29a, miR-185 and miR-424, specifically: 5'-

GCCAATCAAGATGATCCTAGCGCCACCTTTTT-3'.

[0016] The above-described Tud RNA expression sequence was artificially synthesized, and BamHI and EcoRI restriction sites were introduced at both ends of the sequence.

[0017] Top strand:

ATCTTTTAGCCAATCAAGATGATCCTAGCGCCACCTTTTTG-3';

Bottom strand:

[0020] 5'-' TCGCGAGATGAGTGCAAAACGTTGATGATCCTAGCGCC-3'.

[0021] The above-described complementary single strands of 3' and 5' are annealed to obtain a double-stranded stencil annealing product of the corresponding target fragment Tud RNA. The annealing procedure is:

[0022] 20μ1 system:

[0023] lOxBuffer: 2μ1;

[0024] Tud DNA-F/Tud DNA-R: 1/1 μl;

H20: 16 μl.

Procedure: 95 ° C for 5 min, cooled to 25 ° C at a rate of 0.1 ° C / s, and then stored at 4 ° C.

[0027] The restriction enzyme endonuclease BamHI and EcoRI were double-digested with the vector pHBAd-U6-GFP, and the digestion system was as follows

[0028] 20μ1 digestion system 37 ° C 30 min:

2 μl vector (500 ng/μΐ);

[0030] Ιμΐ BamHI/ΙμΙ EcoRI;

[0031] 2μ1 lOxbuffer;

[0032] 14μ1 Η20.

[0033] After completion of the digestion, 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.

[0035] Tud RNA and carrier-coupled reaction system: 1 μΐ annealing product; 100-200

Ngx linearized pHBAd-U6-GFP vector; 2μ1 T4 DNA Ligase Buffer; Ιμΐ Τ4 DNA Ligase; diluted to 总20 to a total volume of 20μ1. The ligation reaction system was allowed to stand at 4 ° C overnight.

The constructed adenovirus 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, and the bacterial solution was sequenced. The sequencing result was consistent with the target sequence, and the plasmid containing pHBAd-T _U d-29a-185-424 was obtained. Enterobacter.

[0037] Embodiment 2

[0038] Take the logarithmic growth phase of the adenovirus shuttle plasmid solution prepared in the above example 2ml, add 100ml LB medium containing 100μg / ml Amp, shaking at 37 ° C 300 rpm overnight, using plasmid extraction The kit (purchased from Invitrogen) was used to extract plasmid pHB Ad-Tud-29a-185-424.

[0039] One day prior to transfection, 293 cells were seeded in 100 mm culture dishes in DMEM + 10 < 3⁄4 fetal bovine serum (FBS) and cultured overnight in a 37 ° C incubator containing 5% CO 2 .

[0040] After the cells were grown to 70 to 80% of the substrate area, the recombinant adenoviral vector plasmid pHBAd-Tud-29 a-185-424 and the backbone plasmid pHBAd-BHG were used, and Lipofectamine 2000 (purchased from Invitrogen) was used. Dyeing reagents for transfection. The specific steps are:

[0041] a. 2 minutes before transfection, complete medium was replaced. Take 5μ _§ recombinant adenoviral vector plasmid

pHB Ad-Tud-29a-185-424, lO g backbone plasmid pHBAd-BHG, was diluted with 300 μΐ DMEM medium and allowed to stand at room temperature for 5 min.

[0042] b Take 40μ1 Lipofectamine 2000, dilute with 300 μΐ DMEM medium, place at room temperature

5 min.

[0043] c. Mix the two and leave at room temperature for 20 min. The mixture was then added to a 100 mm culture dish, mixed and placed in a 37 ° C incubator containing 5% CO 2 for incubation.

[0044] After 5 h of transfection, fresh cell culture medium was replaced.

[0045] Observe the cells every day, until the cells become large and round, grape-like, start to appear obvious plaques and collect the virus from the bottom, collect all the cells and culture solution in the 100 mm culture dish in a 50 ml centrifuge tube. in.

[0046] 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-29a-185-424-T _U d (P1), which is used as a virus for subsequent amplification of a large number of viruses.

[0047] Take 2 ml from the P1 generation virus supernatant and infect a cell of 100 mm cell culture dish (cell density is guaranteed over 90%). The remaining virus supernatant was placed in an externally-circulated cryotube, kept at -80 ° C, and retained as a poisonous species. After the virus is expanded for two days, all the cells are detached from the bottom surface and can be taken for poisoning. The cells are taken together with the culture solution into a 50 ml centrifuge tube, frozen and thawed three times according to the previous freeze-thaw method, and the supernatant is taken for the next generation amplification or Store at 80 ° C. Each generation of virus amplification and drug collection will be repeated as such. [0048] Four T75 flasks were inoculated with 400,000 293 cells per T75 culture flask, and cultured overnight, until the cells grew to 90%.

吋, P2 generation virus (except for a small amount of virus-retaining species) was inoculated into the culture flask for 24 hours. 60% of the cytopathic lesions were observed under the microscope, and the cells were completely lesioned 48 hours later. Harvest the diseased cell suspension, centrifuge at 2000 rpm for 5 min, discard the supernatant, add 4 ml ST buffer (culture medium + 10% serum + 2.5% glycerol), vortex and mix, freeze at -80 ° C and 37 ° C After three times of centrifugation, centrifugation at 3000 rpm for 5 min, the supernatant was taken as a third generation virus (P3).

[0049] Embodiment 3

[0050] The WRL-68 cells were seeded into a six-well plate at a density of 20,000 cells/well, and the Ad-29a-185-424-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.

[0051] The miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, and then S-Poly(T) hsa-miR-29a qPCR-assay primer set, S-Poly(T) hsa-miR-185 qPCR-assay primer Set and S-Poly(T) hsa-miR-424 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

As a template, the expression levels of miR-29a, miR-185 and miR-424 were detected by real-time PCR, and the experiment was repeated 3 times with 3 parallel samples per well, with snord 44 as an internal reference. As a result, as shown in Fig. 2, it was found that the expression level of miR-29a in TuD-29a-185-424 cells was 57% lower than that of WRL-68 cells, and the expression level of miR-185 was 51% lower than that of WRL-68 cells. The expression level of miR-424 was 65<3⁄4 lower than that of WRL-68 cells. The difference was statistically significant (p<0.01), indicating that the TuD-29a-185-424 cell line was successfully constructed.

Industrial applicability

[0052] Through the design of the above-mentioned tool medicine of the present invention, the interaction of miR-29a, miR-185 and miR-424 in the process of tumor development and development can be further explored, thereby contributing to the research of tumor treatment.

Claims

Claim

[Claim 1] Recombinant Ad-29a-185-424-Tud adenovirus containing interfering human miR-29a, miR-185 and mi

The Tud RNA expressed by R-424, the Tud RNA having the nucleotide sequence shown in SEQ ID No. 1.

[Claim 2] The recombinant Ad-29a-185-424-T _U d adenovirus according to claim 1, wherein the shuttle plasmid used to construct the adenovirus is pHBAd-U6-GFP.

[Claim 3] The recombinant Ad-29a-185-424-T _U d adenovirus according to claim 2, wherein the Tud RNA that interferes with miR-29a, miR-185 and miR-424 expression is linked to The shuttle plasmid is under the U6 promoter.

[Claim 4] A method for constructing a recombinant Ad-29a-185-424-Tud adenovirus, 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 The Tud RNA complementary single-stranded target gene fragment shown by ID No. 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. The adenoviral shuttle plasmid was co-transfected into 293 cells with the backbone plasmid pHBAd-BHG to obtain recombinant adenovirus Ad-29a-185-424-Tud.