ShRNA for targeted interference of IL-33 expression, recombinant adenovirus vectors as well as construction method thereof, and application

A technology of recombinant adenovirus and construction method, applied in the field of shRNA targeting interfering IL-33 expression, can solve the problems of lack of interfering IL-33 expression technology in mice, liver fibrosis, side effects and the like

Pending Publication Date: 2021-01-12
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IL-33 has been considered as an early warning signal of liver damage, so it can be used as a new liver damage marker to prevent liver diseases; in addition, IL-33 can also be used to treat some chronic liver damage diseases, but excessive use can also Cause liver fibrosis, produce side effects
The removal of IL-33 can also reduce the damage of liver fibrosis. Therefore, IL-33 seems to be a new target for ...

Method used

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  • ShRNA for targeted interference of IL-33 expression, recombinant adenovirus vectors as well as construction method thereof, and application
  • ShRNA for targeted interference of IL-33 expression, recombinant adenovirus vectors as well as construction method thereof, and application
  • ShRNA for targeted interference of IL-33 expression, recombinant adenovirus vectors as well as construction method thereof, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Artificially synthesized mouse IL-33(250), IL-33(456), and IL-33(594) interference sequences, and added PstI and BamHI restriction sites at both ends, respectively, to obtain shIL-33(250) The nucleotide sequence is shown in SEQ ID NO:1; the nucleotide sequence of shIL-33(456) is shown in SEQ ID NO:2; the nucleotide sequence of shIL-33(594) is shown in SEQ ID NO:3 shown.

Embodiment 2

[0082] The pDC316-ZsGreen1-shRNA vector was double-digested with restriction endonucleases PstI and BamHI. The enzyme digestion reaction conditions were incubated at a constant temperature at 37°C for 4 hours. The enzyme digestion system was shown in Table 1. The size of the target band was detected by agarose gel electrophoresis, and the linear vector was recovered to obtain the linearized pDC316-ZsGreen1-shRNA vector. see results figure 1 .

[0083] Table 1 enzyme digestion system

[0084] Reagent Volume (μL) CutSmart Buffer(10×) 5 endonuclease PstI 2 endonuclease BamHI 2 pDC316-ZsGreen1-shRNA plasmid 10μg wxya 2 o

Make up to 50μL

[0085] The three artificially synthesized IL-33 interference fragments prepared in Example 1 were respectively connected to the linearized vector by T4. The connection system is shown in Table 2. The reaction conditions for the connection were 16°C for overnight connection to obtain three kind...

Embodiment 3

[0092] A shRNA recombinant adenovirus packaging method that interferes with the mouse IL-33 gene. Three recombinant plasmids pDC316-ZsGreen1-shIL-33 and the shuttle plasmid pBHGlox are co-transfected into HEK293 cells to realize the packaging of recombinant adenoviruses. Specifically prepared by the following steps:

[0093] 1) Seed HEK293 cells in a 6-well plate so that the number of cells per well is about 5×10 5 indivual;

[0094] 2) The three recombinant adenovirus plasmids pDC316-ZsGreen1-shIL-33 and the shuttle plasmid pBHGlox(delata)E1,3Cre prepared in Example 2 were respectively adjusted so that the total mass was 4 μg, and mixed with 500 μL DMEM medium to form a mixture Mix1;

[0095] 3) Mix 12 μL Lipofectamine TM 2000 and 500 μL DMEM medium to form a mixture Mix2, mix Mix1 and Mix2 evenly, and let stand for 10 minutes;

[0096] 4) Add the mixed solution obtained in step 3) into a 6-well plate drop by drop, mix well and place it at 37°C in 5% CO 2 Culture in an inc...

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Abstract

The invention provides a set of shRNA for targeted interference of IL-33 gene or protein expression, recombinant adenovirus vectors as well as a construction method thereof, and application, and belongs to the technical field of gene engineering. According to the set of shRNA for targeted interference of IL-33 gene or protein expression, a nucleotide sequence corresponding thereto is shown as SEQID NOs: 1-3. The set of recombinant adenovirus vectors each contain three shRNA sequences. Eukaryotic cells are infected with the three recombinant adenovirus vectors and shuttle plasmids under the conditions of lipidosome and DMEM culture media, and three recombinant adenoviruses capable of interfering IL-33 gene expression are packaged. The three recombinant adenoviruses are used together, so that the IL-33 gene and protein expression in mice can be effectively reduced, and a technical support is provided for deep research of a mechanism of IL-33 as a hepatic fibrosis treatment target.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a group of shRNA targeted to interfere with the expression of IL-33, a recombinant adenovirus vector and a construction method and application thereof. Background technique [0002] RNA interference technology is a newly developed genetic engineering technology in the past ten years. It can inhibit the expression of target genes with high specificity, and provides a valuable tool for studying the biological function of a gene and personalized treatment. Infecting cells with adenoviral vectors can be used to transfer target genes. Due to its high-efficiency and stable transfection characteristics, it is widely used in the study of gene silencing. There are many members of the adenovirus family, but only two are used to construct adenovirus vectors, both of which belong to subgenus C, one is adenovirus type 2 (Ad2), and the other is adenovirus type 5 (Ad5). ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/861C12N15/66A61K31/7088A61P1/16
CPCC12N15/1136C12N15/86C12N15/66A61P1/16C12N2310/14C12N2710/10343C12N2800/107C12N2320/32
Inventor 张泉贾苗苗曹舒扬朱立麒殷俊
Owner YANGZHOU UNIV
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