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SiRNA for specifically knocking down TNF-alpha gene expression and application thereof

An α gene and species-specific technology, applied in the field of siRNA, to improve the ability to resist enzymolysis and increase the residence time in the body

Active Publication Date: 2022-04-29
SILICON GENE TECH (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In systemic lupus erythematosus, the expression of TNF-α in the blood of patients is higher than that of healthy people, but inhibiting the expression of TNF-α may inhibit its immune system regulation function

Method used

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  • SiRNA for specifically knocking down TNF-alpha gene expression and application thereof
  • SiRNA for specifically knocking down TNF-alpha gene expression and application thereof
  • SiRNA for specifically knocking down TNF-alpha gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Obtaining unmodified or modified siTNF-α

[0030] (1) The siRNA sequence is designed online in 'siRNA Target Finder', and the 5' untranslated region (5'UTR) and 3' untranslated region (3'UTR) and the sequence near the start codon should not be used as the design when designing The template of siRNA should also avoid the junction region between exon and exon. Search for the ideal siRNA starting from the 100bp nucleotides downstream of the start codon AUG of the target gene. Three pairs of siTNF-α were designed. One pair of siRNAs with the highest level of specific knockdown was confirmed by preliminary experiments (the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively).

[0031](2) Using siRNAs with nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively as negative controls, the siRNA with the highest specific knockdown level and the negative control were modified diffe...

Embodiment 2

[0042] Example 2: Unmodified or modified siTNF-α transfected cells, detecting the mRNA expression level of TNF-α in cells

[0043] (1) Transfection

[0044] ①To quantify siRNA, dilute to 20uM with DEPC water.

[0045] ②HaCat cells were passaged to 12-well plates, and the density reached 30%-50% the next day before transfection.

[0046] ③For transfection, add 2.5ul siRNA to 50ul DMEM medium as tube A, add 5ul lip3000 to 50ul DMEM medium as tube B, add tube A to tube B, mix well, and let stand at room temperature for 15 minutes. Add dropwise to a 12-well plate. Set up control NC group, unmodified or modified siTNF-α group, and do 3 parallels for each experimental group.

[0047] (2) RNA extraction

[0048] Use the Tiangen kit 'RNA prep Pure Cell / Bacteria Kit', the article number is DP430, the specific operation is as follows:

[0049] ① Remove the cell culture medium, add 500ul PBS to each well to wash the cells, discard, twice.

[0050] ②Add 350ul lysate RL to each well,...

Embodiment 3

[0072] Example 3: Unmodified or modified siTNF-α transfected cells, detecting the protein expression level of TNF-α in cells

[0073] (1) Transfection

[0074] ①To quantify siRNA, dilute to 20uM with DEPC water. Confirm that the transfection concentration is 50 pmol.

[0075] ②HaCat cells were passaged to 12-well plates, and the density reached 30%-50% the next day before transfection.

[0076] ③For transfection, add 2.5ul siRNA to 50ul DMEM medium as tube A, add 5ul lip3000 to 50ul DMEM medium as tube B, add tube A to tube B, mix well, and let stand at room temperature for 15 minutes. Add dropwise to a 12-well plate. Set up control NC group, unmodified or modified siTNF-α group, and do 3 parallels for each experimental group.

[0077] ④ One additional well was set up, and 50 pmol of modified siTNF-α with FAM fluorescent group was transfected.

[0078] (2) After the cells were cultured for 48 hours, the cells were collected and the protein was extracted

[0079] ① After ...

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Abstract

The invention discloses siRNA for specifically knocking down TNF-alpha gene expression and application thereof.The siRNA sequence provided by the invention can specifically knock down the TNF-alpha transcriptional level, a positive-sense strand is modified through sulfo, on the basis that cytotoxicity is not obviously increased, the anti-enzymolysis capacity in serum is greatly improved, the in-vivo residence time of the siRNA is prolonged, and the siRNA can be used for preparing a medicine for treating the TNF-alpha. And potential application values can be exerted in treatment of various tumor diseases and autoimmune diseases.

Description

technical field [0001] The invention relates to a siRNA for specifically knocking down TNF-α gene expression and application thereof, belonging to the technical field of biomedicine. Background technique [0002] siRNA generally refers to double-stranded RNA with a length of 19-25 bp and complementary base pairing. At present, siRNA drugs are already on the market, and more siRNA drugs are in clinical trials. The rich components in serum, especially the diversity of enzymes, make siRNA antiserum less capable of enzymatic hydrolysis and easy to degrade, which greatly affects the duration of siRNA drugs in the human body. In clinical siRNA drugs, the siRNA sequence is chemically modified to increase the residence time of siRNA in vivo. The currently widely used modification methods are as follows. 1. Phosphate backbone modification. The more classic one is thio-modification, that is, in the phosphodiester bond linked by RNA bases, the oxygen atom of the phosphate group is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P35/00A61P37/02A61P19/06A61P19/08A61P29/00
CPCC12N15/1136A61K31/713A61P35/00A61P37/02A61P19/06A61P19/08A61P29/00C12N2310/141C12N2310/315Y02A50/30
Inventor 李萍唐雪明
Owner SILICON GENE TECH (SHANGHAI) CO LTD
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