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Application of viral nucleic acid sequence strictly conservative area as siRNA design template

A technology of highly conserved regions and viral nucleic acids, applied in the application field of highly conserved regions of viral nucleic acid sequences as siRNA design templates, can solve problems such as inexplicability, and achieve the effect of inhibiting abnormal proliferation of new blood vessels

Active Publication Date: 2012-02-29
百源科创分子医学研究所(南通)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem is that it is impossible to explain how these naked siRNAs enter the cell and degrade the target mRNA through the RISC bypass to inhibit CNV

Method used

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  • Application of viral nucleic acid sequence strictly conservative area as siRNA design template
  • Application of viral nucleic acid sequence strictly conservative area as siRNA design template
  • Application of viral nucleic acid sequence strictly conservative area as siRNA design template

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Establishment of AMD Animal Model

[0080] 1. Main instruments, reagents and materials

[0081] 1.1 Instruments: 5.4mm hand-held contact lens (Ocular Instruments, Bellevue, WA), krypton ion laser machine (Coherent, Nuvus2000), fluorescence microscope (Eclipse TS100; Nikon, Tokyo, Japan), anterior segment slit lamp photomicrograph system, Confocal laser retinal tomography (Heidelberg retinatomo-graph II, HRT II; Heidelberg, Germany).

[0082] 1.2 Experimental animals: C57BL / 6J mice, weighing 20-30g.

[0083] 1.3 Materials and reagents: 0.3% sodium pentobarbital (45mg / kg), compound tropicamide (Japan Santen Pharmaceutical Co., Ltd.), 1% methylcellulose, 10% sodium fluorescein (Guangxi Wuzhou Pharmaceutical Co., Ltd. ), 4% paraformaldehyde (diluted with pH 7.3 phosphate buffer, Sinopharm Chemical Reagent Co., Ltd.), 10mg 4', 6-diamidino-2-phenylindole hydrochloride (DAPI), 0.2u / μl Phalloidin-Alexa Fluor 488 and 1 μg / μl Isolectin-B4 (IsolectinB4-Alexa Fluor 568) (Invitr...

Embodiment 2

[0099] Preparation of siRNA

[0100] 1 Main instruments, reagents and materials

[0101] 1.1 Main instruments: nucleic acid synthesizer (GE), microinjector (HAMLTON), etc.

[0102] 1.2 Materials and reagents: chemically synthesized siRNA, antibody drug Avastin (Genentech), 5% glucose solution, etc.

[0103] 1.3 siRNA sequence:

[0104] siRNA code siRNA sequence (5'→3')

[0105] siEv70_1 sense CAAUGCAUUGAGAGGCCUGGAUdTdT SEQ ID NO: 4

[0106] Antisense AUCCAGGCCUCUCAAUGCAUUGdTdT SEQ ID NO: 5

[0107] siEv70_2 * Sense UAGGUACUUCGAGAAGCCUAGUAdTdT SEQ ID NO: 6

[0108] Antisense UACUAGGCUUCUCGAAGUACCUAdTdT SEQ ID NO: 7

[0109] siEv70_3 Sense CCUGAGAGUCCUCCGGCCCCUdTdT SEQ ID NO: 8

[0110] Antisense AGGGGCCGGAGGACUCUCAGGdTdT SEQ ID NO: 9

[0111] siEv70_4 sense AGAGAGAAAAGUAGAGAAGGGCdTdT SEQ ID NO: 10

[0112] Antisense GCCCUUCUCUACUUUCUCUCUdTdT SEQ ID NO: 11

[0113] siEv70_5 sense UAGGAACCCAAGAACCCCUCCdTdT SEQ ID NO: 12

[...

Embodiment 3

[0125] Screening of effective siRNA by immunofluorescence labeling

[0126] 1 Main instruments, reagents and materials

[0127] 1.1 Instruments: 5.4mm hand-held contact lens (Ocular Instruments, Bellevue, WA), krypton ion laser machine (Coherent, Nuvus2000), fluorescence microscope (Eclipse TS 100; Nikon, Tokyo, Japan), confocal laser retinal tomography scanner ( Heidelberg retinal graph II, HRT II; Heidelberg, Germany) et al.

[0128] 1.2 Materials and reagents: 0.3% sodium pentobarbital (45mg / kg), compound tropicamide (Japan Santen Pharmaceutical Co., Ltd.), 1% methylcellulose, 10% sodium fluorescein (Guangxi Wuzhou Pharmaceutical Co., Ltd. ), 4% paraformaldehyde (diluted with pH 7.3 phosphate buffer, Sinopharm Chemical Reagent Co., Ltd.), etc.;

[0129] Fluorescent antibody: 10 mg 4′,6-diamidino-2-phenylindole hydrochloride (DAPI), 0.2 U / μl phalloidin (phalloidin-Alexa Fluor 488) and 1 μg / μl isolectin-B4 (isolectin B4-Alexa Fluor 568) (Invitrogen-Molecular Probes, Eugene...

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Abstract

The invention relates to application of a viral nucleic acid sequence strictly conservative area as a siRNA design template. The designed siRNA sequence can be identified by a TLR (Toll Like Receptor) 3 of a cell membrane to be used as a high-efficiency exogenous ligand for activating a signal path of the TLR 3 so as to inhibit new blood vessel anomalies. The invention also relates to applicationof the siRNA sequence in preparing medicine for curing diseases relevant to the new blood vessel anomalies.

Description

technical field [0001] The present invention relates to the application of highly conserved fragments of viral nucleic acid sequences as siRNA design templates, especially the application of highly conserved fragments of enterovirus nucleic acid sequences as siRNA drug design templates. Active ingredients in medicines. Background technique [0002] siRNA (small interfering RNA: small fragment interfering ribonucleic acid) is a short nucleic acid base fragment with a specific gene code, and the general functional length is 21-23bp. Its principle of action is to combine with specific target gene mRNA to degrade it and lose its function. This gene silencing phenomenon of efficient transcription mediated by double-stranded RNA molecules is called RNAi (RNAinterference: ribonucleic acid interference), which is a defense ability naturally present in cells: double-stranded RNA enters cells and is cut into 21-25 The siRNA of the nucleic acid base fragment first combines with other...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11A61K48/00A61P27/02A61P35/00C12R1/93
Inventor 朱远源陈莉李铁军陆毅祥孙云成王晋康
Owner 百源科创分子医学研究所(南通)有限公司
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