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Deoxyzyme of anti-respiratory syncytial viruses and its medicinal use

A syncytial virus and anti-respiratory technology, which is applied to the anti-respiratory syncytial virus deoxyribozyme and its application in medicine, and can solve the problems of no reliable vaccine and anti-RSV preparations.

Inactive Publication Date: 2007-09-05
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite years of effort, so far there is no reliable vaccine against RSV or specific anti-RSV agents available

Method used

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  • Deoxyzyme of anti-respiratory syncytial viruses and its medicinal use
  • Deoxyzyme of anti-respiratory syncytial viruses and its medicinal use
  • Deoxyzyme of anti-respiratory syncytial viruses and its medicinal use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Design of deoxyribozyme constructs. The selection of the DZ cleavage site in the target RNA mainly considers three factors:

[0063] First, this site is critical for viral replication. The genome of RSV is a non-phasic negative-strand RNA containing approximately 15,000 nucleotides with a structure of 3'-NS1-NS2-N-P-M-SH-G-F-M2(M2-1-M2-2)-L-5' , encoding the corresponding 11 viral proteins: nucleocapsid protein N, phosphoprotein P, polymerase subunit L, transcription elongation factor M2-1, fusion protein F, adhesion protein G and small hydrophobin SH, nonstructural protein NS1 and NS2, matrix protein M and M2-2. During virus replication, nucleocapsid protein N plays an important function. It is an important part of the viral polymerase complex (N, P, L proteins). The viral mRNA is transcribed from the negative-strand genomic RNA under the action of the polymerase complex. This polymerase complex also uses the positive-strand intermediate RNA A new negati...

Embodiment 2

[0071] Example 2: In Vitro Cleavage of RNA Targets by DNAzymes

[0072] Hep-2 human laryngeal epithelial carcinoma cells were routinely cultured in the limit essential essential oil containing 10% inactivated fetal bovine serum (FBS, Hyclon, Logan, UT), 2mM L-glutamine, 100u / ml penicillin and 100μg / ml streptomycin Medium (Dulbecco's modified Eagle's medium Dulbecco, DMEM), culture conditions: 37 ° C, 5% CO 2 .

[0073] Human respiratory syncytial virus subtype B standard strain RSV CH18537 was subcultured in Hep-2 cells cultured in DMEM (virus maintenance solution) containing 2% fetal bovine serum and antibiotics. When the Cytopathic Effect (CPE) reaches 75%-100%, that is, CPE+++-++++ (infected cytopathic degree evaluation method: 0%-25% cells have lesions, which are recorded as CPE+; 25%-50% cells have lesions Lesions, recorded as CPE++; 50%-75% of cells with diseased, recorded as CPE+++; 75%-100% of cells with pathological changes, recorded as CPE++++) Collect and aliquot ...

Embodiment 3

[0077] Example 3: Inhibitory effect of deoxyribozymes on viral replication in infected cells

[0078] Hep-2 cells were routinely cultured in DMEM medium containing antibiotics and 10% FBS. The RSV A2 standard strain, CH 18537 strain, and six wild strains in Chongqing were isolated from the snuff secretions of hospitalized patients; the other four wild RSV strains were isolated in the 2001-2002 epidemic season in Beijing, and these four clinical isolates were not tested. Typing. All the above viruses were subcultured in Hep-2 cells at 37° C., and the culture medium was DMEM (virus maintenance solution) containing antibiotics and 2% FBS. The supernatant was collected when the cytopathy reached 75%-100% (CPE+++-++++), and stored at -80°C after aliquoting. Virus titers were determined on Hep-2 cultured cells using a plaque formation assay. The titer of the virus used was 1.7×10 5 to 6.5×10 7 / ml.

[0079] A monolayer of Hep-2 cells in a 6-well culture plate was infected with...

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Abstract

A deoxyribonse of anti-respiratory tract syncytium virus and its medicinal use are disclosed. The deoxyribonse has purine in RSV RNA nucleotide sequence; pyramine site cracks RNA catalytic active domain specially, the No.1 combined domain adjacent to catalytic domain 5' end is supplemented combined with base sequence specificity at one side of RSV RNA incision site; the No.2 combined domain adjacent to catalytic domain 3' end is supplemented combined with base sequence specificity at one side of RSV RNA incision site. It contains 0.01mg-20mg / 50 mu l anti-respiratory tract syncytium virus deoxyribonse solution. It is non-toxic, has better medicine sensitivity and can inhibit respiratory tract syncytium virus duplication specially.

Description

technical field [0001] The invention relates to a deoxyribozyme against respiratory syncytial virus and its application in medicine. technical background [0002] Deoxyribozyme (DNAzyme, abbreviated as: DZ) is a new and effective means capable of inhibiting the expression of RNA (ribonucleic acid) in cells. It obtains DNA (deoxyribonucleic acid) molecules with RNA cutting function by screening random sequence libraries synthesized in vitro, and can theoretically specifically cut RNA molecules of any sequence. Among them, the one with the strongest function and the most research is 10-23 deoxyribozyme for short: 10-23DZ. 10-23DZ consists of a catalytic core of 15 nucleotides and flanking sequences of 7-11 nucleotides on both sides (see Figure 1). RNA substrates are cleaved between unpaired purine (A, G) and paired pyrimidine residues (C, U). This cleavage is highly sequence-specific and theoretically can cut any target RNA. Since the first case of DZ was reported, biologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/22
Inventor 杨锡强赵晓东蒋利萍解元元周娟崔玉霞刘崇海王莉佳赵耀
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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