SiRNAs for inhibiting epidemic encephalitis B viruses

A Japanese encephalitis virus and epidemic technology, applied in the field of small interfering RNA, to prevent escape mutations, increase resistance to Japanese encephalitis virus, and facilitate administration

Inactive Publication Date: 2014-04-09
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this scheme has not been involved in the design of siRNAs against Japanese encephalitis virus in the past.

Method used

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  • SiRNAs for inhibiting epidemic encephalitis B viruses
  • SiRNAs for inhibiting epidemic encephalitis B viruses
  • SiRNAs for inhibiting epidemic encephalitis B viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The selection of embodiment 1siRNA target site

[0068] A total of 64 JE virus genome sequences included in GenBank were analyzed, including 34 Chinese isolates. Using SeqMan software for homology comparison, select the sequence conserved in more than 80% of the global isolates and more than 95% of the Chinese isolates as the target sequence to design siRNAs. The target sequence is designed using the corresponding online design software provided by INVITROGEN according to its siRNAs expression vector

[0069] ( http: / / rnaidesigner.invitrogen.com / rnaiexpress / setOption.do?hl=en des ignOption=mirna&pid=509133211138749536 ). After the design is completed, use BLAST ( www.ncbi.nlm.nih.gov ), compare potential target sequences to the Human Genome Database, excluding those that are homologous to other coding sequences or ESTs. At the same time, a set of negative control siRNAs (negative control, NC) was designed. Table 1 shows the target sequences of siRNAs targeti...

Embodiment 2

[0072] Construction of embodiment 2 siRNAs expression plasmid

[0073] In the present invention, use invitrogen company ( http: / / zh.invitrogen.com / site / cn / zh / home.html ) provided by the pcDNA6.2-GW / EmGFP-miR vector to express the screened siRNAs, according to the requirements of this vector for expressing the sequence structure of siRNAs, entrust Qingke Biological Company ( www.tsingke.com ) The synthetic double-stranded DNA sequences are shown in Table 2, and the underlined bases in the table are the sense strands and antisense strands of siRNAs corresponding to the corresponding target sequences. The sense and antisense strands were mixed in equal proportions, incubated at 95°C for 4 minutes, and then incubated at room temperature to anneal to form double strands. The double strands were ligated into the sticky ends of the pcDNA 6.2-GW / EmGFP-miR vector by T4DNA ligase-mediated ligation ( figure 1 ) constitutes an expression vector for siRNAs required for expression usi...

Embodiment 3

[0077] Example 3 Construction of Fusion Target Sequence Expression Vector

[0078] In order to analyze the interference effect of siRNAs on each target sequence, the inventors constructed a series of reporter plasmids, that is, the nine target sequences listed in Example 1 were respectively cloned into a firefly luciferase (Firefly Luciferase, Fluc) expression vector after chemical synthesis (purchased from Genordia AB, Sweden) (such as figure 2 shown), the specific method is to chemically synthesize the DNA sequences of the sense and antisense strands of the target sequence respectively and then anneal to form a double strand, and then clone the target sequence into the 5' end of the Fluc gene of firefly luciferase to form a fusion gene (such as 1- Fluc, 2-Fluc), the plasmid is transfected into the cell, and the fusion Fluc can be expressed in the cell. If the target sequence is interfered by siRNAs, the expression level of Fluc will decrease, so it can be judged whether ...

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Abstract

The invention relates to siRNAs for inhibiting epidemic encephalitis B viruses and a design method and use thereof. Particularly, based on the whole genome sequence of the epidemic encephalitis B, conserved sequences of separated strains of a genotype 1, a genotype 2, a genotype 3 and a genotype 4 are selected as target sequences. After the siRNAs designed according to the target sequences are transferred into cells, the expression of the genes of the encephalitis B viruses can be inhibited effectively; and when different siRNA molecules are connected in series and transferred into cells, a better inhibition effect can be obtained. The invention also relates to the use of the siRNA molecules in the inhibition of the expression of the gene of the epidemic encephalitis B viruses and in the preparation of medicines for treating and / or preventing epidemic encephalitis B. The invention provides a new approach for inhibiting infection caused by encephalitis B viruses of different genotypes.

Description

technical field [0001] The invention relates to small interfering RNA (siRNA) capable of inducing RNA interference, in particular to siRNA capable of inhibiting Japanese encephalitis virus, and its design method and application. Background technique [0002] Epidemic Japanese encephalitis virus, referred to as Japanese encephalitis virus, is a zoonotic acute infectious disease transmitted by mosquitoes and is one of the most serious causes of human encephalitis worldwide. In 1871, the epidemic of the disease was first reported in Japan. In 1935, Japanese scholars isolated the virus from the brain tissue of a dead encephalitis patient, so the pathogen was named Japanese encephalitis virus (JEV) internationally. According to WHO statistics, there are an average of 50,000 cases and 15,000 deaths worldwide each year. About one-third of patients will die as a result, and patients with neurological sequelae account for 40%-70% of the surviving patients. The disease is mainly prev...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/10C12N15/11A61K48/00A61P31/14
Inventor吴志强金奇
OwnerINST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI