CN103966213A

CN103966213A - Design of siRNA sequences possessing interference effect on influenza A virus M gene and identification on interference effect

Info

Publication number: CN103966213A
Application number: CN201310048356.8A
Authority: CN
Inventors: 任政华; 霍晋
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-02-06
Filing date: 2013-02-06
Publication date: 2014-08-06

Abstract

The invention discloses three kinds of siRNA with inhibition effect on copying of influenza virus M gene, and by employing a method of determining HA titer through chicken embryo experiments, the interference effectiveness of siRNA carrier plasmid on influenza virus is determined.

Description

A type influenza virus M gene has the design of siRNA sequence and the evaluation of interference effect of interference effect

Technical field

This patent relates to the design of siRNA sequence and the evaluation of interference effect that A type influenza virus M gene is had to interference effect, belongs to biological technical field.

Background technology

Influenza (Influenza), is called for short influenza, is called as one of the most ancient and the most fatal mankind's pestilence.According to historical records, as far back as Britain in 1510, just there is the prevailing disease being caused by influenza by global the first.Nowadays, the U.S. has 20,000 people to die from influenza every year, and Russia also has 10,000 people to die from influenza every year, and Britain has 50,000 people to develop into pneumonia by influenza every year, wherein approximately 20% death.According to estimates, the annual influenza death number in the whole world is up to 600,000 people, more than the number of AIDS patient's death.The U.S. has classified influenza as life-threatening the fifth-largest " killer " after heart trouble, cancer, apoplexy, pulmonary emphysema.

The infringement to people to flu-prevention at present; by dead virus, attenuation strain or restructuring surface glycoprotein, formed etc. at present all kinds of vaccines of flu-prevention; but these vaccines can protect approximately 70～80% normal adults to avoid ill effectively; but vaccine can only be for sub-fraction strain, for the not effect of strain of new potential outburst.And as the crowd of baby, old man, pregnant woman and other various hypoimmunities, the provide protection of vaccine is very limited for High risk group, protection ratio is lower than 40%.Because most death is all to occur in High risk group, the people that vaccine needs most them for these but can not play a very good protection.Symptom when several Tamiflu of applying also can reduce the generation of influenza infection and alleviate influenza infection.But Side effects of pharmaceutical drugs, patient's ability to bear, the variation of drug resistance that may occur have limited the extensive use of these medicines.So, for the method for prevention and treatment influenza infection, still have very eager needs, particularly in High risk group and while breaking out influenza.Therefore, the control of influenza at least remained serious problem in 50 years from now on, and finding more effective prevention and treatment approach is the key subjects of medical research.

As far back as nineteen ninety, the people such as Napoli and Jorgensen find in the experiment of doing transgenic plant, to petunia, inject ectogenic chromogene, find that this does not only make pattern deepen, and makes pattern occur mottled even completely colourless on the contrary.This means that not only foreign gene is inactivated, endogenous homologous gene has also been subject to inhibition.They call co-suppression (co-suppress) this phenomenon.

1994, Macino and Cogoni found, the synthetic required gene of carotenoid is imported in coarse Neurospora, can cause the gene inactivation of mould itself in 30% transformant, and were referred to as the tranquillization effect (quelling) of gene ^[8].

Nineteen ninety-five, during the expression of the Guo of Cornell University and the Kemphues par-1 gene in utilizing the beautiful new rhabditis axei of Antisense RNA Technique blocking-up (C.elegans), be surprised to find that, in control experiment, to nematode, inject just RNA and not only do not increase the expression of this gene, as sense-rna, also can block the expression of this gene on the contrary ^[9].Traditional explanation facility of this and Antisense RNA Technique is disagreed completely, and this phenomenon cannot be explained always by this group.

Until in February, 1998, the Craig C.Mello of Cancer center of the graduate Andrew Z.Fire of Washington Ka Naiji He He Ma Sazhusai university attempts injecting nematode with double-stranded RNA for the first time ^[10], found that dsRNA can efficiently block the expression of corresponding gene specifically, suppression efficiency is at least high 10 times than single stranded RNA.They further point out just RNA that Guo etc. the finds inhibition to genetic expression, and utilize in the past the blocking-up of Antisense RNA Technique to genetic expression, are all that the micro-dsRNA polluting in the RNA being prepared by in-vitro transcription causes.Fire etc. proposed that a new concept---RNA disturbs (RNA interference, RNAi) first afterwards, the sequence-specific gene silencing being mediated by dsRNA (gene silencing).

The RNA interference phenomenon that this dsRNA causes is also found respectively subsequently in the biologies such as fruit bat, insect, fungi, plant Arabidopis thaliana and Mammals and the mankind's various kinds of cell.

Originally generally believe that co-suppress, quelling and RNAi are the gene silencing phenomenons of different mechanisms, but they have common molecular basis along with the deep discovery of studying, all PTGS (post-transcriptional gene silencing, PTGS) different expression form of mechanism is that ubiquitous opposing poisoning intrusion in eukaryote, inhibition transposon are movable, the monitoring mechanism of regulate gene expression.

RNAi, once discovery, becomes rapidly one of focus the most active in biological study field, and Science magazine is classified as one of ten big science achievements in calendar year 2001, within 2002, is classified as again first of ten large science and technology; Nature magazine is also chosen as little RNA one of most important science and technology discovery of 2002 years; Andrew Z.Fire and Craig C.Mello have obtained Nobel Prize in Physiology or Medicine in 2006 because of the discovery that RNA disturbs.Along with the carrying out of experimental study extensively and profoundly, people have had preliminary understanding to the mechanism of RNAi.

In a series of famous experiments, Zamore and colleague find that the dsRNA of injection drosophila cell is cut into the RNA fragment of 21～23 base length, they find simultaneously: with the mRNA of the native gene of dsRNA homology, be only cut into the long segment of 21-23 Nucleotide with position corresponding to dsRNA ^[11].This has raised the machine-processed prelude that discloses RNAi.

According to the difference of organism, the inductor of RNAi can be various molecules, comprises long-chain dsRNA, shRNA or the little RNA of endogenous hair clip sample (hairpin microRNA, miRNA) that plasmid produces.Think at present the main process of RNAi ^[12]for: 1. siRNA formation stages.The inductor of RNAi is cut into small molecules interference RNA (the small interfering RNA of 21-23nt in tenuigenin by RNaseIII Dicer, siRNA), the constitutional features of siRNA is 5 ' end monophosphate, 3 ' terminal hydroxy group, and also 3 ' end has the base of 2～3-nt outstanding; The formation stages of the reticent mixture (RNA-induced Silencing Complex, RISC) that 2. RNA induces.SiRNA and RNAi specific enzymes (as A go-2) combination, forms RISC, has the mRNA that specific endonuclease activity can be degraded specifically with siRNA homology; 3. effective stage.SiRNA sex change in RISC, two strands is untied, unload just RNA, sense-rna is still combined on mixture, and guides RISC to be combined with the target mRNA of homology, under the effect of endonuclease, by target mRNA cut-out, (cutting position is in the central authorities of siRNA, distance 5 ' end 10-nt), thereby blocked it, translate into protein, show as gene silencing.MiRNA causes the mechanism of gene silencing slightly different therewith, and they and said target mrna 3 ' non-coding region (untranslated region, UTR), by incomplete complementary combination, suppress the synthetic of translation process and protein.But also there are some researches show, miRNA can cut-out by mediation mRNA the same as siRNA carry out inhibition of gene expression ^[13].

On RNAi path, Dicer and RISC are two most important protein complexes.The structure and function of understanding these two complex bodys has vital effect for the mechanism of understanding RNAi.

Dicer enzyme is one of member of rnase iii (RNase III) family, and about 200kD finds the earliest in fruit bat, can specifically dsRNA be cut into 21～23nt5 ' phosphorylation, and 3 ' end has the siRNA of the outstanding end of 2～3 Nucleotide.Dicer enzyme contains 4 structural regions, from N, hold C end to be followed successively by: the RNase III region of DEAD/H uncoiling district, PAZ region, 2 series connection (RNase IIIa and RNase IIIb) and double-stranded RNA land (double-stranded RNA-binding domain, dsRBD).Near the conservative region DUF283 of the unknown function being formed by 100 left and right amino acid most of Dicer albumen PAZ region ^[15].Research shows, the function of PAZ structural domain is in conjunction with strand or double-stranded siRNA or DNA, and this combination and nucleotide sequence are irrelevant and avidity is lower.

RNAi be a kind of on evolving conservative mechanism, can see genomic " immunity system " as, its effect is mainly reflected in the following aspects: 1. RNAi is a kind of Antiviral Mechanism.Much virus is usingd RNA as genetic material, and can form dsRNA in some links of life cycle, and this dsRNA will be identified by the Dicer of organism, cuts into the segment of 21-23nt, starts RNAi process and then blocking virus protein expression.2. RNAi safeguards genomic stability by suppressing the movement of transposon and tumor-necrosis factor glycoproteins.3. MicroRNAs regulates and safeguards normal gene expression dose, as can be regulated the expression of albumen and determine the growth selection on opportunity.

As a kind of instrument of quick, effective, special inhibition of gene expression, be widely used in treatment and prevent each virosis.

Summary of the invention

The invention provides three A type influenza virus has been copied to inhibiting siRNA and encoding sequence thereof.As sequence table 400<1>, 400<2>, shown in 400<3>.

The vector expression thing of three siRNA of the present invention shows in the experimental result of animal level, RNAi efficiency inhibition H1N1, H2N3, the virus replication of H9N2.

Embodiment

Embodiment 1: chicken embryo experimental verification siRNA disturbs H9N2 proliferation of influenza virus effect checking

H9N2 virus virulence ED ₅₀10 ^8.0/ 0.1ml, contains 100 ED by H9N2 viral dilution ₅₀virus liquid, volume 0.1ml, is expelled in 9 age in days SPF chick embryo allantoic cavities, injects SiRNA vector plasmid 6 micrograms simultaneously, cultivates 72 hours for 33 ℃, gets allantoic fluid, surveys hemagglutinative titer.

Embodiment 2: chicken embryo experimental verification recombinant plasmid disturbs H1N1 proliferation of influenza virus effect checking

H1N1 virus virulence ED5010 ^8..6/ 0.1ml, contains 100 ED by H1N1 virus dilution ₅₀virus liquid, volume 0.1ml, is expelled in 9 age in days SPF chick embryo allantoic cavities, injects SiRNA vector plasmid 6 micrograms simultaneously, cultivates 72 hours for 33 ℃, gets allantoic fluid, surveys hemagglutinative titer.

Embodiment 3: chicken embryo experimental verification siRNA disturbs H3N2 proliferation of influenza virus effect checking

H3N2 virus virulence ED5010 ^8..0/ 0.1ml, contains 100 ED by H3N2 viral dilution ₅₀virus liquid, volume 0.1ml, is expelled in 9 age in days SPF chick embryo allantoic cavities, injects SiRNA vector plasmid 6 micrograms simultaneously, cultivates 72 hours for 33 ℃, gets allantoic fluid, surveys hemagglutinative titer.

Embodiment 4: allantoic fluid virus titer detects

(1) in every hole of Microhemagglutination plate, drip PH7.2PBS100 μ L, (2) draw tested allantoic fluid sample 100 μ L, doubling dilution 12 holes, and the 12nd hole discards 100 μ L.In every hole, add 0.1% red cell suspension 100 μ L.

(2) establish negative control and influenza virus positive control.

(4) putting the 1min that vibrates on microoscillator mixes.

(5) put (18～20 ℃) 30min under room temperature, observations.

Through three times, repeat experiment, three sequence <400>1 described in sequence table, <400>2, the inhibiting rate that <400>3 infected by influenza copies is as follows:

Claims

1. infected by influenza M gene replication has the siRNA of interference effect, its nucleotide sequence is as sequence table 400<1>, 400<2>, shown in 400<3>.

2. claim 1, the application to described siRNA as the target gene of inhibition influenza virus M gene silencing.