CN101275132B - SiRNA capable of inhibiting influenza A virus replication and coding sequence thereof - Google Patents

Abstract

The present invention provides three pairs of siRNA which inhibits the duplication of influenza virus; an identification technique aiming at the construction of shRNA expression vector pU6-siH1N1 of H1N1 and interference effect for A type influenza virus. Firstly, the invention constructs several expression vectors of siRNA by experimental technique of molecular biology; then determining the validity of interference of siRNA to influenza virus by method of HA titer determined by chick embryo experiment.

Description

A type influenza virus inhibiting siRNA and encoding sequence thereof have been duplicated

Technical field

This patent relates to has the design of siRNA sequence of interference effect and the evaluation of interference effect to influenza virus

Background technology

Influenza (Influenza) is called for short influenza, is called as one of ancient and the most fatal human pestilence.According to historical records, the prevailing disease that has just taken place to cause by influenza as far back as Britain in 1510 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 about 20% death.According to estimates, global annual influenza death number 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.

Influenza is the acute respiratory transmissible disease that is caused respectively by first (A), second (B), third (C) three type influenza viruss (Influenzavirus).The first and second the third have not only reflected viral found age and front and back order, and main is the order that has reflected human hazard rating.The change of first type often occurs with popular form, causes worldwide flu outbreak, and is widely distributed in animal, and also can cause influenza pandemic and cause a large amount of animal deads animal.

Although the anti influenza vaccine of being made up of dead virus, attenuation strain or reorganization surface glycoprotein can protect about 70～80% normal adults to avoid ill effectively; but vaccine can only be at the sub-fraction strain, for the not effect of strain of new potential outburst.And for the crowd of excessive risk crowd such as baby, old man, pregnant woman and other various hypoimmunities, the provide protection of vaccine is very limited, and protection ratio is lower than 40%.Because most death all is to occur among the excessive risk crowd, vaccine but can not play a very good protection for the people that these need most them.Symptom when several Tamiflu of using 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 very eager needs are arranged still, particularly in the excessive risk crowd and outburst during influenza.Therefore, the control of influenza remained a serious problem at least in 50 years from now on, and seeking more effective prevention and treatment approach is the key subjects of medical research.

(RNA interference RNAi) is the sequence-specific PTGS mechanism of at first finding nematode in 1998 by double chain RNA mediate in the RNA interference.RNAi becomes one of focus the most active in the biological study field rapidly once discovery, and the Science magazine is classified it one of as ten big sciences achievement in calendar year 2001, again it is classified as first of the ten big science and technology in 2002; The Nature magazine also is chosen as little RNA one of most important science and technology discovery of 2002 years; Andrew Z.Fire and Craig C.Mello find to have obtained Nobel Prize in Physiology or Medicine in 2006 because of the RNA interferential.Along with the carrying out of extensively and profoundly experimental study, people have had more understanding to the mechanism of RNAi.

Think that at present the main process of RNAi is: 1. siRNA forms the stage.The inductor of RNAi is cut into small molecules interference RNA (the small interferingRNA of 21-23nt by RNaseIII Dicer in tenuigenin, siRNA), the constitutional features of siRNA is 5 ' holds single phosphoric acid, 3 ' terminal hydroxy group, and also 3 ' end has the base of 2～3-nt outstanding; 2. RNA inductive reticent mixture (RNA-induced Silencing Complex, formation stage RISC).SiRNA combines with RNAi specific enzymes (as Ago-2), forms RISC, has specific endonuclease activity and can degrade specifically and siRNA homologous mRNA; 3. effective stage.SiRNA sex change among the RISC, two strands is untied, unload just RNA, sense-rna still is combined on the mixture, and guides RISC to combine with homologous target mRNA, under the effect of endonuclease, (cutting position is in the central authorities of siRNA with target mRNA cut-out, distance 5 ' end 10-nt), translates into protein, show as gene silencing thereby blocked it.MiRNA causes the mechanism of gene silencing slightly different therewith, and (untranslated region UTR) by incomplete complementary combination, suppresses translation process and proteinic synthetic for they and said target mrna 3 ' non-coding region.

RNAi can be applied to the research of multiple biology and function rapidly, and the experimental technique that has benefited from it is simple relatively, and the cycle is short, and phenotype is easy to observe.The factor of most critical is the selection of disturbance target point, preparation and the method for gene introduction of siRNA in the RNAi experiment

As a kind of instrument of quick, effective, special inhibition of gene expression, find especially that after also there is this mechanism in mammalian cell RNAi is widely used in the research of gene function and the treatment research of tumour, virus disease and heredopathia.

Since two thousand three the anti-IAV's of RNAi has a lot, cell levels is arranged, the animal level is also arranged, useful synthetic siRNA's, also useful carrier.Ge etc. are at eight gene design of A type influenza virus virus and synthesized 20 siRNA, with H1N1 cotransfection mdck cell and instar chicken embryo on the ten, results suggest is that the siRNA of target spot has good inhibitory effect to A type influenza virus virus infection with NP and PA gene.Eric Ka-Wai Hui etc. point out that by research M gene with virus is that the chemosynthesis siRNA of target spot can suppress the expression of the matrix prote m1 of influenza virus in the 293T cell specifically, and the siRNAs that utilizes lentiviral vectors to express at the M gene can suppress the expression of M1 in the mdck cell and duplicating of influenza virus equally specifically.Chinese scholar Guo Xiao just waits and adopts the computer program coupling system of oneself writing to grow method for reconstructing, and 6101 siRNA molecules have been carried out the computer screening, and the result shows that the molecule of the siRNA of design should be the most effective at NP and PB1.In the animal level experimentally, Stephen etc. have proved that the special siRNA at the NP of A type influenza virus H1N1 and PA high conservative zone can suppress virus and duplicate in that mouse is intravital, and can suppress to duplicate in the body of multiple H5 and H7 subtype virus.In addition, Qing Ge etc. find the eye socket behind the mouse with the siRNAs of chemosynthesis and a kind of polycation carrier PEI intravenous injection together, can suppress the virus replication in the lung of mouse of virus infection.Same result is found in also that mouse can be expressed the lentiviral vectors of siRNAs precursor and PEI injects altogether or beat into mouse lung altogether with Infasurf.

Summary of the invention:

The object of the present invention is to provide 3 A type influenza virus duplicated inhibiting siRNA and encoding sequence thereof.Described 3 are duplicated the sequence of inhibiting siRNA respectively as sequence table 400＜1 to A type influenza virus 〉, 400＜2 and 400＜3 shown in.

The experimental result of the animal level of three siRNA of the present invention shows, RNAi not only can effectively suppress the virus replication of H1N1, and can suppress duplicating of other hypotype of A type influenza virus (as H3N1), for its treatment and prevention that is used for influenza provides experimental basis.

Embodiment

Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.

The design of embodiment 1:siRNA sequence and the selection of target spot

Design and the rule that should follow of synthetic siRNA are: 1. should avoid selecting 5 ' and 3 ' end UTR and initiation codon district; 2. should select the encoding sequence of its AUG initiator codon downstream 50～100nt location according to the mRNA of goal gene; 3. GC content should be between 30%～50%; 4. avoid 3 same Nucleotide to occur continuously as far as possible; 5. 3 of every chain ' end will have 2 bases (preferably TT) outstanding; 6. last, the dna fragmentation of selection is inquired about through BLAST, should not have homology with other Human genome, to avoid non-specific inhibition.

Adopt this principle, design described three siRNA sequence＜400 of sequence table〉1,＜400〉2 and＜400〉3, and adopt ordinary method to synthesize, carry out following test.

The structure of embodiment 2:shRNA expression vector

With the 129Sv/Ev mouse gene group DNA is that template is carried out PCR, amplifies the U6 promoter sequence of 276bp.The PCR product is through EcoRI and XbaI double digestion, be connected with pBluescript carrier (U.S. Stratagene company) and obtain carrier pU6P, transformed into escherichia coli competent cell, picking positive colony through same double digestion, extract plasmid and check order, to confirm the exactness of insertion sequence.

The structure of embodiment 3:pU6-siH1N1 carrier

Carrier pU6P EcoR V-Xba I double digestion, the plasmid enzyme restriction product separates with 1% agarose gel electrophoresis, cuts glue and carries out the glue recovery with glue recovery test kit, carries out ligation with synthetic siRNA oligonucleotide chain.Transform back picking positive colony.

Embodiment 4: chicken embryo experimental verification pU6-siH1N1 disturbs the validity of H1NI proliferation of influenza virus

Inject H1N1 influenza virus (1000 times of diluent 0.1ml) and pU6-siH1N1 (5 microgram) simultaneously and advance allantoic cavity under the nine age in days chick embryo air sacs, cultivated 72 hours for 33 ℃, get allantoic fluid, survey hemagglutinative titer.

The allantoic fluid virus titer is measured in hemagglutination test, and detailed process is as follows:

(1) drip diluent 1 * PBS 50 μ L in every hole of Microhemagglutination plate, fixed institute adds row's number according to test sample quantity.

(2) draw tested allantoic fluid and drip respectively in the first row hole, each sample 50 μ L, order doubling dilution to the 11 is listed as holes from left to right then, respectively draws 50 μ L from the 11st row hole again and abandons it.Last row do not add sample and make blank.

(3) in every hole, add 0.5% red cell suspension, 50 μ L.

(4) put the 1min that vibrates on the microoscillator, or hand-held blood-coagulation-board is around the circle mixing.

(5) put (18～20 ℃) 30min under the room temperature, or 15～20min under 37 ℃, observations.

Through three repeated experiments, described three sequence＜400 of sequence table〉1,＜400〉2 and＜400〉3 pairs of inhibiting rates that influenza virus is duplicated are as follows:

Sequence	Suppress back virus titer/do not suppress virus titer
		Sequence＜400〉1	8/512
Sequence＜400〉2	16/512
		Sequence＜400〉3	64/512

Sequence table

＜110〉Zhongshan University

＜120〉A type influenza virus inhibiting siRNA and encoding sequence thereof have been duplicated

<160>3

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

<400>1

aatccgaccg ctcttaata 19

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<400>2

aatctggcgc caagctaata a 21

<210>3

<211>19

<212>DNA

＜213〉artificial sequence

<400>3

gaaatctcac tcagttatt 19

Claims (3)

1. one kind is duplicated the siRNA of interference effect to influenza virus, and its nucleotide sequence is as sequence table 400＜1〉shown in.

2. the application of the described siRNA of claim 1 in the medicine of preparation treatment influenza virus.

3. the expression vector pU6-siH1N1 that contains the described siRNA of claim 1.