CN101760457B - SiRNA inhibiting duplication of A type influenza virus and coding sequence thereof - Google Patents

Abstract

The invention discloses a piece of siRNA inhibiting duplication of A type influenza virus, which relates to the authentication technology aiming at architecture of expression vector pU6-siH1N1 of shRNA of H1N1 and interference action on A type influenza virus thereof. In the invention, the experimental technology of molecular biology is utilized for the first time to structure dozens of pieces of expression vectors of siRNA; then the method for testing HA titer by chick embryo experiment is used for confirming the effectiveness of siRNA to influenza virus interference.

Description

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

The application is dividing an application of following application for a patent for invention:

The applying date: on March 6th, 2008

Application number: 200810026651.2

Denomination of invention: A type influenza virus has been copied inhibiting siRNA and encoding sequence thereof

Technical field

This patent relates to infected by influenza the design of siRNA sequence of interference effect and the evaluation of interference effect

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 is caused by influenza by global the first has just occured as far back as Britain in 1510.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, 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 infectious 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.

70～80% normal adults avoids ill although the anti influenza vaccine that is comprised of dead virus, attenuation strain or restructuring surface glycoprotein can be protected approximately effectively; but vaccine can only be for the sub-fraction strain, for the not effect of strain of new potential outburst.And for the crowd of High risk group 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 in the High risk group, 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 High risk group 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.

It is the sequence-specific PTGS mechanism by double chain RNA mediate of at first finding nematode in 1998 that RNA disturbs (RNA interference, RNAi).RNAi becomes rapidly one of focus the most active in the biological study field 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 large 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 have obtained Nobel Prize in Physiology or Medicine in 2006 because of the discovery that RNA disturbs.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 formation stages.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 ' end monophosphate, 3 ' terminal hydroxy group, and also 3 ' end has the base of 2～3-nt outstanding; 2. the formation stages of the RNA reticent mixture (RNA-induced Silencing Complex, RISC) of inducing.SiRNA and RNAi specific enzymes (such as Ago-2) combination form RISC, have can degrade specifically mRNA with the siRNA homology of specific endonuclease activity; 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 be combined with the target mRNA of homology, 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 thereby blocked it, 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) suppress the synthetic of translation process and protein by incomplete complementary combination.

RNAi can be applied to rapidly the research of multiple biology and function, and the experimental technique that has benefited from it is relatively simple, 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, especially find 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, and the animal level is also arranged, useful synthetic siRNA, also useful carrier.Ge etc. are for 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, the siRNA of results suggest take NP and PA gene as target spot has preferably restraining effect to A type influenza virus virus infection.Eric Ka-Wai Hui etc. points out can suppress specifically as the chemosynthesis siRNA of target spot take the M gene of virus the expression of the matrix prote m1 of influenza virus in the 293T cell by research, and the siRNAs that utilizes lentiviral vectors to express for the M gene can suppress the expression of M1 in the mdck cell and copying 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 for NP and PB1.In the animal level experimentally, Stephen etc. have proved that the special siRNA for the NP of A type influenza virus H1N1 and PA high conservative zone can suppress virus copying in Mice Body, and can suppress to copy 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 together intravenous injection, 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 altogether mouse lung with Infasurf.

Summary of the invention:

The object of the present invention is to provide 3 A type influenza virus copied inhibiting siRNA and encoding sequence thereof.Described 3 are copied the sequence of inhibiting siRNA respectively such 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 establishment H1N1 virus replication, and can suppress copying of other hypotype of A type influenza virus (such 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 in any form the present invention.

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 according to the mRNA of goal gene, select the encoding sequence of its AUG initiator codon downstream 50～100nt location; 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 with other Human genome without homology, 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

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

Embodiment 3:pU6-siH1N1 Vector construction

Carrier pU6P separates with 1% agarose gel electrophoresis with EcoRV-Xba I double digestion, plasmid enzyme restriction product, cuts glue and carries out the glue recovery with glue recovery test kit, carries out ligation with the siRNA oligonucleotide chain that synthesizes.Picking positive colony after transforming.

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

Inject simultaneously H1N1 influenza virus (1000 times of diluent 0.1ml) and pU6-siH1N1 (5 microgram) 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 according to test sample quantity.

(2) draw tested allantoic fluid and drip respectively in the first row hole, each sample 50 μ L, then order doubling dilution to the 11 is listed as holes from left to right, 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 37 ℃ of lower 15～20min, observations.

Through three repeated experiments, described three sequence＜400 of sequence table〉1,＜400〉2 and＜inhibiting rate that 400〉3 infected by influenzas copy is as follows:

Sequence	Virus titer after suppressing/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 copied

<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. siRNA who A type influenza virus is copied interference effect, its nucleotide sequence is shown in sequence table sequence 3.

2. the application of siRNA claimed in claim 1 in preparation treatment A type influenza virus medicine.

3. the expression vector pU6-siH1N1 that contains siRNA claimed in claim 1.