CN102643815A

CN102643815A - Method for designing siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus

Info

Publication number: CN102643815A
Application number: CN2012100632375A
Authority: CN
Inventors: 周俊芳; 华修国; 崔立; 房文红
Original assignee: East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences
Current assignee: East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences
Priority date: 2012-03-12
Filing date: 2012-03-12
Publication date: 2012-08-22

Abstract

The invention relates to a method for designing a siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus. The method comprises the following steps of: (1) selecting RNA polymerase, which is in RNA virus, has a conserved sequence and is RNA-dependent, as a target of siRNA; (2) utilizing siRNA design software to generate small RNA molecules resisting the RNA virus; (3) screening theoretically efficient siRNA for experiment research according to a siRNA design rule; and (4) adopting expression plasmid containing RNA polymerase III promoter U6 as an antiviral application form of the small RNA molecules. The method is applied to select an antiviral target sequence, so that the probability of obtaining efficient and antiviral small RNA molecules can be improved obviously, the selection range of an RNA interference target point is greatly reduced, the working efficiency is improved, and the cost for RNA interference technology research and application is obviously reduced.

Description

A kind of method of design of RNA virus resisting siRNA molecule

Technical field

The invention belongs to RNA perturbation technique field, particularly a kind of method of design of RNA virus resisting siRNA molecule.

Background technology

RNA viroid kind is a lot, distributes extensively, and aquatic all have infection with the terrestrial animal and the mankind.Comprise huge influenza virus (Flu virus), viruses of human hepatitis A (HAV), the virus of AIDS (HIV) of human harm like the Taura syndrome (TSV) that aquatic shrimps in culture caused serious threat, Macrobrachium rosenbergii promise da virus (MrNV), infectivity muscle necrosis virus (IMNV) with to terrestrial animal, and various coronavirus such as severe acute respiratory syndrome virus (SARS-CoV), transmissible gastro-enteritis virus (TGEV) etc.Because RNA viruses is in the reproduction process of RNA, the activity of the enzyme of its wrong repair mechanism is very low, does not almost have, so its variation is very fast; And vaccine be will according to the fixed base of virus because of or albumen develop making, so the difficult exploitation of RNA viruses vaccine!

The RNA perturbation technique; As a kind of PTGS mechanism that replaces the classical inverse phosphorothioate odn with short dsrna (siRNA); From 2002 by since " Science " be chosen as annual 10 quantum jumps; Be widely applied to popular domains such as gene function, gene expression regulation mechanism rapidly, and for disease gene is treated, anti-virus infection has been opened up new approach.With respect to traditional gene therapy to knocking out on the gene level; Whole flow scheme design is easier, acts on rapidlyer, and effect is more obvious; Efficiently special, the economy that becomes that the researchist generally acknowledges is research tool easily, in cell levels and animal and plant body, all shows efficient special anti-virus ability.Because the RNA perturbation technique is a PTGS mechanism; Directly target is the mRNA that virus transcription produces; So for RNA viruses especially positive chain RNA virus; The RNA perturbation technique virogene of just can directly degrading, so the excellent choosing beyond doubt for the control of RNA viroid of RNAi technology.Many results of study confirm that also the achievement of expection has been obtained in the antiviral field of RNA perturbation technique.

But, since antiviral only be the short and small sequence of 21 base pairs with siRNA, according to the little RNA principle of design of routine, most viral genome will produce hundreds and thousands of candidate siRNA.Especially the huge RNA viruses of the genome as the coronaviridae member; According to the conventional design principle needs are spent great amount of manpower and material resources; Be used for screening the siRNA (, causing many siRNA invalid or poor efficiencys in practical application that meet " effectively " principle in theory) of ability highly efficient anti-virus in practical application owing to the reasons such as space structure of target RNA.Therefore; The screening scope of how effectively to dwindle high efficiency siRNA is the hot issue that RNA perturbation technique researchist probes into always; Also be to be related to the research and the application cost that can effectively reduce the RNA perturbation technique, make it finally can in production practice, obtain popularization, key in application problem.

Summary of the invention

Technical problem to be solved by this invention provides a kind of method of design of RNA virus resisting siRNA molecule; The siRNA molecule of this method design; Will be under the prerequisite that guarantees acquisition highly efficient anti-virus siRNA; Significantly dwindle RNA molecular screening scope, significantly reduce antiviral research and application cost, help of the promotion and application of RNA perturbation technique in the RNA virus resisting field.

A kind of method of design of anti-RNA viroid siRNA molecule among the present invention comprises:

(1) in the genome of RNA viruses, selects the target of the encoding sox of the conservative viral enzyme of sequence as siRNA;

(2) utilize the siRNA design software to generate the small RNA molecular of anti-RNA viroid;

(3) according to the siRNA design rule, on the screening theory efficiently siRNA be used for experimental study.

The conservative RNA viruses enzyme of sequence in the said step (1) is the RNA polymerase (RdRP) that RNA relies on.

The antiviral application form of the small RNA molecular in the said step (3) is to be built into small RNA molecular the expression plasmid that contains rna plymerase iii promotor U6.

Table 1 is the target sequence of shRNA on TGEV different structure gene and RdRP gene.Wherein, shP1 and shP2 target TGEV RdRP gene, and the different structure gene of shS1, shS2, shM, shN1 and shN2 difference target TGEV; ShT target and TGEV gene do not have one section arbitrary sequence of homology, as showing that shRNA disturbs specific contrast.

Table 1

We are through to RNA viruses---and TGEV carries out the RNA The Study of Interference of cell levels and finds, in the siRNA of target virus RdRP gene and structural protein gene respectively, the little RNA of two target RdRP shows the antiviral effect that is higher than other siRNA far away.The antiviral applied research that level is carried out in the miniature pig body has subsequently further confirmed this two antiviral high efficiency of siRNA molecule and specificity.

Beneficial effect

The present invention is locked in the RNA polymerase that RNA relies on to the action target of siRNA; Two meanings are arranged: (1) RdRP is conservative, and degree is high; Very important for the RNA viruses of easy variation, because according to the siRNA of the sequences Design among the Genbank, this sequence variations occur in this place virus that might target; And research shows, even the variation of 1 base all can cause the antiviral effect of siRNA significantly to reduce.(2) part RNA viruses such as coronavirus genome are huge.With TGEV is example, and the siRNA of " effectively " in theory that obtains will be greater than 1000.If all carry out screening study to these siRNA, undoubtedly with the human and material resources of labor at cell levels.And the siRNA molecule that designs according to the present invention; Will be under the prerequisite that guarantees acquisition highly efficient anti-virus siRNA; Significantly dwindle RNA molecular screening scope, significantly reduce antiviral research and application cost, help of the promotion and application of RNA perturbation technique in the RNA virus resisting field.

Description of drawings

Fig. 1 is that CPE analyzes the restraining effect of different shRNA expression vectors to TGEV.Wherein, m indication window middle part cell, s indication window border cell; Normal is negative control (a untreated normal cell); Mock is positive control (only infecting TGEV); Fig. 2 is that the MTS method is analyzed the provide protection of different shRNA expression vectors to the ST cell.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Embodiment 1

The design of small RNA molecular is with synthetic

(1) chooses the encoding sox of TGEV structural protein and RdRP respectively;

(2) utilize software to generate the siRNA molecule of anti-TGEV.

(3) according to the little RNA sequences Design rule of having announced, siRNA molecule efficiently on the Choice Theory, and be built into hair fastener type little RNA (shRNA) molecule (table 1).

(4) the shRNA molecule inserts the plasmid expression vector that vector construction becomes to contain the U6 promotor.

Embodiment 2

ShRNA expression plasmid transfection ST cell

(1) ST cell (2-3 * 10 ⁴Individual/hole) insert 48 orifice plates and in complete DMEM, cultivate;

(2) to transfection reagent TransFast ^TMAdding 400 μ l among the transfection reagent does not have DNase water, whirlpool concussion dissolving ,-20 ℃ of preservations;

(3) dissolving plasmid DNA1.2 μ g is dissolved in the unparalleled anti-substratum of 300 μ l serum-frees;

(4) room temperature is dissolved transfection reagent and vortex, gets 3.6 μ l and adds in the DNA/DMEM mixture vortex mixing; Incubated at room 10-15 minute, form transfection composite;

(5) remove substratum in the ST cell, add transfection composite 100 μ l/ holes, continue at 37 ℃ of 5%CO ₂Hatched in the incubator 1 hour;

In the ST cell, add the unparalleled anti-perfect mediums of 400 μ l (containing 5%NBS); At 37 ℃, 5%CO ₂Incubator in continue to cultivate.

Embodiment 3

TGEV infects the ST cell

Remove cell culture medium behind the shRNA expression plasmid transfection ST cell 28h, press 200CCID ₅₀Amount inoculation TGEV.Behind the 40h, analysis of cells pathology, cytotoxicity (MTS) or extracting viral RNA are done quantitative RT-PCR and are detected.。

Embodiment 4

Cytopathy (CPE) is analyzed

The cytopathy that causes because TGEV duplicates propagation is the typical case very, can know under simple microscope whether demonstration the degree of cytopathy and pathology occurred.So at pair cell transfection shRNA expression vector and after infecting TGEV, we can be through the observation of pair cell pathology, more different shRNA expression vectors suppress virus replication, resist cytopathic ability.From Fig. 1; We can find out; After meeting malicious 40h; The shP2 of target RdRP gene and shP1 expression plasmid transfection hole cytopathy do not occur or only present faint doubtful " pathology " (explain: the TGEV pathology generally begins to stretch to the middle part, hole from bore edges) at the culture hole edge, and the shRNA of other targets TGEV structure gene disturbs the hole TGEV characteristic pathology all to occur, and just lesion degree is different.Wherein, the disease-resistant change ability of shS1 and shN1 expression plasmid is strong slightly, only pathology in various degree occurs at the edge in hole, and shS2, shM and shN2 disturbs the lesion degree in hole heavier, and pathology appears in almost whole porocyte.And the pathology in shS2 and shN2 interference hole and the situation of non-special interference hole shT are approaching.The CPE analytical results shows that the shRNA of design has all shown the inhibition of proliferation effect in the ST cell to TGEV mostly, and still, the shRNA of target RdRP gene obviously is better than the shRNA of target structure gene to the inhibition effect of TGEV.

Embodiment 5

Cytotoxicity (MTS) test

ST cell (1-1.5 * 10 ⁴Individual/hole) carry out plasmid transfection and virus infection test by the same method of 48 orifice plates at 96 orifice plates, every hole plasmid and virus quantity are respectively the half the of 48 orifice plates.After meeting malicious 36h, press the explanation of MTS reagent, in 100 μ l/ hole substratum, add 20 μ l/ hole MTS continued, hatched in the incubator of 5%,CO2 4 hours at 37 ℃.Measure each hole liquid light absorption value at last at the 492nm place.Accurately measure the viable cell quantity of respectively disturbing the hole after TGEV infects through the MTS test, can accurately show the degree of protection of each shRNA expression vector, reflect the ability that anti-separately TGEV infects the ST cell.

Fig. 2 ordinate zou representes that different shRNA disturb the light absorption value in hole.We can clearly be seen that two shRNA of target virus RdRP gene disturb the light absorption value in hole to be only second to negative control (normal cell) hole, and are higher than the value that other shRNA disturb the hole far away.That is to say that viable cell is maximum in two shRNA interference holes of target virus RdRP gene.Obviously, each shRNA expression vector disturbs the quantitative relation of viable cell in the hole and the basically identical as a result that CPE analyzes.

The analytical results of CPE and MTS proves that all the antiviral effect of the shRNA of target virus RdRP gene significantly is better than the target viral structural gene.

Claims

1. the method for design of a RNA virus resisting siRNA molecule comprises:

(2) utilize the siRNA design software to generate the small RNA molecular of RNA virus resisting;

2. the method for design of a kind of RNA virus resisting siRNA molecule according to claim 1 is characterized in that: the conservative RNA viruses enzyme of the sequence in the said step (1) is the RNA polymerase that RNA relies on.

3. the method for design of a kind of RNA virus resisting siRNA molecule according to claim 1 is characterized in that: the antiviral application form of the small RNA molecular in the said step (3) is: be built into small RNA molecular the expression plasmid that contains rna plymerase iii promotor U6.