CN1569233A - Method for manufacturing gene medicine for preventing and treating SARS and its medicinal prescription - Google Patents

Abstract

The invention integrates the RNA interference technology, human gene pool search technology, biological computer information technology, liposome technology and gene engineering technology. It provides a practical method to better design and prediction of high specific gene fragment for acquiring a highly effective gene medicine to deactivating a group of related pathopoiesia genes. The invention in particular relates to a method for choosing a pathopoiesia SARS target gene and determining isogenous siRNS or anti nucleotide sequence and other related auxiliary component to assemble the novel gene medicine for the prevention and treatment of the SARS virus infection. The invention concerns a prescription of gene medicine in further, in particular to a specific nucleotide containing, 21 pairs nucleotide constituting monochain or double chains oligonucleotide molecule.

Description

Prevent and treat the manufacture method and the pharmaceutical formulation of the genomic medicine of the severe acute respiratory syndrome that SARS virus causes

1. invent affiliated or direct applied technical field

The field of the invention is a kind of micromolecular double-stranded oligonucleotide and a kind of method and prescription of making genomic medicine.

2. the most approaching prior art situation

The market background

Begin severe acute respiratory syndrome (the SevereAcute Respiratory Syndrome of eruption and prevalence in Guangdong Province since in November, 2002, SARS), be also referred to as severe acute respiratory syndrome (Atypical pneumonia), in the time of short some months, epidemic situation has involved 28 countries and regions, and has caused the countries in the world government and the people's great attention and close attention.According to the report of The World Health Organization (WHO), whole world SARS patient was 8456 people totally by on June 26th, 2003, and the accumulative total death toll is 809.China is the most serious country of current SARS morbidity, and continent patient accumulative total is 4884 people so far, and the accumulative total death toll is 235.

Popular China and other national people's the physical and mental health of not only giving of SARS has been brought serious disaster, but also has a strong impact on morbidity area and even global economic development.Some external economy scholars estimate that the cost that pay for SARS in various countries, East Asia and area is: 2,200,000,000 dollars in China, 1,700,000,000 dollars in Hong Kong, 400,000,000 dollars in Indonesia, 2,000,000,000 dollars of Korea Ss, 6.6 hundred million dollars in Malaysia, 2.7 hundred million dollars in Philippine, 9.5 hundred million dollars in Singapore, 8.2 hundred million dollars in Taiwan, Vietnam 1,500 ten thousand, 1,100,000,000 dollars in Japan.The loss of estimating of expansion of currency slope not being adjusted is 10,600,000,000 dollars.The final geographic loss in Asia is greatly about about 50,000,000,000 dollars, and the loss that the whole world suffers because of SARS will be above 1,500 hundred million dollars.Because the SARS disaster, China's economic growth rate in 2003 will reduce by 0.1 to 0.5 percentage point, and more than lasting half a year, GDP will reduce 1% as if epidemic situation.Fortunately, China has been fully recognized that this severity of disease and hazardness up and down.The main leader of party and state will monitor the diffusion of SARS and the emphasis that the emphasis of Party and government's work is classified in control as.The Hu Jintao General Secretary emphasizes and will laxly strengthen the SARS preventing and controlling not, takes resolute measure, the dependence science, and the control eqpidemic disease spreads.

SARS is the severe acute respiratory syndrome that is caused by a kind of newfound coronavirus (being also referred to as SARS virus).According to the nearest statistical data of World Health Organization (WHO), the SARS mortality in said patients may be up to 10-15%.Still lack the specific medicament of treatment SARS and the vaccine of prevention SARS at present.In order to control the epidemic situation of SARS as early as possible, Chinese Government has transferred great amount of manpower and material resources and financial resources.For example, the central government has determined to invest 2,000,000,000 RMB and has been used for preventing and treating SARS and rapid disease surveillance and the medical health system that improves China.Simultaneously, Chinese Government has organized scientific research rapidly, researches and develops early diagnosis reagent, control medicine and vaccine fast, and has obtained certain progress.In short several weeks, China national food, drug administration (i.e. Zhong Guo SDA) have extremely fast ratified the new drug (new drug of these two anti-SARS all is to utilize the synthetic interferons medicine of technique for gene engineering) of two anti-SARS.Because these two kinds of new drugs have just entered clinical trial, its clinical prevention effect remains to be observed.

Technical background

Since 12 days March in this year of World Health Organization (WHO) after the SARS alarm is sent in the whole world, 13 laboratorys of China, Hong Kong, Germany, Canada, France, the U.S., Japan, Holland, 10 countries and regions of Britain and Singapore have carried out particular study to the cause of disease of SARS, consistently in the meeting that scientists 16 days is held in Geneva determine that the mutation coronavirus is exactly the pathogen of SARS.World Health Organization (WHO) be responsible for SARS research chief scientist Crouse, execute Bristol and say, the discovery of pathogen " extremely important ", this makes scientist can concentrate research virus, exploitation vaccine and new drug or screening have medicine now.In short 3-4 month time, the research of etiology, Clinical symptoms and the epidemiologic feature of SARS has just been obtained progress.Peiris etc. Lancet (2003, point out that SARS is relevant with a kind of new coronavirus of never finding on 362:9365) in the mankind or animal.Research worker adopts classical Virus culture method, serological technique and modern molecular gene technology, detects and determined Hong Kong 50 routine patients'SARS pathogenic factor.On the contrary, those are suffered among the patient of other respiratory system diseases, and none routine patient's respiratory tract specimen contains the RNA of coronavirus.Moreover, there is not discovery to resist the antibody of this novel coronavirus in 200 parts of serum specimens from the blood donor yet.The cause of disease relation that this result has supported the research worker of Ya Telan disease prevention and control centre (CDC) and Toronto to report forcefully infers that they have also isolated a kind of new coronavirus from patient SARS.Cross section Cheng Now 3 Strip of ， Seed virus are all of a sudden as the perverse Like thing of Phaleria macrocarpa Xia the Zai micro mirror. " coronavirus " thereby gain the name.

In the some months after this disease of SARS is identified, finish in the gene ordering of April 13,16 and 17 days declaration SARS virus respectively from the scientist of Beijing, Hong Kong, Canada and the U.S..This achievement has been used for the disconnected and treatment SARS infectious disease of card significantly.In the detected SARS infectious disease that whether infects of the thousands of people of the U.S..Simultaneously, the ordering having begun to be used to of the gene of the SARS virus development and the screening of this disease Molecular Study and vaccine and medicine.BBC reports that obtaining the SARS virus sample from the regional patient of difference shows that the genome sequence of SARS virus is also incomplete same, exists certain difference, and the gene difference of different SARS virus can be up to 15 alkali bases.Some articles have reported that SARS virus can undergo mutation, and this gives and researches and develops specifically that the vaccine of anti-SARS virus has brought difficulty.

Britain's ' The Times ' revealed on the 25th that U.S. biological medicine mechanism was obtaining great achievement aspect the research SARS, and first kind of SARS specific drug can just can be used for clinical treatment in some months in the world.American National medical treatment association represents, they carry out analytic demonstration to the new drug to anti-sars that AVI Biopharma, Inc. of Ore. provides.Initial stage the analysis showed that, this new drug can effectively kill the coronavirus that causes the SARS disease.The director Denis of this pharmacy mechanism Boolean lattice doctor also claims optimistically, as long as this efficacy of new drug can be affirmed by authoritative institution in ensuing two weeks, they will drop into clinical practice immediately with new drug, in some months new drug are delivered in victims of SARS's hands of various countries.The AVI company introduction says that the action principle of this medicine is by biochemical reaction, stops the procreation process of duplicating of SARS coronavirus gene, and its effect is very obvious.Specifically, this new drug has used the new technique of a kind of being named as " antisense therapy ", can effectively attack the nbccs gene strand of infectious hepatitis virus, west Nile virus and SARS coronavirus etc.Because these viruses all are to rely on ribonucleic acid transhipment autogene information to come synthetic protein in human body, therefore contain in the new drug with viral gene and arrange mutually complementary rna single strand, can stop the crucial part of causing a disease of above-mentioned virus in human body cell, to be duplicated, thereby stop the generation and the deterioration of disease.

Boolean lattice doctor has played an analogy: " everybody has imagined a slide fastener; the SARS virus rna single strand is exactly half in this slide fastener; if the rna single strand that we find an energy and SARS virus rna single strand to fit like a glove, the toxicity of the virus that just can thoroughly neutralize." in addition, genetic test result shows that this medicine can't influence the process that the normal ribonucleic acid transhipment is duplicated in the human body cell when acting on the SARS virus part.

Shanghai Inst. of Life Science, CAS president Pei Gang announced on the 16th: through the tackling key problem of uniting of a plurality of institutes of this academy, research worker has successfully been cloned 6 kinds of major protein genes such as the S in the SARS virus, M, N, E and RNA polymerase, (3CL) proteolytic enzyme, and in viral key protein expression of gene, separation, purification experiment, obtained E albumen in the SARS virus, N albumen and (3CL) three kinds of proteic expression samples of proteolytic enzyme, expressed sample and carried out the medicine virtual screening.

Personnel introduce according to the study: E albumen is little peplos associated protein, plays crucial effects in the self-assembling process of coronavirus; N albumen is a kind of important structural protein in the coronavirus, contains the signal sequence that does not exist consideration convey to move in other coronavirus N protein, research infers that SARS virus is later on to enter into nucleus by this consideration convey shifting signal sequence invading host cell, and integrates its biological action of performance with host DNA; (3CL) proteolytic enzyme and SARS virus duplicate closely relatedly, be the desirable target spot of SARS resisting medicine screening.

The research worker of Shanghai life science institute is said, they are to clone's success of 6 kinds of albumen genes of SARS virus, express the acquisition of sample with three kinds of albumen, to carrying out the biological function research of SARS virus in a deep going way, further see clearly the hateful appearance of SARS, vaccine and medicine for seeking anti-SARS have great progradation.

Scientists is for discovering that the SARS viral gene difference occurs and do not feel surprised, because SARS virus is a kind of RNA viruses, the variation that they take place when carrying out self replication is usually more than the variation of DNA viruses.Yet the variation of this virus does not have that some are estimated so big.In 14 kinds of SARS viruses investigating, research worker has been found 129 kinds of genovariations, and wherein 16 kinds occur repeatedly.

Have scientist to point out, these genovariations may make the effort of development SARS viral vaccine complicated, and length consuming time is just as the development of HIV viral vaccine.But doctor Sha Funa points out, has reason to be filled with unbounded confidence.He says, although there is gene difference in poliovirus all over the world, and Yan Zhi vaccine " all effective " for it in the whole world.We can say that Measles Vaccine also is like this.He thinks that the situation of SARS virus is more as poliomyelitis and Measles virus, rather than picture HIV virus.

3. Fa Ming the purpose and the problem that will solve

SARS is severe acute respiratory syndrome (the Severe Acute Respiratory Syndrome that is caused by a kind of newfound coronavirus (being also referred to as SARS virus), SARS), be also referred to as severe acute respiratory syndrome (Atypicalpneumonia), in the time of short some months, epidemic situation has involved 31 countries and regions, and has caused the countries in the world government and the people's great attention and close attention.According to the nearest statistical data of World Health Organization (WHO), the SARS mortality in said patients may be up to 10-15%.Still lack the specific medicament of treatment SARS and the vaccine of prevention SARS at present.In order to control the epidemic situation of SARS as early as possible, great amount of manpower and material resources and financial resources have been transferred in countries in the world.For example, the central government of China has determined to invest 2,000,000,000 RMB and has been used for preventing and treating SARS and rapid disease surveillance and the medical health system that improves China.Simultaneously, Chinese Government has also organized scientific research rapidly, researches and develops early diagnosis reagent, control medicine and vaccine fast, and has obtained certain progress.In short several weeks, China national food, drug administration (i.e. Zhong Guo SDA) have extremely fast ratified the new drug (new drug of these two anti-SARS all is to utilize the synthetic interferons medicine of technique for gene engineering) of two anti-SARS.Because these two kinds of new drugs have just entered clinical trial, its clinical prevention effect remains to be observed.

It is one that the present invention collects RNA perturbation technique, human body gene library searching technology, biocomputer information technology, liposome technology and technique for gene engineering, researches and develops and prepare a kind of natural genomic medicine.Two main purposes of the present invention are:

Provide practicality and the method for system.These methods relate to development anti-SARS virus genomic medicine process, they comprise how screening, predict, identify, synthesize, prepare and assemble a kind of natural genomic medicine, and how to treat the corresponding scheme that animal and human's SARS virus sexuality is dyed.Wherein, a kind of prediction of special recommendation and selected efficiently, short and small interference two strands or the short-cut method of single strain oligonucleotide.

The long double-stranded oligonucleotide of preparation, a particularly 19-25 nucleotide of the active ingredient of the genomic medicine of description anti-SARS virus.The architectural feature of siRNA contains the double-stranded oligonucleotide of or several CGG motifs.Special siRNA molecular energy SARS virus nucleic acid interaction homologous with it, thus the expression of corresponding RNA molecule suppressed, the duplicating and the particulate formation of SARS virus of SARS virus gene.

The present invention also at large sets forth the efficacy component of anti-SARS virus genomic medicine, and the method for treatment and prevention SARS also is provided further.SARS can reach the purpose of treatment and prevention by said a kind of or the double-stranded oligonucleotide that several 19-25 nucleotide are long of the present invention who gives various dose and different types.

The problem to be solved in the present invention mainly comprises the specificity of genomic medicine, high efficiency and stability.Genomic medicine of the present invention is the natural siRNA molecule that is present in the living cells, and it is a kind of oligonucleotide of short and small two strands, has high specificity and high efficiency.When multiple siRNA molecule with difference in functionality and other auxiliary composition were assembled into genomic medicine, these genomic medicines were compared with other bio-pharmaceutical, can show following multiple advantage:

● brand-new design and development are theoretical: exist a kind of natural antiviral genetic immunization system in the cell.Effect composition in this system can be amplified and be strengthened by bionic method external, then with in its defeated object of bringing back to life.It can special and those homologous viral genes of deactivation efficiently.Sequence pattern CGG in the effect composition of the present invention or their derived sequence are that design and the selected molecule of siRNA efficiently are as the important indicator of genomic medicine.

● the short new drug development cycle: by computer to the analysis of the firsts and seconds structure of nucleic acid molecules with to the research of SARS virus genomics, conservative part in the selected specific SARS virus RNA sequence is as the target of genomic medicine, and its homologous sequence is as the active ingredient of genomic medicine.This new research and development strategy can reduce the time of the chemistry that is used to study a kind of drug molecule and physical property widely and find out the time of its action site on target molecule.

● lower medicament research and development cost: study a new drug and and its target molecule between interaction usually need the reasearch funds of great number and a large amount of search times, the free use of Computing method fast and common gene database can reduce the cost of researching and developing novel genomic medicine significantly.

● high specificity: the present invention can filter out the target fragment of the tool potential in the specific mRNA sequence, with its homologous siRNA as genomic medicine.They are discerned mutually and act on by the principle of the Watson-Crick base pairing of classics.Moreover the fragment of selecting is only common by homologous genes family members' member, and with other gene family members' member few similarity is only arranged, thereby the specificity of medicine is guaranteed fully.

● extremely low malicious seondary effect: the main active ingredient in the genomic medicine of the present invention is a kind of natural nucleic acid fragment, and it is present in the cell from the lower animal to the human body.Because its high specific and high efficiency make its consumption be lower than other medicine significantly.Obviously, its toxic and side effects also is lower than other medicine out and away.

● advantages of higher stability: siRNA molecule of the present invention is a kind of double-stranded oligonucleotide, can interact with some protein and other micromolecule, has nucleic acid such as cDNA than other strand that much better stability is arranged.It can resist the degraded of nuclease effectively, and easy and some protein form stabilized complex.Its some base also is easy to modify, and is used to strengthen the performance of nuclease-resistant.

● multiple use: genomic medicine of the present invention is the mixture of a kind of dissimilar, various dose and multiple siRNA molecule.They can be adjusted according to patient's the concrete condition and the order of severity of disease, and can be made into the preparation of different types, be used for different by way of, will start the New Times of diagnosing a disease and writing a prescription for each patient.

● high efficiency: can interrupt corresponding mRNA molecule efficiently can several different Disease-causing genes of while deactivation be characteristics of genomic medicine of the present invention also, and the breakthrough on this methodology is particularly useful for the treatment and the prevention of viral infection.

● high mutation: according to the principle of mathematics theory of probability, but the energy rate that the sudden change of a nucleotide takes place in a short and small sequence will be widely less than the probability in one section very long sequence.SARS virus can be undergone mutation, and this vaccine for the research and development anti-SARS virus has brought certain difficulty.

4. Fa Ming concrete technical scheme

From the infection of ectogenic virus, antibacterial and fungus, show all that to endogenic cancer, hyperlipidemia, hypertension, senile dementia and some other heritability disease abnormal gene expression is the main cause that causes numerous disease in the human body.The duplicating and expressing in human body cell of external SARS virus gene caused a large amount of breedings of SARS virus, finally causes the death of cell and the deterioration of the state of an illness.Control SARS most important purpose be exactly put forth effort to find various science and efficient ways, stop these abnormal gene expression, so that the development of control disease and propagation effectively, until final kill virus and cure SARS for a full due.

The international biotechnology of technical merit----forward position

Continuous 2 years of U.S.'s " science " magazine is assessed as the discovery about siRNA one of ten big technological achievements in 2001 and 2002; After nanoelectronics, rank the second at calendar year 2001 RNA perturbation technique, and little RNA bravely got first in 2002.About basic research and the industry development of siRNA exclusiveness becomes the most important commanding elevation that countries in the world are seized in the biotechnology field.Since finding that RNA disturbed that it has become very clear in (RNAi) this phenomenon several years so far in 1998: the natural functions of this process is a kind of system of ancient biological gene group, it is used to resist the infringement such as movable hereditary materials such as transposon, viruses, the space-time of gene expression in the regulating cell, propagated and multiformity.RNAi, this adjustment system the most ancient, the most polyenergic cellular level is to be kept at from plant, fungus, insecticide, batrachia, birds, rat, mice, monkey until the human cell through selection and the evolution in 10000000 year.These the Nature offer the immune important of this cellular level to people's complete evidence to be described.Obviously, if by external by way of strengthen with magnocell in the monitoring capacity of this genetic immunization system, the expression of those alien genes (SARS virus gene) in the human target cell of just more effectively disinthibiting.Be used for the treatment of those existing medicines and irremediable viral infection of method such as SARS, to benefit the whole mankind.

Technical characterstic:

In this Item Sets world today the tightest new life science and technology, RNA perturbation technique, human body gene library searching technology, biocomputer information technology, liposome technology and the technique for gene engineering of high-new point be one, research and develop and prepare a series of genomic medicine.These natural genomic medicines are different as " antisense therapy " technology with other biologics and existing gene therapy, and the benefit of its degrading genes product approximately is tens of times and even the hundreds of times of " antisense therapy ".In addition, it has following all characteristics:

Treatment is theoretical novel,

Take stopgap measures and effect a permanent cure,

Safety, nontoxic seondary effect

Natural active ingredient

The specific aim prescription

Science and technology pharmacy, credible reliable

Technology contents:

(1) identifies the mRNA molecule relevant with disease

Scientists has been decoded the gene ordering of SARS virus and has been identified that crucial functional gene is as the RNA polymerase that RNA instructs, the protease of 3C-sample, PLP protease, unwindase, M, E, S and N albumen etc.The gene of existing different SARS virus strains can both be found in gene bank.When these hereditary function RNA molecules are used as the treatment target position, homologous with it small fragment RNA will be the most effective medicine.To discerning mutually and act on by the principle of Watson-Crick base pairing between these medicines and the target molecule.This is the theoretical basis that we can in depth inquire into the mechanism of action of medicine and develop effective new drug apace.

SARS virus is a kind of RNA molecule of positivity strand.Its RNA is exactly a kind of fairly individual target spot, because it does not still have the gene information of carrying function and can be directly as mRNA, so it is the biomolecule of a kind of pair of characteristic.Simultaneously, mRNA also be one from gene to proteic important bridge, keep guard over normally carrying out of hereditary information.With RNA is the siRNA genomic medicine of target spot, is the double-stranded oligonucleotide of homology of a small fragment among the mRNA.The double-stranded oligonucleotide molecule of each that is designed can both interact with the specific regions of targeting SARS virus RNA and its mRNA, suppresses the generation of targeted rna encoded protein matter.The oligonucleotide that we design all has the efficient function of potential high selectivity therapeutic agent.By disease being formed the intervention of commitment, genomic medicine can prevent the generation of pathogenic protein, as some above-mentioned functional proteins.

Nucleic acid molecules involved in the present invention is including, but not limited to the mRNA molecule, and they are:

● the mRNA of the RNA polymerase that coding RNA instructs,

● the mRNA of the protease of coding 3C-sample,

● the mRNA of coding PLP1/2 protease,

● the mRNA of coding unwindase,

● the mRNA of coding N nucleoprotein,

● the mRNA of coding M memebrane protein,

● the proteic mRNA of coding S,

● the proteic mRNA of coding E,

● the sequence of coding TRS,

(2) design and selection SARS virus resisting siRNA

Though the siRNA molecular energy specifically with one or more homologous nucleic acid interactions, have advantages of higher stability and effectiveness, but aim of the present invention mainly is to suppress the function of those genomic RNA molecules, dyes thereby can take precautions against and treat the SARS virus sexuality.Concrete grammar is seen the patent (0114117.1) before me

SARS virus resisting siRNA molecule involved in the present invention, they are:

● the siRNA of the mRNA of the RNA polymerase that anti-RNA instructs,

● the siRNA of the mRNA of the protease of anti-3C-sample,

● the siRNA of the mRNA of anti-PLP1/2 protease,

● the siRNA of the mRNA of anti-unwindase,

● the siRNA of the mRNA of anti-N nucleoprotein,

● the siRNA of the mRNA of anti-M memebrane protein,

● the siRNA of the proteic mRNA of anti-S,

● the siRNA of the proteic mRNA of anti-E,

● the siRNA of the sequence of anti-TRS.

The overwhelming majority in these siRNA molecules contains one or several CGG motifs, the position of CGG motif can be in the siRNA molecule any position as from the negative 3 to positive 22 nucleotide.The nucleotide sequence of 80 siRNA molecules of concrete representative is seen the nucleotide sequence part of present patent application.SiRNA can obtain with chemical synthesis and biological synthesis process; The chemical synthesis of SiRNA is referring to our another patent of invention (1).Biological synthesis process means with engineered method one section dna segment that can form siRNA inserted a kind of bio-carrier such as plasmid or virus, and these contain the recombiant plasmid of dna segment of siRNA or virus can stablize in cell and express corresponding siRNA molecule chronically.

(3) check the specificity of the siRNA molecule predicted

In this section, it is not only common by the different members of same gene family to be necessary to make clear which high conserved region territory, and is not shared by other gene family.The siRNA of the mRNA of the RNA polymerase that instructs as an anti-RNA, the different members of it and SARS gene family all has 100% homology, but with the different members of human body gene family without any homology, or be no more than 75% with the minority member's of human body gene family homology.To this, the method for an analytical sequence is earlier this sequence to be divided into different sections, and these different sections belong to different gene families, and they on the structure, or are correlated with on the function on evolving, and are keeping their total feature or patterns.The DNA sequence of existing known road high conservative necessarily relates to a certain important function, and the pattern of this sequence can be used to distinguish the member of family and the difference between non-member.The discovery operation method of binding sequence pattern and strict multisequencing are arranged formula, and the zone that has in sequence that a kind of effective method identifies reflection family differences and the family can be provided.At last, this is present in the basis that a kind of intragenic constant sequence pattern will be used as the active ingredient of selection genomic medicine among the present invention.In order to detect homologous DNA sequence, available BLAST and FASTA search engine are retrieved and they matching databases, as Genebank, and Swiss-port EMBL.These data bases pass through special arrangement and favorable tissue, have all nucleotide sequences of delivering at present, retrieve very convenient.But still can not only rely on a note among the data base to find all homologous sequences of same gene so far.Recently effective method is, takes out a certain member of same gene family, as the enquirement sequence, open the corresponding calculated formula then, compared with each sequence among the data base, finally obtain all homologous sequences.

(4) check the siRNA molecule predicted accessibility to its RNA target

The present invention proposes the high conserved sequence among the RNA, particularly do circulus and can be used as an important identification mark, it not only can be used to identify some accessible target sequences among the RNA, also can be used to the little rna gene of coding RNA interference sequence in the screening DNA genome.Because the RNA interference sequence must contain one section reverse complementary sequence, the present invention uses existing computer software method can predict the special computers software of the dried circulus of RNA is searched for RNA molecule fragment and the various mRNA of SARS virus with dried circulus in SARS virus genomic information storehouse secondary structure as folding ranking method (FOLDALIGN) and other.In addition, other several different methods also are useful, seek the structure with the common property special seized with terror of microRNA as computer, microarray assays between gene and they include complementary series in the subarea and some regulate proteic binding sequence.Then, the fragment that whether search exists corresponding homology nucleotide in the human genome information bank is a critical step of the template of selected special siRNA molecule.

(5) check the immunostimulating of the siRNA molecule predicted

Recognized that now some oligonucleotides can cause nonspecific immunostimulation, as the GGGG---nucleotide sequence more than three, these sequences can cause immune problem.In addition, the nucleotide sequence that contains purine-purine-C-G-pyrimidine-pyrimidine can activate the B cell non-specificly.So, in the design and selection of siRNA, should avoid these sequences.

(6) modify the siRNA molecule of being predicted

The terminal nucleotide of natural siRNA molecule can change their molecular structure and spatial orientation through chemical modification, thereby on the basis of the biological function that keeps natural siRNA molecule, obtains the ability of the anti-RNA nuclease in more highland of siRNA molecule again.Like this, the natural siRNA molecule of the siRNA molecular proportion of modified has higher benefit, as in vivo with intracellular metabolism cycle stretch-out.

(7) the two-way specificity and the concertedness of siRNA molecule

The two-way specificity of siRNA molecule means two different mRNA molecules of deactivation and inhibition that a siRNA molecule can be special.SARS virus contains two can instruct the synthetic RNA molecule of multiple protein and a leading sequence, and these are characterized as SARS virus us and design multi-functional siRNA molecule necessary condition is provided.In addition, the present invention also unites the more than one siRNA molecule (see figure 5) of use, makes the siRNA genomic medicine suppress SARS virus and duplicates, and ability synthetic and assembling improves greatly.

(8) form of genomic medicine

Genomic medicine can be mixed with different forms, as: water preparation, powder, oil preparation, ointment, tablet, pill, liquid medicine, colloid, propellant, subcutaneous embedded block.Perhaps, traditional drug effect carrier, hydras, powder or grease or other shape are essential or wish.The composition of oral medicine and preparation comprise powder, granular substance, finely particulate, the emboliform mixture of sodium, watery or non-watery suspension, capsule, tablet or small pill.In addition, intensifier, spice, diluent, emulsifying agent, dispersant or bonding agent perhaps be need or require.

(9) route of administration

Active ingredient of the present invention or compatibility comprise the double-stranded oligonucleotide that 21-23 nucleotide is long, i.e. 21-23ntsiRNA, 21-23nt RNA, or 21-23nt DNA.Except double-stranded oligonucleotide, other relevant composition comprises the carrier of accepting on the drug effect, or other is commonly used to strengthen and promote the auxiliary composition of drug absorption.Route of administration is including, but not limited to following all kinds of:

(1) external: comprise eye and nose,

(2) suck: comprise in the trachea, in the oral cavity, cross skin or outer emulsifying agent or the propellant of skin.

(3) oral

(4) injection or drop: comprise intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular etc.

(5) intracranial administration: sheath is interior, Intraventricular.

(10) usage of genomic medicine

About the existing a large amount of report of the research of treatment compatibility and relevant administration number of times, the size of dosage depends on the order of severity of disease, compliance and patient's health status.Therapeutic process can be cured or obviously be alleviated from several days to several weeks, several months, several years or up to disease, and ideal dosage yardstick can decide in the situation of intravital distribution of people and accumulation by the tolerance medicine.Brainstrust is easy to determine ideal dosage usually.

Perhaps, ideal dosage can change along with the variation of the relative efficiency of concrete different double-stranded oligonucleotide, evaluates according to EC50S usually.Because the EC50S zootype inside and outside why is a kind of reliable index in studying, in general, the dosage range of siRNA molecule is 5ng～200mg/kg body weight, this dosage can be once a day for several times or weekly or for several times, every month once or for several times, annual or for several times.Brainstrust usually can be according to measuring institute's medicine of giving in people intravital residence time, medicine at body fluid or the concentration in organizing decide the number of times and the frequency of administration.After the treatment along with success, patient is kept treatment also usually to be needed with the recurrence that wards off disease.At this moment, the double-stranded oligonucleotide of a maintenance dose is necessary, and usually dosage range is 5ng～200mg/kg body weight, this dosage can be once a day for several times or weekly or for several times, every month once or for several times, once a year or give patient for several times.The optimal dose scope is 5ug～5mg/kg body weight/day.

4. compare the advantage that is had with prior art

1) .SARS is the severe acute respiratory syndrome that is caused by a kind of newfound coronavirus (being also referred to as SARS virus).Up to the present, the medicine that does not still have effective anti-SARS virus in the world.

2). natural anti-virus small-molecule substance: siRNA is the natural small-molecule substance that is present in plant, fungus and the zooblast, and it not only plays an important role to normal gene regulation, and is the genetic immunization system of viral infection resisting natural in the life entity.

3). the mechanism of action is more clearly: lot of experiments shows that siRNA is by combining with the complementary RNA of its sequence and cause the RNA chain break, thereby suppresses the synthetic of respective egg white matter.Therefore, its mechanism of action is more clearly.

4). specificity is very high: the specificity problem is one of sixty-four dollar question in the drug research.The many medicines that use clinically as antiviral drugs, cause toxicity and a lot of side effect exactly in default of specificity at present.But siRNA only combines with the complementary RNA of its sequence and causes the RNA chain break, promptly only acts on its special " target spot ".In this research, we design and synthetic siRNA only acts on the RNA of SARS virus, and the RNA of human body cell is not influenced, so specificity is very high.

5). no overt toxicity and side effect: up to now, the siRNA that all external and intravital results of study all show effective dose (0.1-10ug/Kg) does not have tangible toxicity and side effect to the cell and the laboratory animal of In vitro culture, so siRNA is the foolproof medicine of a class.

6). high mutation: SARS virus can be undergone mutation, and vaccine of anti-SARS virus has brought difficulty to researching and developing specifically for this.And the siRNA genomic medicine has higher mutation.

7). use widely: siRNA is one of popular topic in current biomedical research field, from the basic research to the drug development, all is extensive use of the siRNA perturbation technique.And the siRNA perturbation technique has successfully applied to following aspect:

Special and efficiently suppress duplicating and breeding of HIV (human immunodeficiency virus) (Human Immunodeficiency Virus)

Special and efficiently suppress duplicating and breeding of hepatitis B virus

Special and efficiently suppress duplicating and breeding of hepatitis C virus

Special and efficiently suppress duplicating and breeding of poliovirus

Special and efficiently suppress duplicating and breeding of herpesvirus

Suppress the growth (anticancer) of tumor cell

8). the huge investment of drugmaker:, the research-and-development activity of the huge siRNA of being engaged in of many big drugmaker's investments has been arranged in the U.S. and other developed country.Recently, there are a plurality of drugmakers also carrying out the work of relevant siRNA anti-SARS virus in the world.This shows the application prospect of siRNA anti-SARS virus genomic medicine.

Certainly, this has reflected that also this field competitiveness is very strong simultaneously.

9). it is reported that the preliminary study result of AVI Biopharma, Inc. of the U.S. shows that " antisense (anti-sense) " technology can obviously suppress duplicating and breeding of SARS virus external, this achievement in research may enter clinical trial very soon.Because " target spot " of " antisense (anti-sense) " technical role also is SARS virus RNA, this is very similar to we siRNA of design.But, the result of study of other virus (as HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) and hepatitis virus) is shown the antiviral effect of siRNA perturbation technique is generally all than strong dozens or even hundreds of times of " antisense (anti-sense) " technology.In addition, our controlled trial shows that the antiviral effect of siRNA is than antisense (anti-sense) technology and the strong manyfold of polypeptide sealing virus receptor technology.

6. about explanation and accompanying drawing

Totally 5 width of cloth accompanying drawings and 5 description of drawings.

The RNA polymerase siRNA that Fig. 1 .RNA-relies on is to the inhibition or the killing effect of SARS virus BJ01 strain.

Fig. 2. unwindase siRNA is to the inhibition or the killing effect of SARS virus BJ01 strain.

Fig. 3. nucleoprotein-N siRNA is to the inhibition or the killing effect of SARS virus BJ01 strain.

Fig. 4. proteolytic enzyme siRNA is to the inhibition or the killing effect of SARS virus BJ01 strain.

Fig. 5 proteolytic enzyme and nucleoprotein-N siRNAs is to the collaborative inhibition or the killing effect of SARS virus BJ01 strain.

7. working of an invention example and optimum implementation

Strain: the 5th generation of the isolating SARS virus BJ01 strain of Beijing patient's lung tissue.

Is 10-10 with DMEM culture fluid 10 by continuous rare pool with SARS virus BJ01 strain.The viral liquid of the rare damp degree of difference is inoculated in 96 well culture plates of the Vero-E6 cell that grows up to monolayer every hole 0.1ml, each concentration 3 hole.Establish the normal cell contrast simultaneously.Put 37 ℃, 5%CO ₂Cultivate in the incubator, continuous 7 day every day observation of cell form and the variation of number.The test triplicate, the half that calculates SARS virus BJ01 strain according to the REED-MUENCH method infects metering (TCID ₅₀).

With the sterilized water of no RNA enzyme siRNA being configured to concentration is: 20umol/ml, with liposome technology the Vero-E6 cell is advanced in the siRNA transfection then, and liposome is 1-3ul/l-3ug with the ratio of siRNA.After the transfection 6 hours, infect the SARS virus BJ01 strain of metering with half the Vero-E6 cell is attacked.Establish normal cell, liposome, SARS virus BJ01 strain simultaneously and do not have target siRNA contrast.Put 37 ℃, 5%CO ₂Cultivate in the incubator, continuous 7 day every day observation of cell form and the variation of number, test repeats two to three times.The result is as follows:

The RNA polymerase siRNA that example 1RNA-relies on is to the inhibition or the killing effect of SARS virus BJ01 strain.

See Fig. 1.The SiRNA sequence is: CAUCAUCCGG UGAUGCUAC dTdT.

The result shows: the siRNA of the RNA polymerase that anti-RNA-relies on can suppress or kill about 50% SARS virus.

Example 2 unwindase siRNA are to the inhibition or the killing effect of SARS virus BJ01 strain.See Fig. 2.

The SiRNA sequence is: UAGUGUAUA CGGCAUGCUC dTdT

The siRNA of anti-unwindase can suppress or kill about 70% SARS virus.

Example 3 nucleoprotein-N siRNA is to the inhibition or the killing effect of SARS virus BJ01 strain.See Fig. 3.

The SiRNA sequence is: GUGCGU GCAGACGGUU CGU dAdC

The siRNA of anti-nucleoprotein-N can suppress or kill about 95% SARS virus.

Example 4 proteolytic enzyme siRNA are to the inhibition or the killing effect of SARS virus BJ01 strain.See Fig. 4.

The SiRNA sequence is: CGU AGUCGCGGUA AUUCAA dTdT

The siRNA of anti-proteolytic enzyme can suppress or kill about 90% SARS virus.

Example 5 proteolytic enzymes and nucleoprotein-N siRNAs is to the collaborative inhibition or the killing effect of SARS virus BJ01 strain.See Fig. 5.The siRNA of anti-nucleoprotein-N and proteolytic enzyme unites use and can suppress or kill 100% SARS virus.

8. list of references

1 Yin Dynasty's winter gave birth to and is solicitous big, (2001) design and method and the pharmaceutical formulation of selecting natural siRNA as genomic medicine.People's Republic of China's number of patent application: 01144177.1

2. Yin Dynasty's winter gives birth to and is solicitous big, the prescription and the manufacture method of (2002) gene cosmetic for skin whitening.People's Republic of China's number of patent application: 02155254.1

3.Ruan?YJ，Wei?CL，Ee?AL，Vega?VB，Thoreau?H，Su?ST，Chia?JM，Ng?P，Chiu?KP，Lim?L，Zhang?T，Peng?CK，Lin?EO，Lee?NM，Yee?SL，NgLF，Chee?RE，Stanton?LW，Long?PM，Liu?ET.(2003)Comparative?full-length?genome?sequence?analysis?of14?SARS?coronavirus?isolates?and?common?mutations?associated?with?putative?originsof?infection.Lancet.361(9371)：1779-85.

4.[No?authors?listed](2003)Gene?patents?and?the?public?good.

5.Nature.423(6937)：207.

6.Holmes?KV.(2003)SARS-associated?coronavirus.N?Engl?J?348(20)：1948-51.

7.Enserink?M，Vogel?G.(2003)Infectious?diseases.Hungry?for?details，scientistszoom?in?on?SARS?genomes.Science.300(5620)：715-7

8.Vogel?G.(2003)SARS?outbreak.Modelers?struggle?to?grasp?epidemic′s?potentialscope.Science.300(5619)：558-9.

9.Enserink?M.(2003)Infectious?diseases.Calling?all?coronavirologists.Science.300(5618)：413-4.

10.Ksiazek?TG，Erdman?D，Goldsmith?CS，Zaki?SR，Peret-T，Emery?S，Tong?S，Urbani?C，Comer?JA，Lim?W，Rollin?PE，Dowell?SF，Ling?AE，Humphrey?CD，Shieh?WJ，Guarner?J，Paddock?CD，Rota?P，Fields?B，DeRisi?J，Yang?JY，Cox?N，Hughes?JM，LeDuc?JW，Bellini?WJ，Anderson?LJ；SARS?Working?Group.(2003)?A?novelcoronavirus?associated?with?severe?acute?respiratory?syndrome.N?Engl?J?Med.348(20)：1953-66.

11.Qin?L.Xiong?B，Luo?C，Guo?ZM，Hao?P，Su?J，Nan?P，Feng?Y，Shi?YX，Yu?XJ，LuoXM，Chen?KX，Shen?X，Shen?JH，Zou?JP，Zhao?GP，Shi?TL，He?WZ，Zhong?Y，JiangHL，Li?YX.(2003)Identification?of?probable?genomic?packaging?signal?sequencefrom?SARS-CoV?genome?by?bioinformatics?analysis.Acta?Pharmacol?Sin.24(6)：489-96.

9. nucleotide sequence

＜110〉solicitous big and solicitous industry

＜120〉prevent and treat the manufacture method and the pharmaceutical formulation of the genomic medicine of the severe acute respiratory syndrome that SARS virus causes

<130>01144177.1

<140>00000000

<141>2003-04-26

<150>01144177.1

<151>2001-12-14

<150>02155254.1

<151>2002-12-12

<160>79

<170>PatentIn?version?3.1

<210>1<211>21<212>RNA/DNA<213>SARS?Virus <400>1

cggctgcatg?cctagtgcac?c 21

<210>2<211>21<212>RNA/DNA<213>SARS?Virus <400>2

cggtttcgtc?cgtgttgcag?t 21

<210>3<211>21<212>RNA/DNA<213>SARS?Virus <400>3

ucuguguagc?ugucgcucgt?t 21

<210>4<211>21<212>RNA/DNA<213>SARS?Virus <400>4

cgggtgtgac?cgaaaggtaa?g 21

<210>5<211>21<212>RNA/DNA<213>SARS?Virus <400>5

cggggactct?gtggaagagg?c 21

<210>6<211>21<212>RNA/DNA<213>SARS?Virus <400>6

cggaggcacg?tgaacacctc?a 21

<210>7<211>21<212>RNA/DNA<213>SARS?Virus <400>7

cggccacaag?gtcgttgagc?t 21

<210>8<211>21<212>RNA/DNA<213>SARS?Virus <400>8

cggcattcag?tacggtcgta?g 21

<210>9<211>21<212>RNA/DNA<213>SARS?Virus <400>9

cggtaataag?ggagccggtg?g 21

<210>10<211>21<212>RNA/DNA<213>SARS?Virus <400>10

cgggcaagtc?aatgtgcact?c 21

<210>11<211>21<212>RNA/DNA<213>SARS?Virus <400>11

cggtaactat?aaagttacca?a 21

<210>12<211>21<212>RNA/DNA<213>SARS?Virus <400>12

cggtgaactc?gaagcactcg?a 21

<210>13<211>21<212>RNA/DNA<213>SARS?Virus <400>13

cggtaccgaa?gttactgagt?t 21

<210>14<211>21<212>RNA/DNA<213>SARS?Virus <400>14

cggtacagag?gatgattatc?a 21

<210>15<211>21<212>RNA/DNA<213>SARS?Virus <400>15

cggttcgtac?acaggtttat?a 21

<210>16<211>21<212>RNA/DNA<213>SARS?Virus <400>16

cggtgaggtt?ctttcacttg?a 21

<210>17<211>21<212>RNA/DNA<213>SARS?Virus <400>17

cgggaggtta?agactataaa?a 21

<210>18<211>21<212>RNA/DNA<213>SARS?Virus <400>18

cgggtgtaga?agctgtgatg?t 21

<210>19<211>21<212>RNA/DNA<213>SARS?Virus <400>19

cggagctcac?cttacaaaga?t 21

<210>20<211>21<212>RNA/DNA<213>SARS?Virus <400>20

cggcattaat?tatgtgaagt?c 21

<210>21<211>21<212>RNA/DNA<213>SARS?Virus <400>21

cgggtactgt?gctgagagca?a 21

<210>22<211>21<212>RNA/DNA<213>SARS?Virus <400>22

cgggagtcta?ctcagtcttt?t 21

<210>23<211>21<212>RNA/DNA<213>SARS?Virus <400>23

cggatagcaa?gattgttcaa?c 21

<210>24<211>21<212>RNA/DNA<213>SARS?Virus <400>24

cggctggtac?cacacaaaca?g 21

<210>25<211>21<212>RNA/DNA<213>SARS?Virus <400>25

cggaatgtgg?aaaggttatg?g 21

<210>26<211>21<212>RNA/DNA<213>SARS?Virus <400>26

cggatgcatc?aacgttttta?a 21

<210>27<211>21<212>RNA/DNA<213>SARS?Virus <400>27

cgggtttgcg?gtgtaagtgc?a 21

<210>28<211>21<212>RNA/DNA<213>SARS?Virus <400>28

gaaacgagta?actcgtccct?c 21

<210>29<211>21<212>RNA/DNA<213>SARS?Virus <400>29

cggcacaggc?actagtactg?a 21

<210>30<211>21<212>RNA/DNA<213>SARS?Virus <400>30

cggttgctgt?ccatgacttt?t 21

<210>31<211>21<212>RNA/DNA<213>SARS?Virus <400>31

cggtgatttc?gtacaagtag?c 21

<210>32<211>21<212>RNA/DNA<213>SARS?Virus <400>32

cggagttcct?attgtggatt?c 21

<210>33<211>21<212>RNA/DNA<213>SARS?Virus <400>33

cggaagagag?actttgtctc?t 21

<210>34<211>21<212>RNA/DNA<213>SARS?Virus <400>34

cgtgaatttt?aataaagact?t 21

<210>35<211>21<212>RNA/DNA<213>SARS?Virus <400>35

cggtggctgg?cataatatgt?t 21

<210>36<211>21<212>RNA/DNA<213>SARS?Virus <400>36

cggctcacta?tatgttaaac?c 21

<210>37<211>21<212>RNA/DNA<213>SARS?Virus <400>37

cggtgatgct?acaactgctt?a 21

<210>38<211>21<212>RNA/DNA<213>SARS?Virus <400>38

cggctcaagg?tttagtagct?a 21

<210>39<211>21<212>RNA/DNA<213>SARS?Virus <400>39

cggtactggg?aacctgagtt?t 21

<210>40<211>21<212>RNA/DNA<213>SARS?Virus <400>40

cggtgcctgt?attaggagac?c 21

<210>41<211>21<212>RNA/DNA<213>SARS?Virus <400>41

cggcatgcaa?aagtactcta?c 21

<210>42<211>21<212>RNA/DNA<213>SARS?Virus <400>42

cggacttgct?ctctattacc?c 21

<210>43<211>21<212>RNA/DNA<213>SARS?Virus <400>43

cggcatgctc?tcatgcagct?g 21

<210>44<211>21<212>RNA/DNA<213>SARS?Virus <400>44

cgggtaacct?tcagatgaac?c 21

<210>45<211>21<212>RNA/DNA<213>SARS?Virus <400>45

cggatattga?ttatgttcca?c 21

<210>46<211>21<212>RNA/DNA<213>SARS?Virus <400>46

cggcgaagca?cctgtttcca?t 21

<210>47<211>21<212>RNA/DNA<213>SARS?Virus <400>47

cggcattatt?caacagttgc?c 21

<210>48<211>21<212>RNA/DNA<213>SARS?Virus <400>48

cggtcttcat?ttaatgatag?g 21

<210>49<211>21<212>RNA/DNA<213>SARS?Virus <400>49

cggctaataa?atgggacctt?a 21

<210>50<211>21<212>RNA/DNA<213>SARS?Virus <400>50

cggaagcatt?tttaattggg?g 21

<210>51<211>21<212>RNA/DNA<213>SARS?Virus <400>51

cggtgattat?tattaacaat?t 21

<210>52<211>21<212>RNA/DNA<213>SARS?Virus <400>52

cggccacggt?ttgtggacca?a 21

<210>53<211>21<212>RNA/DNA<213>SARS?Virus <400>53

cggagattct?actgaatgtg?c 21

<210>54<211>21<212>RNA/DNA<213>SARS?Virus <400>54

cggaggtaca?aattgacagg?t 21

<210>55<211>21<212>RNA/DNA<213>SARS?Virus <400>55

cggcttcatt?gctggactaa?t 21

<210>56<211>21<212>RNA/DNA<213>SARS?Virus <400>56

cggatggctt?gttattggcg?t 21

<210>57<211>21<212>RNA/DNA<213>SARS?Virus <400>57

cggcatttca?acaccaaaac?t 21

<210>58<211>21<212>RNA/DNA<213>SARS?Virus <400>58

aatcgacggc?tcttcaggag?t 21

<210>59<211>21<212>RNA/DNA<213>SARS?Virus <400>59

cggtttacgt?ctactcgcgt?g 21

<210>60<211>21<212>RNA/DNA<213>SARS?Virus <400>60

cggtactatt?accgttgagg?a 21

<210>61<211>21<212>RNA/DNA<213>SARS?Virus <400>61

cggaacaggt?ttttgtacat?a 21

<210>62<211>21<212>RNA/DNA<213>SARS?Virus <400>62

cgggattgcg?attgcaatgg?c 21

<210>63<211>21<212>RNA/DNA<213>SARS?Virus <400>63

cgggggacaa?ttgtgaccag?a 21

<210>64<211>21<212>RNA/DNA<213>SARS?Virus <400>64

cggacactcc?ctagggcgct?g 21

<210>65<211>21<212>RNA/DNA<213>SARS?Virus <400>65

cggtagcaac?gacaatattg?c 21

<210>66<211>21<212>RNA/DNA<213>SARS?Virus <400>66

cggagttaga?tgatgaagaa?c 21

<210>67<211>21<212>RNA/DNA<213>SARS?Virus <400>67

cggtactcga?catacctatc?a 21

<210>68<211>21<212>RNA/DNA<213>SARS?Virus <400>68

cggcaaaatg?aaagagctca?g 21

<210>69<211>21<212>RNA/DNA<213>SARS?Virus <400>69

cggcgctaac?aaagaaggca?t 21

<210>70<211>21<212>RNA/DNA<213>SARS?Virus <400>70

cggcagtcaa?gcctcttctc?g 21

<210>71<211>21<212>RNA/DNA<213>SARS?Virus <400>71

cggtaattca?agaaattcaa?c 21

<210>72<211>21<212>RNA/DNA<213>SARS?Virus <400>72

cggggaccaa?gacctaatca?g 21

<210>73<211>21<212>RNA/DNA<213>SARS?Virus <400>73

cgggaacatg?gctgacttat?c 21

<210>74<211>21<212>RNA/DNA<213>SARS?Virus <400>74

cggctgacat?ggatgatttc?t 21

<210>75<211>21<212>RNA/DNA<213>SARS?Virus <400>75

cggagtacga?tcgagggtac?a 21

<210>76<211>19<212>RNA/DNA<213>SARS?Virus <400>76

caucauccgg?ugaugcuac 19

<210>77<211>19<212>RNA/DNA<213>SARS?Virus <400>77

uaguguauac?ggcaugcuc 19

<210>78<211>19<212>RNA/DNA<213>SARS?Virus <400>78

gugcgugcag?acgguucgu 19

<210>79<211>19<212>RNA/DNA<213>SARS?Virus <400>79

cguagucgcg?guaauucaa 19

Claims (12)

1. The right that the present invention will declare is:

   1. prevent and treat the prescription of SARS (SARS) gene medicine, it is including, but not limited to following all or part composition:

   ● one or more oligonucleotide compositions that can suppress or kill SARS virus;

   ● receptible carrier on one or more drug effects;

   ● one or more special targets are led protein or polypeptide;

   ● other active ingredient, auxiliary composition or additive.
2. 2. at the oligonucleotide composition that can suppress or kill SARS virus of statement in (1), it is the long double-stranded oligonucleotide (dsRNA of a 19-25 nucleotide, dsDNA, or RNA/DNA) or the long single strain oligonucleotide of 10-30 nucleotide, as RNA, DNA, or the heterozygote of RNA/DNA, and can suppress effectively or expression of gene that deactivation of SARS virus is relevant.
3. 3. in the prescription of statement (1 and 2), the oligonucleotide composition is meant the homologous sequence and/or the anti-sequence of anticipating of any one section gene relevant with SARS virus, and these genes are including, but not limited to RNA polymerase, unwindase, proteolytic enzyme, nucleoprotein N, memebrane protein M, S albumen (S protein) and E albumen (the small membrane protein).
4. 4. in statement (1,2 and 3) the oligonucleotide composition in, it be one group by one or more strands and or double-stranded oligonucleotide form, contain one or more specific motifs (Motif), the molecule that can hybridize efficiently with the specific target sequence in the corresponding RNA molecule.
5. 5. at the oligonucleotide composition of statement in (1 and 2), it is the oligonucleotide that 10-30 nucleotide of the corresponding mRNA molecule of a series of deactivation is effectively grown.They are including, but not limited to the list and/or the double-stranded oligonucleotide molecule of following sequence number.They are: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78.
6. 6. in the prescription of statement (1), the oligonucleotide composition is a kind of mixture, by one or more multi-form strands and/or double-stranded oligonucleotide molecule, mode with equivalent or inequality, or formed with any other compatibility form, be specially adapted to have the patient of different SARS (SARS) symptom.
7. 7. at the oligonucleotide molecule of declaring in (1,2,3,4,5 and 6), it is a kind of natural strand chain and/or double-stranded oligonucleotide molecule, and it is a kind of oligonucleotide molecule that contains the modified nucleotide composition of heterozygosis formula also.
8. 8. at the heterozygosis number polynucleotide molecule of statement in (2 and 7), it is meant the strand or the double-stranded oligonucleotide molecule of the heterozygosis formula that nucleotide and Deoxydization nucleotide form, or refer to mononucleotide in strand or the double-stranded oligonucleotide molecule or monodeoxyribonucleotide be nature and/or through the monomer or the binary of distinct methods modified.
9. 9. at the oligonucleotide molecule of declaring in (1,2,3,4,5 and 6), it is a kind of siRNA molecule that obtains with chemical synthesis or biological synthesis process (as plasmid or virus).
10. 10. in the prescription of statement (1), the composition of oligonucleotide composition can be and finally prevents and treats 0.0000001% to 99% of SARS (SARS) gene medicine total amount.
11. 11. a method of preventing and treating SARS (SARS), it is meant the method that gene medicine simple or compound anti-sars (SARS) is used for crowd prevention and treatment with different dosage forms such as water preparation, spray or unguentum etc. by different spray and eye water and eye ointment etc. by way of as intravenous injection, nasal cavity and oral cavity.
12. 12. method that can suppress or kill SARS virus effectively, come transfection human or animal's cell to suppress the formation of SARS virus thereby it is meant with a certain amount of oligonucleotide composition that can suppress or kill SARS virus, reach the purpose of preventing and treating SARS (SARS).

Images