CN102337263B

CN102337263B - siRNA (Small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application

Info

Publication number: CN102337263B
Application number: CN 201010235942
Authority: CN
Inventors: 梁子才; 张鸿雁
Original assignee: SHENZHEN RIBO BIOTECHNOLOGY CO Ltd
Current assignee: Suzhou Ruibo Biotechnology Co., Ltd
Priority date: 2010-07-21
Filing date: 2010-07-21
Publication date: 2013-05-01
Anticipated expiration: 2030-07-21
Also published as: CN102337263A

Abstract

The invention provides siRNA (small interfering ribonucleic acid) which is capable of inhibiting expression of an enterovirus 71 type gene. The invention also provides a pharmaceutical composition which contains the siRNA provided by the invention as an active component. The invention further provides application of the siRNA in the aspect of preparing a medicament for inhibiting the expression of the enterovirus 71 type gene and application of the siRNA in the aspect of preparing a medicament for treating and/or preventing diseases. The siRNA provided by the invention has high inhibition rate on the expression of the enterovirus 71 type gene and is capable of effectively preventing and/or treating various diseases including hand-foot-and-mouth diseases.

Description

Suppress siRNA and composition and the application of enterovirns type 71 genetic expression

Technical field

The invention relates to a kind of for suppressing enterovirns type 71 genetic expression siRNA and the pharmaceutical composition that contains this siRNA, also relate to this siRNA for the preparation of the application in the medicine that suppresses enterovirns type 71 genetic expression with for the preparation of the application in the medicine that treats and/or prevents disease.

Background technology

(entorovirus 71 for enterovirns type 71, EV71) be Picornaviridae (Picornaviridae) enterovirus genus (Enterovirus) member, it is one of former to be that hand foot mouth disease (hand-foot-and-mouth disease, HFMD) main causes a disease.The infection of EV71 also can cause the multiple diseases relevant with neural system such as paralytic disease of herpangina, aseptic meningitis, BBE and poliomyelitis sample except causing patient's hand foot mouth disease, disable and case fatality rate higher.

The hand foot mouth disease that is caused by EV71 virus is the common transmissible disease of a kind of infant, and take Infants Below morbidity in three years old as main, it is actually rare that the adult infects viral sequela, but can transmitted virus.Virus can be propagated through gi tract (fecal oral route), also can propagate through respiratory tract (spittle, cough, sneeze etc.), also can cause propagation because of contact patient mouth and nose secretory product, skin or herpes mucosae liquid and contaminated hand and article etc.It fash, ulcer etc. occur with positions such as heating and hand, foot, oral cavities clinically and shows as cardinal symptom, and individual patient can cause the mortality complication such as aseptic meningitis, encephalitis, AFP Cases, neurogenic pulmonary edema and myocarditis.On May 2nd, 2008 played China Ministry of Health and includes this disease in the Class C Notifiable disease.

In recent years, EV71 virus has arrived several times on a large scale outbreak of epidemic and in rising trend in the Asian-Pacific area, and Bulgaria was very popular in 1975, has 705 infants and infects dead 44 examples; Malaysia in 1997 is very popular and infects 2628 people, dead 30 examples; Outbreak of epidemic occured in the Taiwan in 1998, approximately had 120,000 people of surpassing infected, dead 78 examples.Especially from 3～April in 2008, after the epidemic situation of acute infection hand foot mouth disease had been broken out in Fuyang, China Anhui Province, a plurality of provinces and cities of China all successively found the popular of hand foot mouth disease on a large scale.Particularly Outbreak occurs especially in this year, according to statistics, this year, whole nation accumulative total reported hand foot mouth disease 38.6 ten thousand many cases by April 30; Dead 236 people; Only national reported cases in one month April of this year are 24.9 ten thousand, and death is 175 examples, and compared with the same period of last year, national reported cases increase by 100,000 many cases, increase by 37.49%; Death increases by 139 examples, increases by 143%.

Therefore, the hand foot mouth disease that causes of EV71 virus has become the popular the most widely seriousness disease of serious threat infantile health.

At present without specificity therapeutic method, just take supportive treatment as main, the main medicine of using has Interferon, rabbit and other spectrum antiviral (acyclovir, ganciclovir, ribavirin etc.), but the shortcoming of these medicines is that specific aim is poor, specificity is low, and toxic side effect is high or be still not clear.March in this year, health ministry indicates further to accelerate the paractical researchs such as hand foot mouth disease diagnostic reagent, vaccine, medicine research and development in national sanitation system hand foot mouth disease preventing and controlling teleconference, for the hand foot mouth disease control provides powerful science and technology support.

Therefore, develop a kind of new way that prevents and/or treats hand foot mouth disease and become problem in the urgent need to address.

Summary of the invention

The object of the present invention is to provide a kind of new way that prevents and/or treats hand foot mouth disease.

An a new direction has been opened up in treatment enterovirns type 71 (EV71) infection that appears as of RNA perturbation technique.RNA disturbs (RNA interference, RNAi) to be closed the expression of corresponding gene or make the process of this gene silencing in the mRNA level by double-stranded RNA (double-stranded RNA, dsRNA) molecule.The RNA perturbation technique is called again Knockdown (knock-down) or gene silencing (gene silencing) visually, it is a kind of typical post-transcriptional gene regulation method, claim again PTGS (post-transcriptional gene silencing, PTGS).The report of relevant RNA interference appears at nineteen ninety the earliest, report simultaneously the RNA interference phenomenon in the transgenic plant by two different research groups, in nearly all eukaryotes such as nematode, fruit bat, zebra fish and mouse, observed the RNA interference phenomenon again later on.1999, Hamilton and Baulcombe have detected length in the plant that the RNA interference occurs be the RNA fragment of 21-25 Nucleotide, and it is necessary that these RNA segments are proved to be the RNA interference, is called small molecules interference RNA (siRNA).Double-stranded siRNA and the relevant enzyme of cell source and the silencing complex (RNA-induced silencing complex, RISC) that Protein formation RNA induces.In the RNA interfering process, the positive-sense strand among the double-stranded siRNA is excluded from complex body, and antisense strand instructs RISC to be attached to the corresponding site of said target mrna, then by the degraded of the rnase iii in mixture said target mrna, thereby closes the expression of target gene.

The genome of EV71 virus is the sub-thread positive chain RNA that contains about 7411 Nucleotide.Provided by the invention for heterogeneic siRNA in the EV71 viral genome, can effectively suppress by the expression of EV71 virogene and copy, thereby at gene level the EV71 virus of invading body is worked, play the effect that prevents and/or treats hand foot mouth disease.

According to a first aspect of the invention, the invention provides a kind of siRNA, wherein, this siRNA can suppress the expression of enterovirns type 71 gene.

According to a second aspect of the invention, the invention provides a kind of pharmaceutical composition be used to preventing and/or treating hand foot mouth disease, wherein, this pharmaceutical composition contains siRNA provided by the invention as activeconstituents.

According to a third aspect of the invention we, the invention provides described siRNA for the preparation of the application in the medicine that suppresses enterovirns type 71 genetic expression.

According to a forth aspect of the invention, the invention provides described siRNA for the preparation of the application in the medicine that treats and/or prevents disease, wherein, described disease is disease, acute respiratory disease, myocarditis or the arbitrarily combination of two or more in them of hand foot mouth disease, central nervous system infection.

SiRNA provided by the invention is high to the inhibiting rate that suppresses enterovirns type 71 genetic expression, can effectively prevent and/or treat the various diseases that comprises hand foot mouth disease.

Description of drawings

Fig. 1 has shown among the embodiment 2 and to utilize two fluorescence report systems to detect the inhibiting rate (in HEK 293a cell, the working concentration of 10nM) of siRNAEV71-T1 to EV71-T50 sequence provided by the present invention.

Fig. 2 has shown that the sequence one of utilized respectively in the embodiment of the invention 3 among the siRNA EV71-T1 to EV71-T50 is to the inhibiting rate (in human rhabdomyosarcoma cells, the working concentration of 10nM) of EV71 viral gene expression.

Fig. 3 shown one of utilize respectively in the embodiment of the invention 4 among the siRNA EV71-T1 to EV71-T50 sequence to the inhibition of the mRNA content of the EV71 virus in the suckling mouse intestinal tissue of EV71 virus infection.

Embodiment

A kind of siRNA is characterized in that, this siRNA can suppress the expression of enterovirns type 71 gene.

(entorovirus 71, EV71) are Picornaviridae (Picomaviridae) enterovirus genus (Enterovirus) members, and its genome is the sub-thread positive chain RNA that contains about 7411 Nucleotide for enterovirns type 71.An open reading frame (ORF) is only arranged among the RNA; Its coding region comprises structural protein gene group P1 and nonstructural protein gene group P2, P3 Three regions.The EV71 genome at first translates one and contains 2194 amino acid whose polyproteins, and polyprotein constantly is hydrolyzed the proteolytic enzyme that produces by itself and is cut in synthetic, produces P1, P2 and three precursor proteins of P3.

The P1 genome comprises 1A (polypeptide VP4), 1B (polypeptide VP2), 1C (polypeptide VP3), four genes of 1D (polypeptide VP1).The P1 precursor protein finally forms VP1 (293 amino acid), VP2 (254 amino acid), VP3 (245 amino acid) and four virus capsid proteins of VP4 (69 amino acid) through a series of processing.

The P2 genome comprises 2A (specific protease), 2B, three genes of 2C.

The P3 genome comprises 3A, VPg (5 ' end is in conjunction with albumen), 3C (specific protease), 3D (RNA polymerase component).The P3 precursor protein utilizes the mechanism such as self cutting of non-concentration dependent to produce 3C (to have protease activity, 184 amino acid) or 3CD (having protease activity), 3AB (have initial viral RNA synthetic function) or 3A (86 amino acid) and 3B (end is in conjunction with albumen VPg, 22 amino acid) and 3D (RNA polymerase, 462 amino acid).

Therefore, siRNA provided by the invention suppresses the expression of one or several gene of P1 gene regions, P2 gene regions and P3 gene regions in the enterovirns type 71 genome by specificity, thereby reduction virus load, at gene level the enterovirns type 71 of invading body is worked, reach the purpose of the hand foot mouth disease that prevention and treatment enterovirns type 71 cause.

Under the preferable case, nucleotide sequence shown in described siRNA provided by the invention one of has among the EV71-T1 to EV71-T50, perhaps have with EV71-T1 to EV71-T50 in one of shown in the nucleotide sequence of nucleotide sequence with sequence identity of at least 85%, wherein

EV71-T1

Positive-sense strand: 5 '-GUUUGCAAAUCCUGUUAAAdTdT-3 '

Antisense strand: 5 '-UUUAACAGGAUUUGCAAACdTdT-3 '

EV71-T2

Positive-sense strand: 5 '-GGUUAUGGUGAGUGGCCUUdTdT-3 '

Antisense strand: 5 '-AAGGCCACUCACCAUAACCdTdT-3 '

EV71-T3

Positive-sense strand: 5 '-GCCCGGAUGUUUCAGUGAAdTdT-3 '

Antisense strand: 5 '-UUCACUGAAACAUCCGGGCdTdT-3 '

EV71-T4

Positive-sense strand: 5 '-CCGGAUGUGUUAACUGAAAdTdT-3 '

Antisense strand: 5 '-UUUCAGUUAACACAUCCGGdTdT-3 '

EV71-T5

Positive-sense strand: 5 '-GUAAAUUCCACCAAGGAGCdTdT-3 '

Antisense strand: 5 '-GCUCCUUGGUGGAAUUUACdTdT-3 '

EV71-T6

Positive-sense strand: 5 '-CCACACCAGUGGAUUAAUUdTdT-3 '

Antisense strand: 5 '-AAUUAAUCCACUGGUGUGGdTdT-3 '

EV71-T7

Positive-sense strand: 5 '-CCAACAAUUGUGCUACAAUdTdT-3 '

Antisense strand: 5 '-AUUGUAGCACAAUUGUUGGdTdT-3 '

EV71-T8

Positive-sense strand: 5 '-GGAGCGACGCCAGUAAUCCdTdT-3 '

Antisense strand: 5 '-GGAUUACUGGCGUCGCUCCdTdT-3 '

EV71-T9

Positive-sense strand: 5 '-CCACGAAUGCCACUAGCUUdTdT-3 '

Antisense strand: 5 '-AAGCUAGUGGCAUUCGUGGdTdT-3 '

EV71-T10

Positive-sense strand: 5 '-GGUCAGGAUCAUUGGAAGUdTdT-3 '

Antisense strand: 5 '-ACUUCCAAUGAUCCUGACCdTdT-3 '

EV71-T11

Positive-sense strand: 5 '-CCCUUGUAAUACCAUGGAUdTdT-3 '

Antisense strand: 5 '-AUCCAUGGUAUUACAAGGGdTdT-3 '

EV71-T12

Positive-sense strand: 5 '-GUAUAUGGUAUCAGACAAAdTdT-3 '

Antisense strand: 5 '-UUUGUCUGAUACCAUAUACdTdT-3 '

EV71-T13

Positive-sense strand: 5 '-GCAAGGAUGCUAGUGAUAUdTdT-3 '

Antisense strand: 5 '-AUAUCACUAGCAUCCUUGCdTdT-3 '

EV71-T14

Positive-sense strand: 5 '-AGAUAGGGUGGCAGAUGUAdTdT-3 '

Antisense strand: 5 '-UACAUCUGCCACCCUAUCUdTdT-3 '

EV71-T15

Positive-sense strand: 5 '-GCUGAGACCACUCUUGAUAdTdT-3 '

Antisense strand: 5 '-UAUCAAGAGUGGUCUCAGCdTdT-3 '

EV71-T16

Positive-sense strand: 5 '-CAACUGGGACAUAGAUAUAdTdT-3 '

Antisense strand: 5 '-UAUAUCUAUGUCCCAGUUGdTdT-3 '

EV71-T17

Positive-sense strand: 5 '-CCACAUUCGGAGAACACAAdTdT-3 '

Antisense strand: 5 '-UUGUGUUCUCCGAAUGUGGdTdT-3 '

EV71-T18

Positive-sense strand: 5 '-CCUUUAGUGGUUAGGAUUUdTdT-3 '

Antisense strand: 5 '-AAAUCCUAACCACUAAAGGdTdT-3 '

EV71-T19

Positive-sense strand: 5 '-GGAUUUACAUGAGAAUGAAdTdT-3 '

Antisense strand: 5 '-UUCAUUCUCAUGUAAAUCCdTdT-3 '

EV71-T20

Positive-sense strand: 5 '-GCAACUUUAGAGUGGUCAAdTdT-3 '

Antisense strand: 5 '-UUGACCACUCUAAAGUUGCdTdT-3 '

EV71-T21

Positive-sense strand: 5 '-UGUAGAGGCUAGCGAGUAUdTdT-3 '

Antisense strand: 5 '-AUACUCGCUAGCCUCUACAdTdT-3 '

EV71-T22

Positive-sense strand: 5 '-GCAAUGGGCUCGUUGGCUUdTdT-3 '

Antisense strand: 5 '-AAGCCAACGAGCCCAUUGCdTdT-3 '

EV71-T23

Positive-sense strand: 5 '-CCGACUACAUCAAGGGUCUdTdT-3 '

Antisense strand: 5 '-AGACCCUUGAUGUAGUCGGdTdT-3 '

EV71-T24

Positive-sense strand: 5 '-GCUCUCAAGAACUAUCUUAdTdT-3 '

Antisense strand: 5 '-UAAGAUAGUUCUUGAGAGCdTdT-3 '

EV71-T25

Positive-sense strand: 5 '-UGAAGGAGCAGUUGAGAAAdTdT-3 '

Antisense strand: 5 '-UUUCUCAACUGCUCCUUCAdTdT-3 '

EV71-T26

Positive-sense strand: 5 '-CCUUAGCGCUGAUAGGUUGdTdT-3 '

Antisense strand: 5 '-CAACCUAUCAGCGCUAAGGdTdT-3 '

EV71-T27

Positive-sense strand: 5 '-UCAAGAAGUUCAAUGACAUdTdT-3 '

Antisense strand: 5 '-AUGUCAUUGAACUUCUUGAdTdT-3 '

EV71-T28

Positive-sense strand: 5 '-UUGAUUGGCUUAAGGAGAAdTdT-3 '

Antisense strand: 5 '-UUCUCCUUAAGCCAAUCAAdTdT-3 '

EV71-T29

Positive-sense strand: 5 '-GGAACAAUCUGCUGCCUCAdTdT-3 '

Antisense strand: 5 '-UGAGGCAGCAGAUUGUUCCdTdT-3 '

EV71-T30

Positive-sense strand: 5 '-GCAAGUUCCAACCGCUAUAdTdT-3 '

Antisense strand: 5 '-UAUAGCGGUUGGAACUUGCdTdT-3 '

EV71-T31

Positive-sense strand: 5 '-AUAUGCAGUUCAAGAGCAAdTdT-3 '

Antisense strand: 5 '-UUGCUCUUGAACUGCAUAUdTdT-3 '

EV71-T32

Positive-sense strand: 5 '-GAGCAAACACCGAAUUGAAdTdT-3 '

Antisense strand: 5 '-UUCAAUUCGGUGUUUGCUCdTdT-3 '

EV71-T33

Positive-sense strand: 5 '-GCUCGAGCAAUCGCUGAUAdTdT-3 '

Antisense strand: 5 '-UAUCAGCGAUUGCUCGAGCdTdT-3 '

EV71-T34

Positive-sense strand: 5 '-GUGACAGACUCGUACAAAAdTdT-3 '

Antisense strand: 5 '-UUUUGUACGAGUCUGUCACdTdT-3 '

EV71-T35

Positive-sense strand: 5 '-GCCCAUUAGUGUGUGGGAAdTdT-3 '

Antisense strand: 5 '-UUCCCACACACUAAUGGGCdTdT-3 '

EV71-T36

Positive-sense strand: 5 '-GCUAUCCAACUUAGAGAUAdTdT-3 '

Antisense strand: 5 '-UAUCUCUAAGUUGGAUAGCdTdT-3 '

EV71-T37

Positive-sense strand: 5 '-CCACCCAAGUUCAGGCCAAdTdT-3 '

Antisense strand: 5 '-UUGGCCUGAACUUGGGUGGdTdT-3 '

EV71-T38

Positive-sense strand: 5 '-GGUUGUCUCGUUGGUGUAUdTdT-3 '

Antisense strand: 5 '-AUACACCAACGAGACAACCdTdT-3 '

EV71-T39

Positive-sense strand: 5 '-GCUCCUAAGCAAGUGCUUAdTdT-3 '

Antisense strand: 5 '-UAAGCACUUGCUUAGGAGCdTdT-3 '

EV71-T40

Positive-sense strand: 5 '-GCAAGUCCAAACAGACCAGdTdT-3 '

Antisense strand: 5 '-CUGGUCUGUUUGGACUUGCdTdT-3 '

EV71-T41

Positive-sense strand: 5 '-GCAAGGAGUCAACCUGGAAdTdT-3 '

Antisense strand: 5 '-UUCCAGGUUGACUCCUUGCdTdT-3 '

EV71-T42

Positive-sense strand: 5 '-GCACCAUGAUGUACAACUUdTdT-3 '

Antisense strand: 5 '-AAGUUGUACAUCAUGGUGCdTdT-3 '

EV71-T43

Positive-sense strand: 5 '-GGAGUUACUUUGCUAGUGAdTdT-3 '

Antisense strand: 5 '-UCACUAGCAAAGUAACUCCdTdT-3 '

EV71-T44

Positive-sense strand: 5 '-GCUAUUGAUCUUCACACUAdTdT-3 '

Antisense strand: 5 '-UAGUGUGAAGAUCAAUAGCdTdT-3 '

EV71-T45

Positive-sense strand: 5 '-CCACUUAUGUCAAGGACGAdTdT-3 '

Antisense strand: 5 '-UCGUCCUUGACAUAAGUGGdTdT-3 '

EV71-T46

Positive-sense strand: 5 '-GCCUUUGACUACUCAGGUUdTdT-3 '

Antisense strand: 5 '-AACCUGAGUAGUCAAAGGCdTdT-3 '

EV71-T47

Positive-sense strand: 5 '-GGAGAUAGGGUAUAGUGAAdTdT-3 '

Antisense strand: 5 '-UUCACUAUACCCUAUCUCCdTdT-3 '

EV71-T48

Positive-sense strand: 5 '-CUCAACAUGGUCGCUUAUGdTdT-3 '

Antisense strand: 5 '-CAUAAGCGACCAUGUUGAGdTdT-3 '

EV71-T49

Positive-sense strand: 5 '-GGACCAAGGACGCACGAAAdTdT-3 '

Antisense strand: 5 '-UUUCGUGCGUCCUUGGUCCdTdT-3 '

EV71-T50

Positive-sense strand: 5 '-GCAAUUGGCUCGAGUUAUUdTdT-3 '

Antisense strand: 5 '-AAUAACUCGAGCCAAUUGCdTdT-3 '.

Advance-go on foot to be preferably, described siRNA one of has among EV71-T1, EV71-T3, EV71-T4, the EV71-T6 to EV71-T10, one of one of one of among the EV71-T12 to EV71-T15, among the EV71-T17 to EV71-T34, among the EV71-T37 to EV71-T47, the nucleotide sequence shown in EV71-T49 or the EV71-T50, perhaps have the nucleotide sequence that has at least 85% sequence identity with one of these nucleotide sequences.

Further be preferably, described siRNA has the nucleotide sequence shown in EV71-T4, EV71-T6, EV71-T7, EV71-T9, EV71-T12, EV71-T13, EV71-T15, EV71-T18, EV71-T19, EV71-T21, EV71-T24, EV71-T25, EV71-T29, EV71-T30, EV71-T34, EV71-T39, EV71-T44 or the EV71-T45, perhaps has the nucleotide sequence that has at least 85% sequence identity with one of these nucleotide sequences.

In the most preferred situation, described siRNA has the nucleotide sequence shown in EV71-T7, EV71-T19, EV71-T21, EV71-T30, EV71-T34, EV71-T39, EV71-T44 or the EV71-T45, perhaps has the nucleotide sequence that has at least 85% sequence identity with one of these nucleotide sequences.

A kind of preferred embodiment in, described siRNA has the nucleotide sequence shown in the EV71-T7, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T19, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T21, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T30, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T34, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T39, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T44, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

In another preferred embodiment, described siRNA has the nucleotide sequence shown in the EV71-T45, perhaps has the nucleotide sequence that has at least 85% sequence identity with this nucleotide sequence.

Described siRNA is double stranded rna molecule, comprise positive-sense strand and antisense strand, and the length of positive-sense strand and antisense strand is 21 Nucleotide, described positive-sense strand and antisense strand 3 ' end separately is two continuous deoxythymidylic acids, and two continuous deoxythymidylic acids, 19 Nucleotide complementations in addition that positive-sense strand and antisense strand are removed 3 ' end form double-stranded.

According to the present invention, described nucleotide sequence preferably has chemically modified, and this chemically modified is at least a in the following modification:

(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of described siRNA;

(2) to the modification of 2 '-OH of the ribose in the nucleotide sequence of described siRNA;

(3) to the modification of the base in the nucleotide sequence of described siRNA.

Described chemically modified is conventionally known to one of skill in the art, and the modification of described phosphodiester bond refers to the oxygen in the phosphodiester bond is modified, and comprises the thiophosphoric acid modification, as shown in Equation 1; With the borine phosphate modified, as shown in Equation 2.Two kinds of modifications can both be stablized the siRNA structure, keep high specific and the high-affinity of base pairing.

Formula 1 formula 2

Described ribose is modified the modification that refers to 2 '-OH in the Nucleotide pentose,, introduces some substituting group in the hydroxy position of ribose that is, and for example, 2 '-fluoro is modified, as shown in Equation 3; 2 '-oxygen methyl is modified, as shown in Equation 4; 2 '-oxygen ethylidene methoxyl group is modified, as shown in Equation 5; 2,4 '-dinitrophenol(DNP) is modified, as shown in Equation 6; Lock nucleic acid (LNA), as shown in Equation 7; 2 '-amido modified, as shown in Equation 8; 2 '-deoxidation is modified, as shown in Equation 9.

Formula 3 formulas 4

Formula 5 formulas 6

Formula 7 formulas 8 formulas 9

Described base modification refers to the base of Nucleotide is modified, for example, 5 '-the bromouracil modification, as shown in Equation 10; 5 '-the iodouracil modification, the N3-methyl uracil is modified as shown in Equation 11, as shown in Equation 12; 2,6-diaminopurine is modified, as shown in Equation 13.

Formula 10 formulas 11

Formula 12 formulas 13

These modifications can increase the bioavailability of described siRNA, and the affinity of raising and target sequence strengthens the ability that the opposing nuclease is hydrolyzed in cell.

In addition, in order to promote siRNA to enter cell, can be on the basis of above modification, at the terminal group of introducing the lipotropy such as cholesterol, albumen, polypeptide, polymer, aptamers (aptamer), VITAMIN, folic acid, cholic acid or lipid molecule or helping permeable membrane of siRNA; Or be beneficial to have an effect by the cytolemma and the intracellular mRNA that are consisted of by lipid bilayer with liposome, polymer, lipid molecule or nanoparticle parcel siRNA.

The preparation method of siRNA provided by the invention comprises the design of siRNA sequence and synthesizing of siRNA sequence.

Select each recent years of China to come isolated EV71 whole genome sequence to carry out the gene comparison, choosing higher conservative EV71 viral genome (the Genbank registration number is FJ713137) is template, again respectively for the conserved regions of EV71 virogene, choose the nucleotide sequence of 19bp, design siRNA.

Described P1, P2, P3 genome are respectively 745-3330bp, 3331-5064bp, 5065-7323bp at EV71 viral genome (the Genbank registration number is FJ713137) relative position.

Describedly undertaken by following principle for P1 genome (VP4, VP2, VP3 and VP1), P2 genome (2A, 2B and 2C), the design of P3 genome (3A, 3B, 3C and 3D) siRNA sequence:

50-100 base begins behind the gene promoter of EV71 virus FJ713137 strain, and seek the 19bp nucleotide sequence that meets following condition in the gene order: (1) is initial with G or C, with A or T ending; (2) terminal last 7 bases have at least 5 to be A or T; (3) avoid 4 continuous bases, such as AAAA or CCCC, thus the complexity of increase base; (4) GC content is between the 30-50%.

After choosing according to mentioned above principle, analyze by BLAST again, candidate siRNA target site is carried out sequence analysis with human genome sequencing, eliminating has the sequence of higher sequence homology (16 above bases) with human genome sequencing, guaranteeing that candidate siRNA target site can be to Human genome generation restraining effect, and only the EV71 virogene had special restraining effect.

3 ' end of the 19bp nucleotide sequence that will obtain so at last adds two deoxythymidine acid as the positive-sense strand of siRNA sequence, adds that at 3 ' end of the complementary sequence of this 19bp nucleotide sequence two deoxythymidine acid are as the antisense strand of siRNA sequence.

The synthetic method that can adopt chemosynthesis of described siRNA sequence, it is synthetic that perhaps the synthetic biotech company of nucleic acid is specialized in trust, as entrusting the sharp rich company in Guangzhou to synthesize.

In general, the method for described chemosynthesis comprises following Four processes: (1) oligomerization ribonucleotide synthetic; (2) deprotection; (3) purifies and separates; (4) desalination.

For example, have concrete steps of siRNA chemosynthesis of nucleotide sequence shown in the EV71-T1 as follows:

(1) the oligomerization ribonucleotide is synthetic: automated DNA/the RNA synthesizer (for example, AppliedBiosystems EXPEDITE8909) the upper RNA that sets synthetic 1 mmole, the coupling time of setting simultaneously each circulation is 10-15 minute, initiator is that 5 '-O-of connecting of solid phase is to dimethoxy-thymidine upholder, first circulates in and connects a base on the solid support, then in the n time (19 〉=n 〉=2) circulation, the base that connects the n-1 time circulation connects a base, repeats this circulation until finish the synthetic of whole nucleotide sequences.

(2) deprotection

The solid support that is connected with siRNA is joined in the test tube, and in this test tube, add the ethanol/ethamine (volume ratio is 1: 3) of 1-5 milliliter, then sealing, place 55-70 ℃ of incubator, hatched 2-30 hour, fech connection has the solid support of siRNA and with distilled water drip washing 2-3 time (each approximately 1 milliliter), collects elutriant, and at room temperature dry 30-40 minute.Then, add the tetrahydrofuran solution (1M) of 1-5 milliliter tetrabutyl ammonium fluoride, room temperature was placed 4-12 hour, added 2-5 milliliter ethanol again, and collecting precipitation namely obtains the crude product of siRNA.

(3) purifies and separates

The crude product of the siRNA that obtains is dissolved in the ammonium acetate solution that the 2-5 ml concn is 1-5 mole/milliliter, then separates by the C18 high pressure liquid chromatography, obtain the siRNA product of purifying.

(4) desalination

Be siRNA product 2-4 time (each 2-5 milliliter) of the aqueous ethanolic solution washing purifying of 75 % by weight with concentration, removing salt, and drying under the room temperature.Then with oligomerization Yeast Nucleic Acid mixed dissolution (10mM Tris in the damping fluid of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl), this solution is heated to 90-95 ℃, then slowly this solution is cooled to room temperature, and kept room temperature 16-22 hour, obtain containing the solution of siRNA.

The present invention also provides a kind of pharmaceutical composition be used to preventing and/or treating hand foot mouth disease, and wherein, this pharmaceutical composition contains at least a siRNA among the siRNA provided by the invention as activeconstituents.

Preferably, pharmaceutical composition contains at least two or more siRNA among the siRNA provided by the invention as activeconstituents.

More preferably, described pharmaceutical composition can also contain one or more in pharmaceutically acceptable thinner, pharmaceutically acceptable carrier, pharmaceutically acceptable assistant agent and other chemotherapeutics.

Among the present invention, the formulation of described pharmaceutical composition can be the various formulations in the pharmacy field, for example, can be tablet, capsule, powder agent, injection liquid, emulsion, suppository, paste, lotion, aerosol or paster.

In one embodiment, described pharmaceutical composition can be injection liquid.

Among the present invention, described injection liquid contains pharmaceutically acceptable carrier and siRNA provided by the invention, the content of described siRNA and pharmaceutically acceptable carrier can in very large range change, under the preferable case, with respect to the siRNA of 100 weight parts, the content of described pharmaceutically acceptable carrier is the 100-10000000 weight part.

Among the present invention, described pharmaceutically acceptable carrier is had no particular limits, can be that the pH value is the phosphate buffered saline buffer of 5.5-8.5 for the phosphoric acid buffer of 4.0-9.0, tri methylol amino methane hydrochloride damping fluid, physiological saline or the pH that the pH value is 7.5-8.5, under the preferable case, described pharmaceutically acceptable carrier is that the pH value is the phosphoric acid buffer of 4.0-9.0.

According to the present invention, described injection liquid can also contain protective material and/or osmotic pressure regulator; Take described injection liquid as benchmark, described protectant content is the 0.01-30 % by weight, and described protective material selects one or more in white muscle alcohol, sorbyl alcohol and the sucrose; It is 200-700 m osmole/kilogram that the content of described osmotic pressure regulator makes the osmotic pressure of described injection liquid, and described osmotic pressure regulator is sodium-chlor and/or Repone K.

When injection during medicinal compositions of the present invention, the injection consumption can be this area dosage commonly used, and described dosage can be according to various parameters, especially determine according to the severity of patient's to be treated age, body weight and illness.

On the other hand, the present invention also provides described siRNA for the preparation of the application in the medicine that suppresses enterovirns type 71 genetic expression.

In addition, the present invention also provides described siRNA for the preparation of the application in the medicine that treats and/or prevents disease, wherein, described disease can comprise: the disease of hand foot mouth disease, central nervous system infection, acute respiratory disease, myocarditis and their combination.Disease in the group that the disease of described central nervous system infection can form for the paralytic disease that is selected from by herpangina, aseptic meningitis, BBE, poliomyelitis sample; Described acute respiratory disease can be for being selected from the disease in the group that is comprised of pharyngitis, asthma, bronchiolitis and pneumonia neurogenic pulmonary edema.

On the other hand, the present invention also provides a kind of enterovirns type 71 gene that suppresses in the method for patient's expression in vivo, wherein, the method comprises patient's administration siRNA of the present invention or pharmaceutical composition, described patient refers to the object suffering from disease or have ill danger, be preferably Mammals, more preferably human.

In addition, the present invention also provides a kind of methods for the treatment of of disease, wherein, the method comprises that described disease can comprise to patient's administration siRNA of the present invention or pharmaceutical composition: the disease of hand foot mouth disease, central nervous system infection, acute respiratory disease, myocarditis and their combination.Disease in the group that the disease of described central nervous system infection can form for the paralytic disease that is selected from by herpangina, aseptic meningitis, BBE, poliomyelitis sample; Described acute respiratory disease can be for being selected from the disease in the group that is comprised of pharyngitis, asthma, bronchiolitis and pneumonia neurogenic pulmonary edema.

Further specify the present invention below in conjunction with embodiment, unless stated otherwise, the used reagent of the present invention, substratum are the commercial goods.

Embodiment 1

Select each recent years of China to come isolated EV71 whole genome sequence to carry out the gene comparison, the EV71 viral genome (the Genbank registration number is FJ713137) that acquisition has higher conserved sequence is template, again respectively for the conserved regions of EV71 virus FJ713137 gene, choose the nucleotide sequence of 19bp, design siRNA.

Describedly undertaken by following principle for P1 genome (VP4, VP2, VP3 and VP1), P2 genome (2A, 2B and 2C), the design of P3 genome (3A, VPg, 3C and 3D) siRNA sequence:

The siRNA that designs carries out chemosynthesis through Guangzhou Rui Bo company, obtains siRNA EV71-T1 to EV71-T50, and the nucleotide sequence of described siRNA EV71-T1 to EV71-T50 is as shown in table 1 below.

Table 1

Embodiment 2

The detection of siRNA sequence retarding effect

(1) cultivation of HEK 293a cell

With the DMEM perfect medium that contains 10% foetal calf serum and 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, on 24 porocyte culture plates with 1 * 10 ⁵The density of individual cells/well inoculation HEK293a cell (being derived from Peking University) is 37 and CO in temperature ₂Content is to cultivate in the incubator of 5 volume %, goes down to posterity, changes fresh culture in per 72 hours.In transfection front 24 hours, the trypsin digestion cell with 0.25%, counting is then with 1 * 10 ⁵The concentration of individual cell/ml is inoculated into 24 orifice plates, every hole 500 μ l.

(2) preparation of fusion plasmid

Preparation siRNA attacks the fusion plasmid of site and Fluc (Firefly luciferase) expressed sequence, is used for detecting siRNA it is attacked the inhibition degree in site.

Concrete operation step is as follows: be converted to dna sequence dna according to designed siRNA sequence, and add the sticky end sequence of restriction enzyme at the two ends of its positive-sense strand, wherein 5 ' end is the sticky end sequence of BglII: GATCT-, and 3 ' end is the sticky end sequence of ApaI :-GGGCC.The single stranded DNA sequence that designs is synthetic by the handsome company in Shanghai (Invitrogen).

DNA is dissolved in respectively in the sterilized water without the DNA enzyme, is configured to the dna solution that concentration is 20 μ M.Each 3 μ l of paired single stranded DNA are added in the sterilized water of 14 μ l and be made into the annealing reaction system that cumulative volume is 20 μ l.Each annealing reaction kept 10 minutes at 90 ℃, whenever fell 5 ℃ again and kept 3 minutes, finally was down under 25 ℃ of conditions that also keep and finished.The double-stranded DNA final concentration is 3 μ M.

The siQaunt plasmid with luciferase expressed sequence that after product and Bgl II (NEB, R0144V) and Apa I (NEB, R0114V) digestion with restriction enzyme cross of will annealing connects.T4 ligase enzyme (the Invitrogen of the siQuant plasmid that 15ng is crossed through digestion with restriction enzyme, 0.6 μ l DNA annealing product, 2 μ l 5X T4 damping fluids and 0.5 activity unit, 15224-017) add in the sterilized water of respective volume, be made into the reaction system that final volume is 10 μ l, after fully mixing, 25 ℃ of lower placements 30 minutes, obtain connecting product.

The connection product of 5 μ l previous steps is added 50 μ l DH5 α competent cells (TIANGEN, CB101) transform, mixing is placed on 24 minutes on ice, puts into 42 ℃ of heat shocks 60 seconds again, places back 2 minutes on ice rapidly subsequently.With 1ml SOC nutrient solution (prescription: 20g/L Tryptone; 5g/LYeastExtract; 0.5g/L NaCl; 2.5mM KCl; 10ml MgCl ₂20mM glucose) add bacterium liquid, in 37 ℃ of 200rpm vibrations 1 hour.With bacterium liquid with 4000rpm centrifugal 5 minutes, carefully discard 700 μ l supernatants afterwards, residue 300 μ l bacterium liquid evenly are applied to the LB agarose plate (prescription: 10g/L Tryptone that contains 100 μ g/ml penbritins; 5g/L Yeast Extract; 5g/L NaCl; 1.5%Agar).Be inverted in 37 ℃ of incubators, incubated overnight.

3 mono-clonal bacterial plaques of each sample picking add respectively the LB substratum of 30 μ l, and vortex uses as template.With 0.5 μ l Primer 1---SiQuant-forward (20 μ M), the corresponding DNA antisense strand of 0.5 μ l Primer 2---(20 μ M), 2 μ l templates, 2.5 μ l10X PCR damping fluids, 0.5 μ l dNTP (2.5mM) and 0.25 μ l Ex Taq polysaccharase (Takara, DRR001A) be made into the reaction system that final volume is 25 μ l in the sterilized water of adding 18.75 μ l, after fully mixing, carry out the PCR reaction.Reaction conditions is: 95 ℃ of denaturation 30min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 30s, 25 circulations; 72 ℃ are extended 10min.Use 1% sepharose checking PCR product, deposition condition is 120V constant voltage 30 minutes.Demonstration has 150bp product band such as electrophoresis result, is positive findings.Become positive residue mono-clonal bacterium liquid all to be connected in the LB substratum that 4ml contains 100 μ g/ml penbritins PCR result, spend the night in 37 ℃ of 250rpm.

Positive bacteria liquid is extracted plasmid with the little extraction reagent kit of plasmid (Promega, Al460).With the bacterium liquid of 3-5ml in 14,000rpm centrifugal 5 minutes, supernatant discarded.Add the concussion of 250 μ l cell suspending liquids and vortex or piping and druming thin with abundant suspension; Add again 250 μ l cell pyrolysis liquids and put upside down centrifuge tube 4 times with abundant mixing, clarify to cell suspending liquid; Add 10 μ l alkaline protease solutions and put upside down centrifuge tube 4 times with abundant mixing; Add 350 μ l neutralization solutions and put upside down rapidly centrifuge tube 4 times with abundant mixing, in 4 ℃ with cell pyrolysis liquid with about l4, centrifugal 15 minutes of 000rpm rotating speed.For each sample a centrifugal column is inserted in the 2ml collection tube; Cleared lysate carefully is transferred in the ready centrifugal column, avoids any white precipitate and supernatant liquor are together shifted; From collection tube, take out centrifuge tube after 1 minute and discard liquid in the collection tube with maximum velocity centrifugation; Centrifugal column is reinserted in the collection tube; Add the post scavenging solution that 750 μ l had before diluted with 95% alcohol, with maximum velocity centrifugation 1 minute; From collection tube, take out centrifuge tube and discard liquid in the collection tube; Centrifugal column is reinserted in the collection tube, with maximum velocity centrifugation 2 minutes.Centrifugal column is transferred in the new 1.5ml sterilization centrifuge tube, and room temperature left standstill 10 minutes.The sterilized water of 50 μ l nuclease free is added centrifugal column, leave standstill after 2 minutes with maximum velocity centrifugation 1 minute.The plasmid of wash-out in the centrifuge tube is collected, and after measuring concentration, unification is diluted to 200ng/ μ l, is stored in-20 ℃.

(3) transfection of siRNA

Use the Lipofectamine of Invitrogen company ^TMThe siRNA EV71-T1 to EV71-T50 that 2000 liposomes obtain embodiment 1 with its respectively the corresponding fusion plasmid that is obtained by previous step carry out respectively cotransfection, not add siRNA as blank.All samples and blank cotransfection can be expressed the PRL-TK plasmid of Renilla luciferase (Renilla Luciferase) as interior mark simultaneously.

Concrete operation step is as follows: siRNA is dissolved in the sterilized water without the RNA enzyme, and being mixed with concentration is the siRNA solution of 20 μ mol/L.Supernatant in the every porocyte of sucking-off, and the low blood serum medium of the Opti-MEM I of adding 0.5ml (Invitrogen company, 31985-062).With 0.3 μ l, 20 μ mol/LsiRNA solution (final working concentration is 10nmol/L), 100ng fusion plasmid and 50ng PRL-TK plasmid are diluted in low blood serum medium (the Invitrogen company of 50 μ l Opti-MEM I, 31985-062), with 1.0 μ l Lipofectamine ^TM2000 liposomes are diluted in low blood serum medium (the Invitrogen company of 50 μ l Opti-MEM I, 31985-062), then above-mentioned two kinds of solution are at room temperature hatched after 5 minutes and mix, mixing solutions is after room temperature leaves standstill 20 minutes, and 100 these mixing solutionss of μ l are added to inoculation to be had in described 24 orifice plates of cell.The ultimate density of siRNA is 10nM.37 ℃ of cultivations of cell 4 hours add the DMEM perfect medium that 1ml contains 10% foetal calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates again, then cultivate under 37 ℃ 24 hours again.

(4) two fluorescence report system detects the inhibiting rate of siRNA sequence

Use Dual-

Reporter gene detection system (Promega, E1960) detects the cell of transfection.Nutrient solution in 24 hours the cell after the transfection is removed, clean cell with 500 μ l PBS after, remove scavenging solution.Described PBS consists of: NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂HPO ₄4.3mmol/L, KH ₂PO ₄1.4mmol/L.Every hole adds 100 μ l 1X PLB and rocked 20 minutes at horizontal shaking table in room temperature.Cell pyrolysis liquid is transferred in the 0.5ml centrifuge tube, with the centrifugal 1min of the rotating speed of 10000rpm.Draw 10 μ l supernatants in 96 orifice plates, every hole adds 50 μ l LARII reagent, detects Lampyridea fluorescent value, adds 50 μ l Stop﹠amp again;

Reagent detects sea pansy fluorescent value.

SiRNA inhibiting rate (%)=[1-(experimental port Lampyridea fluorescent value/sea pansy fluorescent value)/(blank well Lampyridea fluorescent value/sea pansy fluorescent value)] * 100%, the result as shown in Figure 1.

Can find out from the data of Fig. 1, under the working concentration of 10nM, the inhibiting rate of siRNAEV71-T1 to EV71-T50 sequence provided by the invention is more than 40%.Even except siRNAEV71-T5, siRNA EV71-T11, siRNA EV71-T16, siRNA EV71-T36 and siRNA EV71-T48, the suppression efficiency of remaining siRNA sequence can both arrive more than 70%; Especially one of among EV71-T1, EV71-T3, EV71-T4, the EV71-T6 to EV71-T10, one of among the EV71-T12 to EV71-T15, one of among the EV71-T17 to EV71-T34, one of among the EV71-T37 to EV71-T47, the suppression efficiency of EV71-T49 or EV71-T50 even can reach more than 80%.

Embodiment 3

Inhibiting rate to enterovirns type 71 genetic expression detects

(1) cultivation of rhabdosarcoma (Rhabdomyosarcoma, RD) cell

With the MEM perfect medium (Gibco) that contains 5% foetal calf serum, 1% Sodium.alpha.-ketopropionate, 1.5% sodium bicarbonate and 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, on 24 porocyte culture plates with 4 * 10 ⁴The density inoculation RD cell of individual cells/well is 37 ℃ and CO in temperature ₂Content is to cultivate in 5% the incubator, goes down to posterity, changes fresh culture in per 72 hours.In transfection front 24 hours, the trypsin digestion cell with 0.25%, counting is then with 4 * 10 ⁴The concentration of individual cell/ml is inoculated into 24 orifice plates, every hole 500 μ l.

(2) transfection of siRNA and virus infection

Use the LipofectamineTM2000 liposome of Invitrogen company that the siRNA EV71-T1 to EV71-T50 that embodiment 1 obtains is carried out respectively transfection, not add siRNA as blank.

Bed board after 24 hours cell converge about 50% and get final product transfection.Supernatant in the every porocyte of sucking-off, and the low blood serum medium of the Opti-MEM I of adding 0.5ml (Invitrogen company, 31985-062).With 6pmol siRNA be diluted in the low blood serum medium of 50 μ l Opti-MEM I (Invitrogen company, 31985-062) in, with 1.0 μ l Lipofectamine ^TM2000 liposomes are diluted in low blood serum medium (the Invitrogen company of other 50 μ lOpti-MEM I, 31985-062), then above-mentioned two kinds of solution are at room temperature hatched after 5 minutes and mix, mixing solutions is after room temperature leaves standstill 20 minutes, and 100 these mixing solutionss of μ l are added to inoculation to be had in described 24 orifice plates of cell.The ultimate density of siRNA is 10nM.37 ℃ of cultivations of cell add DMEM (GIBCO, the Lot NO.SH30022.01B) perfect medium that 1ml contains 10% foetal calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates after 4 hours again, continue at 37 ℃ of lower cultivations.After siRNA transfection 12 hours, be 10 with TCID50 ⁹The EV71 virus infection cell is infected.

(3) virus titer detects

The cell infection virus-4 is got the supernatant liquor in 24 orifice plates after 8 hours, be used for virus titer and detect.At first healthy RD cell is inoculated into 96 orifice plates with the concentration of 8000-10000 cells/well, the cell of inoculating each hole after rear 20 hours gets final product virus inoculation when being paved into about 60% abundance of individual layer.With the nutrient solution sucking-off in 96 orifice plates, with the nutrient solution of 100ul serum-free cell surface is washed one time.The supernatant liquor that contains virus of collecting is used the nutrient solution of serum-free with 10 times of ratios dilutions (10 ^-1-10 ^-10).The virus inoculation that dilution is good is in 96 orifice plates, and each extent of dilution is inoculated a tandem totally 8 holes, and the above-mentioned viral dilution liquid of 100 μ l is inoculated in every hole.Wherein the inoculation of two tandem normal cells does not contain the nutrient solution of virus as positive controls.37 ℃ of CO ₂After hatching 1 hour in the incubator, discard virus liquid, add nutrient solution 200 μ l and continue at 37 ℃ of CO ₂Cultivate in the incubator.After 48 hours, microscopically observation of cell pathology (CPE) calculates the TCID50 of this virus liquid by the Reed-Muench method.Such as TCID50=10 ^-9.0/ 0.1ml (being TCID50 (log10)=9.0), i.e. explanation is with this virus liquid dilution 10 ^9.0Times the time inoculate 100 μ l viral dilution liquid and can make 50% cell generation pathology.The result is as shown in table 2.

Table 2

	EV71-T1	EV71-T2	EV71-T3	EV71-T4	EV71-T5	EV71-T6	EV71-T7	EV71-T8
									TCID50(log10)	7.2	7.4	7.2	6.7	7.4	6.7	6.4	7.1

										EV71-T9	EV71-T10	EV71-T11	EV71-T12	EV71-T13	EV71-T14	EV71-T15	EV71-T16
TCID50(log10)	6.7	7.5	7.4	6.7	6.7	7.1	6.7	7.3

	EV71-T17	EV71-T18	EV71-T19	EV71-T20	EV71-T21	EV71-T22	EV71-T23	EV71-T24
									TCID50(log10)	6.9	6.7	6.4	6.9	6.4	6.9	7.2	6.7

										EV71-T25	EV71-T26	EV71-T27	EV71-T28	EV71-T29	EV71-T30	EV71-T31	EV71-T32
TCID50(log10)	6.7	7	6.9	6.9	6.8	6.4	7.1	6.8

	EV71-T33	EV71-T34	EV71-T35	EV71-T36	EV71-T37	EV71-T38	EV71-T39	EV71-T40
									TCID50(log10)	7.5	6.3	7.5	7.5	7.1	7.2	6.4	7

										EV71-T41	EV71-T42	EV71-T43	EV71-T44	EV71-T45	EV71-T46	EV71-T47	EV71-T48
TCID50(log10)	6.8	6.8	6.6	6.3	5.9	6.6	6.5	7.6

	EV71-T49	EV71-T50		Provirus (contrast)
									TCID50(log10)	6.5	6.5		9.0

Can find out from the data of upper table 2, siRNA EV71-T1 to EV71-T50 provided by the invention has significant restraining effect to EV71 Viral infection activity, and especially EV71-T7, EV71-T19, EV71-T21, EV71-T30, EV71-T34, EV7l-T39, EV71-T44 or EV71-T45 can be reduced to the TCID50 (log10) of EV71 virus below 6.5.

(4) fluorescence quantitative PCR method detects the restraining effect that siRNA expresses the EV71 virus mRNA.

By the Real Time-PCR expression amount of EV71 virogene mRNA in the RD of EV71 virus infection cell of siRNA EV71-T1 to EV71-T50 that detected respectively transfection in (2), with untransfected siRNA through the RD of EV71 virus infection cell in contrast.

Concrete steps are: with 1ml Trizol (GIBCOL company) cracking transfection siRNA through the RD of EV71 virus infection cell, and extract total RNA, the concrete steps of extracting total RNA are: be 37 ℃ and CO with the cell after the transfection in temperature ₂Content is to cultivate 48 hours in 5% the incubator, centrifugal collecting cell then, and wash one time with the 2ml PBS of precooling; Described PBS consists of: NaCl137mmol/L, KCl 2.7mmol/L, Na ₂HPO ₄4.3mmol/L, KH ₂PO ₄1.4mmol/L; Every hole adds 1ml Trizol, and room temperature was placed 5 minutes, cell generation cracking; Lysate is transferred in the 1.5mlEP pipe; Add 200 μ l chloroforms, use hand concuss 15 seconds, room temperature was placed 3 minutes; 14000rpm4 ℃ centrifugal 15 minutes; Get honest and upright and thrifty 500 μ l on the liquid phase and be put in the new EP pipe, add 500 μ l Virahols, room temperature was placed 10 minutes; 12000rpm, 4 ℃ centrifugal 10 minutes, remove supernatant, be that 75% ethanol is washed throw out once with 1ml concentration; 7600rpm, 4 ℃ are centrifugal 5 minutes; Remove supernatant, drying at room temperature RNA precipitation 10 minutes; Add 20 μ l ddH ₂O dissolves RNA.

The DNase I (RNase-free) (TakaRa company) of 2 units is added in the DEPC water of the above-mentioned RNA of being dissolved with, and under 37 ℃ of conditions, left standstill 30 minutes, to remove DNA residual among total RNA.After DNase I processing, adopt the PureLink Micro-to-Midi Total RNAPurification Kit of Invitrogen company that total RNA is purified, the concrete steps of purifying are: the concentration that adds 20 μ l in total RNA is 70% ethanol, vibration mixes, mixture is transferred on the purification column, centrifugal 15 seconds of room temperature 12000rpm, discard filtered liquid, add 700 μ l cleaning buffer solution I (TakaRa company), centrifugal 15 seconds of room temperature 12000rpm discards filtered liquid, add 500 μ l cleaning buffer solution II (TakaRa company), centrifugal 15 seconds of room temperature 12000rpm discards filtered liquid, adds 500 μ l cleaning buffer solution II (TakaRa company) again, centrifugal 15 seconds of room temperature 12000rpm, abandon filtered liquid, centrifugal 1 minute of room temperature 12000rpm is transferred to purification column on the RNA collection tube, add 30 μ l DEPC water, room temperature was placed 1 minute, and centrifugal 2 minutes of room temperature 13000rpm places-80 ℃ of preservations with the RNA sample.

The total RNA that obtains after purifying is carried out reverse transcription reaction, in reverse transcription reaction, used reversed transcriptive enzyme is the M-MLV reversed transcriptive enzyme of Promega company, the concrete steps of reverse transcription reaction are: the total RNA after 1ug is purified mixes in test tube with the Oligo dT of 1ul (0.5ug), with DEPC water cumulative volume is complemented to 16.25 μ l, test tube is heated, and the condition of heating comprises that Heating temperature is 70 ℃, and be 5 minutes heat-up time; Then test tube is cooled to rapidly 0 ℃, and adds damping fluid (5 * MLVbuffer, 5 μ l, 10mM Dntp1.25 μ l, RNasin 0.5 μ l, M-MLV 1 μ l), under 42 ℃ of conditions, hatched 1 hour, obtain cDNA.

With the cDNA that the obtains template as the PCR reaction, carry out Real-time PCR reaction.Real-time PCR reaction system is: ddH ₂O 17.5 μ l, 10mM Dntp 0.5 μ l, 10 * Taq buffer2.5 μ l, Taq 0.5 μ l, F primer 0.5 μ l, R primer 0.5 μ l, Syber Green I 1 μ l, cDNA 2 μ l; The condition of PCR reaction is: 94 ℃ 2 minutes, 94 ℃ 15 seconds, 60 ℃ 30 seconds, totally 40 circulations.Set up simultaneously GAPDH as reference gene, calculate the siRNA inhibiting rate according to following formula.

SiRNA inhibiting rate=[1-(the GAPDH copy number after the copy number of the EV71 virogene after the siRNA transfection/siRNA transfection)/(copy number of control wells EV71 virogene/control wells GAPDH copy number)] * 100%.

Totally 10 pairs of Realtime PCR primers are chosen respectively corresponding primer according to adding siRNA sample.Primer sequence such as following table:

The primer numbering	Upstream primer	Downstream primer
			EV71-1	AGCCACTGAGGGTTCCACCATAAA	TGGATCCTGCTTGAGACCCTGTTT
EV71-2	AGCACATGCCCTCAATGTTTGTCC	ACCTTCCCAACAGATGTCACCACT
			EV71-3	GGAGCACATGCCCTCAATGTTTGT	ACCTTCCCAACAGATGTCACCACT
EV71-4	TAACAATGTGCCCACGAATGCCAC	ACCATTGGGTGTAGTATCCGCACA
			EV71-5	ACTGGTGGATGAGCAAGGAGTCAA	TGGCAGTGCTGATACTTTCTGGGA
EV71-6	CGCAGGTTTCAGTGCCATTCATGT	TTGGCATCTAAGGATACCACCGCA
			EV71-7	CGCAGGTTTCAGTGCCATTCATGT	TGGCATCTAAGGATACCACCGCAA
EV71-8	ATCAGGTCGATGAGTCACCGCATT	TGGATCCTGCTTGAGACCCTGTTT
			EV71-9	ATCAGGTCGATGAGTCACCGCATT	ACTTGCGAACCCATGTTTAGCTGC
EV71-10	CGCAGGTTTCAGTGCCATTCATGT	TGCCCAATCATTGTGAGTGGCAAG

The GAPDH:3-glyceraldehyde phosphate dehydrogenase gene.The glyceraldehyde 3-phosphate dehydro-genase gene is the house-keeping gene in the cell, and stably express in cell is not subjected to other impacts that adds factor, the internal reference that therefore reacts as quantitative fluorescent PCR.The result as shown in Figure 2.

As can be seen from Figure 2, under the working concentration of 10nM, siRNA EV71-T1 to EV71-T50 provided by the invention is all inhibited to the EV71 viral gene expression.Especially siRNAEV71-T7, siRNA EV71-T19, siRNA EV71-T21, siRNA EV71-T30, siRNAEV71-T34, siRNAEV71-T39, siRNAEV71-T44, siRNA EV71-T45, suppression efficiency all more than 85%.

Embodiment 4

The experiment of EV71 virus infected mice

Laboratory animal: the Balb/c suckling mouse of an age in days

At first suckling mouse is carried out the EV71 Viral infection.Experimental group suckling mouse (n=5) at first the approach of direct oral cavity administration or abdominal injection to carry out TCID50 be 10 ⁹The EV71 virus infection.

The siRNA EV71-T1 to EV71-T50 that respectively embodiment 1 is obtained carries out chemically modified, the concrete U and the C that are modified to positive-sense strand carry out the modification of 2 '-oxygen methyl, U in the antisense strand is carried out 2 '-fluoro to be modified, siRNA after respectively 1.2mg (0.09 μ mol) being modified afterwards is dissolved in the stroke-physiological saline solution of 1.5ml without the RNA enzyme, being mixed with concentration is the siRNA solution of 60 μ mol/L, and mixes with 1: 1 volume ratio with liposome.

Join in the stroke-physiological saline solution (siRNA concentration is 0.15mg/ml) of 5ml without the RNA enzyme with the siRNA after the liposome, obtain injection liquid.

Suckling mouse behind the EV71 virus infection 24 hours, use respectively injection liquid by the tail vein injection of routine suckling mouse to be injected, the volume of injection is injection liquid/kg body weight of 20ml, the physiological saline of blank group injection equal volume, all only inject 1 time, not add siRNA as blank.With the negative control group of the suckling mouse that does not infect EV71 virus.

Put to death mouse in 14 days after the administration, cut open the belly at ice chest and get intestinal tissue, the standby inspection of homogenate.

MAIN OUTCOME MEASURES:

(1) detection of intestinal tissue EV71-mRNA

After the injected in mice the 14th day, mouse is put to death in dislocation, take out intestinal tissue, cut the tissue block into about 100mg, put into homogenizer and fully grind, then according to the explanation of Trizol (GIBCOL company), use the total RNA of Trizol extracting hepatic tissue, the RNA that extracts is after DNA is removed in the DNase enzymic digestion, and reverse transcription is cDNA, then detects the restraining effect that siRNA expresses the EV71 virus mRNA with fluorescence quantitative PCR method.Concrete steps are identical with corresponding content among the embodiment 3, and the result as shown in Figure 3.

Infect after 6 days, not shown by the suckling mouse of siRNA administration and significantly become thin and hind leg paralysis symptom, all dead after 12-14 days.Experimental group appeal illness through the siRNA administration progressively is eased, and the experimental group animal is all normally survivals after 14 days.

As can be seen from Figure 3, siRNAEV71-T1 to EV71-T50 provided by the invention can suppress the expression of EV71 virogene in the suckling mouse intestinal tissue significantly.Especially, siRNA EV71-T19, siRNA EV71-T21, siRNA EV71-T34, siRNA EV71-T44, siRNA EV71-T45 can reach more than 85% the inhibiting rate of EV71 viral gene expression in the intestinal tissue.

Claims

1. a siRNA is characterized in that, this siRNA can suppress the expression of enterovirns type 71 gene, the nucleotide sequence of this siRNA shown in EV71-T7, wherein,

EV71-T7

Positive-sense strand: 5 '-CCAACAAUUGUGCUACAAUdTdT-3 '

Antisense strand: 5 '-AUUGUAGCACAAUUGUUGGdTdT-3 '.

2. siRNA according to claim 1, wherein, described nucleotide sequence has chemically modified, and described chemically modified is modified for the U of positive-sense strand and C are carried out 2`-oxygen methyl, the U in the antisense strand is carried out the 2`-fluoro modify.

3. a pharmaceutical composition that is used for preventing and/or treating the hand foot mouth disease that enterovirns type 71 causes is characterized in that, this pharmaceutical composition contains claim 1 or 2 described siRNA as activeconstituents.

4. pharmaceutical composition according to claim 3, wherein, this pharmaceutical composition also contains one or more in pharmaceutically acceptable thinner, pharmaceutically acceptable carrier and the chemotherapeutic.

5. according to claim 3 or 4 described pharmaceutical compositions, wherein, the formulation of this pharmaceutical composition is tablet, capsule, powder agent, injection liquid, emulsion, suppository, paste, lotion, aerosol or paster.

6. pharmaceutical composition according to claim 5, wherein, described pharmaceutically acceptable carrier is that the pH value is the phosphate buffered saline buffer of 5.5-8.5 for the phosphoric acid buffer of 4.0-9.0, tri methylol amino methane hydrochloride damping fluid, physiological saline or the pH that the pH value is 7.5-8.5.

7. pharmaceutical composition according to claim 6, wherein, described injection liquid also contains protective material and/or osmotic pressure regulator; Take the gross weight of described injection liquid as benchmark, described protectant content is the 0.01-30 % by weight, and described protective material is selected from one or more in inositol, sorbyl alcohol and the sucrose; It is 200-700 m osmole/kilogram that the content of described osmotic pressure regulator makes the osmotic pressure of described injection liquid, and described osmotic pressure regulator is sodium-chlor and/or Repone K.

8. claim 1 or 2 described siRNA are for the preparation of the application in the medicine that suppresses enterovirns type 71 genetic expression.

9. claim 1 or 2 described siRNA are for the preparation of the application in the medicine that treats and/or prevents the hand foot mouth disease that enterovirns type 71 causes.