WO2015143958A1 - 用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、dna分子、蛋白及应用 - Google Patents

用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、dna分子、蛋白及应用 Download PDF

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WO2015143958A1
WO2015143958A1 PCT/CN2015/072624 CN2015072624W WO2015143958A1 WO 2015143958 A1 WO2015143958 A1 WO 2015143958A1 CN 2015072624 W CN2015072624 W CN 2015072624W WO 2015143958 A1 WO2015143958 A1 WO 2015143958A1
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seq
sequence
primer
dna molecule
nucleic acid
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蒋澄宇
盛苗苗
钟颖
张奇伟
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中国医学科学院基础医学研究所
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a small nucleic acid molecule, DNA molecule, protein and application for preventing and/or treating Ebola viral hemorrhagic fever.
  • Viral hemorrhagic fever refers to a serious disease caused by several types of viruses, often accompanied by bleeding symptoms. According to the pathogen, it belongs to four families, namely, the genus of the genus Erythrophila (Lassa fever, Huning and Pumara), the Bunia virus family (Crimiya-Congo haemorrhagic fever, Rift Valley fever, Hantan hemorrhagic fever), The genus Actinomy (Ebola and Marburg) and the Flaviviridae (yellow fever, dengue hemorrhagic fever, Omsk hemorrhagic fever, Kisana forest disease).
  • Ebola hemorrhagic fever is Ebola virus, also known as Ebola virus disease. It is one of the most toxic viral diseases known to date, and the mortality rate is as high as 50-90%.
  • Ebola virus also known as Ebola virus disease.
  • SEBO Ebo-Zaire
  • SEBO Ebo-Sudan
  • REBO Ebola-Les EBO-R
  • EBO-CI EBO-Côte d'Irium
  • BEBO EBO-Bundibugyo Kenya
  • the Ebola virus can spread through contact and spread quickly. Once the patient is ill, he or she can die within 24 hours. The main symptoms can be high fever, headache, nausea, vomiting, diarrhea, massive bleeding inside and outside the body, body aches and so on. It is a Biosafety Level 4 (BSL-4) virus with a high biohazard.
  • BSL-4 Biosafety Level 4
  • the host of the Ebola virus has not been identified and was originally thought to be a rodent, but was quickly denied after the experiment.
  • Ebola is a single-stranded negative-strand RNA virus with a genome of about 19 kb and seven genes: NP, vp35, vp40, gp, vp30, vp24 and L, of which NP, VP30, VP35 and RNA
  • the dependent RNA polymerase forms the center of the virion, the ribonucleoprotein complex, which is responsible for transcription and replication.
  • VP40 and VP24 are responsible for the formation of the nucleocapsid, the budding and packaging of the virus, and the extent of the host.
  • GP surface glycoprotein may mediate the binding and fusion of Ebola virus and target cells, and plays a very important role in the pathogenesis of Ebola hemorrhagic fever. Transferring GP alone to humanized cells or several other non-humanized cells will cause the cells to round and fall off, and the cell surface adhesion factor will be down-regulated; transferring GP into human or pig blood vessels will increase blood vessels. Permeability, which causes blood vessels to bleed.
  • NPC1 inhibitor The only treatment today is the injection of NPC1 inhibitor, because Ebola virus needs to enter the nucleus through NPC1 for self-replication.
  • NPC1 protein transports cholesterol between cells, but the inhibitors block the cholesterol transport route and cause Niemann's disease. Therefore, more and more research ideas are needed to find drugs for the prevention and treatment of Ebola hemorrhagic fever.
  • the present invention provides a small nucleic acid molecule, a DNA molecule, a protein for preventing and/or treating hemorrhagic fever, and their use in drug screening, and provides a medicament for preventing and/or treating hemorrhagic fever.
  • the present invention is based on the common symptoms of oozing and bleeding caused by various vascular structural and functional abnormalities caused by various viral hemorrhagic fevers mentioned in the background art section, and is obtained through extensive research. the result of. Therefore, various substances which can be inhibited or alleviated by the Ebola virus as the main research object mentioned below are also applicable to the above other viral hemorrhagic fever.
  • vascular endothelial cells are one of the main targets of Ebola virus, vascular endothelial cells play an important role in maintaining vascular permeability and humoral balance. After Ebola virus infects vascular endothelial cells, it usually causes vascular structures. And functional abnormalities, which in turn lead to diffuse intravascular coagulation (DIC) of Ebola hemorrhagic fever.
  • human umbilical vein (HUV) is a common source of vascular endothelial cells, it is rich in source, convenient in obtaining materials, superior in vascular conditions, and less in other cells, and can obtain vascular endothelial cells with low cellular immunogenicity. The experimental conditions and the results obtained can be made more in line with human conditions. Therefore, in the vascular endothelial cell experiment of the present invention, human umbilical vein endothelial cells are used as a cell model for research.
  • the present invention provides the following technical solutions:
  • a small nucleic acid molecule for preventing and/or treating Ebola viral hemorrhagic fever which is an inhibitor of microRNA differentially expressed by Ebola virus-infected cells And/or an inhibitor of the microRNA precursor.
  • the above small nucleic acid molecule is a microRNA having the sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. Precursor of the precursor.
  • sequence of the above small nucleic acid molecule has 80% or more with SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. Homology, preferably having more than 95% homology, more preferably, the sequence of the above small nucleic acid molecule is SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. ID NO. 11 or SEQ ID NO.
  • a small nucleic acid molecule as described above for use in the prevention and/or treatment of Omsk hemorrhagic fever, Kisana forest disease, Crimean hemorrhagic fever, Xinjiang hemorrhagic fever, dengue Application in hemorrhagic fever, chikungunya fever, yellow fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, Lassa fever, epidemic hemorrhagic fever or Marburg disease.
  • a medicament for preventing and/or treating Ebola viral hemorrhagic fever comprising a therapeutically effective amount of the above small nucleic acid molecule is provided.
  • the above medicament further comprises one or more effective substances for preventing and/or treating Ebola viral hemorrhagic fever, preferably, the above-mentioned effective substance for preventing and/or treating Ebola viral hemorrhagic fever From antiviral drugs, immunostimulating drugs and corticosteroids.
  • an application of the above small nucleic acid molecule for inhibiting cell growth shedding caused by Ebola viral hemorrhagic fever infection in vitro wherein the small nucleic acid molecule is transferred before or after the cell is infected with Ebola virus. Dye into the cells.
  • the use of the above small nucleic acid molecule for screening a drug for preventing and/or treating Ebola viral hemorrhagic fever in vitro wherein the small nucleic acid molecule is nucleic acid with the drug to be tested Source alignment.
  • a DNA molecule wherein the expression level of the above DNA molecule in an Ebola virus-infected cell is upregulated by one or more of the above small nucleic acid molecules.
  • sequence of the above DNA molecule is SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32 has more than 80% homology, preferably has more than 95% homology, more preferably
  • sequence of the above DNA molecule is SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.
  • SEQ ID NO. 18 SEQ ID NO. ID NO. 20
  • SEQ ID NO. 21 SEQ ID NO. 22
  • SEQ ID NO. 23 SEQ ID NO. 24
  • SEQ ID NO. 25 SEQ ID NO. 26
  • SEQ ID NO. 27 SEQ ID NO.28
  • SEQ ID NO. 29 SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO.
  • DNA molecule is SEQ ID NO. 14, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. Or SEQ ID NO. 32, the preferred sequence is SEQ ID NO. 14, SEQ ID NO. 23 or SEQ ID NO.
  • a protein encoded by the above DNA molecule the sequence of the above protein being SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45.
  • the sequence of the above protein is SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51 or SEQ ID NO.
  • sequence of the above protein is SEQ ID NO. 34, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. SEQ ID NO. 52, preferred sequence is SEQ ID NO. 34, SEQ ID NO. 43 or SEQ ID NO.
  • kits for detecting Ebola viral hemorrhagic fever comprising a primer capable of specifically amplifying a microRNA inhibited by the small nucleic acid molecule.
  • the reverse transcription primer sequence is SEQ ID NO. 140: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCTGCT
  • the primer used for real-time quantitative PCR FP sequence is SEQ ID NO. 146: CCGCGCAATGGATTTTT, RP The sequence is SEQ ID NO. 147: GTGCACGCTCCGAGGT;
  • the reverse transcription primer sequence is SEQ ID NO. 141: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCCAAC
  • the primer used for real-time quantitative PCR FP sequence is SEQ ID NO. 148: CCGCGCTAGGTAGTTTCCT
  • RP sequence is SEQ ID NO.149: GTGCACGCTCCGAGGT;
  • the reverse transcription primer sequence is SEQ ID NO. 142: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTCGCCC
  • the primer used for real-time quantitative PCR FP sequence is SEQ ID NO. 150: TCGCGCAAAAGCTGG
  • RP sequence is SEQ ID NO.151: GTGCACGCTCCGAGGT;
  • the reverse transcription primer sequence is SEQ ID NO. 143: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACTAGA
  • the primer used for real-time quantitative PCR FP sequence is SEQ ID NO. 152: TCGCGCTCGAGGAGC
  • RP sequence is SEQ ID NO.153: GTGCACGCTCCGAGGT;
  • the reverse transcription primer sequence is SEQ ID NO. 144: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACAACC.
  • the primer used in real-time quantitative PCR is SEQ ID NO. 154: TGCCGCTGGCAGTGT, and the RP sequence is SEQ ID NO. 155: GTGCACGCTCCGAGGT;
  • the reverse transcription primer sequence is SEQ ID NO. 145: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTGCTGT
  • the primer used for real-time quantitative PCR FP sequence is SEQ ID NO. 156: TCGCGCTGCGGG
  • RP sequence is SEQ ID NO. 157: GTGCACGCTCCGAGGT.
  • kits for detecting Ebola viral hemorrhagic fever comprising a primer which specifically amplifies the above DNA molecule.
  • the FP sequence of the primer is SEQ ID NO. 100: AAGCAGGATCTTCTAAGGTT
  • the RP sequence is SEQ ID NO. 101: AGTGTTGTTCTTGGTCTCTC;
  • the FP sequence of the primer is SEQ ID NO. 102: GTCACTTGCTTCCTTACTTAG
  • the RP sequence is SEQ ID NO. 103: TGAGCCATAACCACAGAG;
  • the sequence of the DNA molecule is SEQ ID NO. 15
  • the FP sequence of the primer is SEQ ID NO. :ACCAATCAGTGCTCAGTAT
  • the RP sequence is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 106: TGGTCATCATCTGCTTGTG, and the RP sequence is SEQ ID NO. 107: TTCCTATTCGGTCACTCTCT
  • the FP sequence of the primer is SEQ ID NO. 108: GGTAGAACTGAACAACGATAG
  • the RP sequence is SEQ ID NO. 109: CAGCAATGGTATAGCAACTT; when the sequence of the DNA molecule is SEQ ID NO. , the FP sequence of the primer is SEQ ID NO.
  • the RP sequence is SEQ ID NO. 111: CAGCAATTCCATCTTCATCACT; when the sequence of the DNA molecule is SEQ ID NO. FP is a sequence of primer SEQ ID NO.112: CTAATTCTCCAGCCTCATTG, RP sequence of SEQ ID NO.113: ATGGTAATCGTCCGTTCA; when the DNA When the SEQ ID NO.
  • the FP sequence is SEQ ID NO. 116: GATTCTCTTCCACAGACTATATG, the RP sequence is SEQ ID NO. 117: CCTTCCTCATCCAGTTCATG; when the sequence of the DNA molecule is SEQ ID NO. 22, the FP sequence of the primer is SEQ ID NO.
  • telomere sequence SEQ ID NO. 118 TTCATCTCCGCTTCTTGTG, RP sequence SEQ ID NO. 119: TGTTCTTCAGTGAGTTCCTT; when the sequence of the DNA molecule is SEQ ID NO. 23, the FP sequence of the primer is SEQ ID NO. 120: GCCTGCTGCTTAATCTTG, and the RP sequence is SEQ ID NO. 121: CATCATCCGCCTTGAATG; When the sequence is SEQ ID NO. 24, the FP sequence of the primer is SEQ ID NO. 122: AGGCAAGATAAGCAAGGAGAAG, the RP sequence is SEQ ID NO. 123: AGGTCTATTCTGTGGATGTTCT; when the sequence of the DNA molecule is SEQ ID NO. 25, the FP sequence of the primer SEQ ID NO.
  • RP sequence is SEQ ID NO. 125: GGAAGTCACCTCAGAGTCCAG; when the sequence of the DNA molecule is SEQ ID NO. 26, the FP sequence of the primer Of SEQ ID NO.126:
  • RP sequence is SEQ ID NO. 127: GCTTGGAGATGGCATATAAGA; when the sequence of the DNA molecule is SEQ ID NO. 27, the FP sequence of the primer is SEQ ID NO. 128: CAGAACATACCACGACAAG, and the RP sequence is SEQ ID NO. 129: CTGGAAGGAGTAGAGGATGT; When the sequence of the DNA molecule is SEQ ID NO. 28, the FP sequence of the primer is SEQ ID NO. 130: GAGCCTGAGACTTGTGTATC, the RP sequence is SEQ ID NO. 131: CGTATTCTGGGAAGTGTTG; when the sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 132: GGCGTGGTATCTGTCTGT, the RP sequence is SEQ ID NO. 133: CTGTTGTGTCATCCTGTTC; when the sequence of the DNA molecule is SEQ ID NO. 30, the FP sequence of the primer is SEQ ID NO. 134: TATCTAGAGGGCGGTTCTGG, The RP sequence is SEQ ID NO. 135: TTTCTCTGAGGTGGCATAC; when the sequence of the DNA molecule is SEQ ID NO. 31, the FP sequence of the primer is SEQ ID NO. 136: GGTTGGTGTTGATTCAACC, and the RP sequence is SEQ ID NO.
  • the small nucleic acid molecule of the present invention By using the technical solution of the present invention, by using the small nucleic acid molecule of the present invention, the expression of microRNAs and/or microRNA precursors differentially expressed by Ebola virus-infected cells can be inhibited, and the expression of target genes of microRNAs can be up-regulated. , thereby increasing the resistance of cells to Ebola viral hemorrhagic fever infection.
  • the small nucleic acid molecules, DNA molecules, proteins and their applications for preventing and/or treating Ebola viral hemorrhagic fever provided by the present invention not only provide prevention and/or treatment of Ebola virus hemorrhagic fever The drug molecule, and provides a new research direction for the development of related viral hemorrhagic fever drugs.
  • Figure 1 shows the cell survival rate of human umbilical vein endothelial cells infected with Ad-GP recombinant adenovirus supernatant and Ad- ⁇ E1 recombinant adenovirus supernatant (control virus supernatant) at different time points;
  • FIG. 2 shows the total RNA electrophoresis patterns of Ad-GP recombinant adenovirus supernatant and Ad- ⁇ E1 recombinant adenovirus supernatant (control virus supernatant) infected with human umbilical vein endothelial cells at different time points;
  • Figure 3 shows Ad-GP recombinant adenovirus supernatant and Ad- ⁇ E1 recombinant adenovirus supernatant (control virus supernatant) infected human umbilical vein endothelial cells, transfected with microRNA inhibitors 24 h before infection, and infected
  • the MTS assay was performed to detect the survival rate of cells after 48 hours;
  • Figure 4 shows that Ad-GP recombinant adenovirus supernatant and Ad- ⁇ E1 recombinant adenovirus supernatant (control virus supernatant) infected human umbilical vein endothelial cells, which were transfected with microRNA inhibitors 3 h after infection and infected MTS experiment after 48h Detecting the survival rate of the cells;
  • Figures 5a, 5b, and 5c show that the quantitative PCR method can verify that the inhibitor of microRNA can up-regulate the expression of the target gene
  • Figure 6 and Figures 7a and 7b show that Ad-GP recombinant adenovirus supernatant and Ad- ⁇ E1 recombinant adenovirus supernatant (control virus supernatant) infected human umbilical vein endothelial cells, and transfected with microRNAs 24 h before infection, respectively. And detecting the expression of the microRNA target gene protein 24 h after infection; wherein, Figure 6 is a protein hybridization blot; Figure 7a, 7b is a statistical analysis of Figure 6;
  • Figure 8a and Figure 8b show the effect of analogs of microRNAs on mRNA expression of Renilla fluorescein-labeled wild-type target genes and mutant target genes;
  • Figure 9 shows that the use of double-stranded oligonucleotide interfering RNA to reduce the expression of microRNA target genes reduces the survival rate of Ad-GP infected HUVEC cells;
  • Figure 10 shows that overexpression of microRNA target genes by overexpression significantly reduces the rate of cell shedding and floating caused by GP overexpression
  • Figure 11 shows that overexpression of a plurality of different microRNA target genes at the same time by overexpression significantly reduces the rate of cell shedding and floating caused by GP overexpression.
  • the present invention discloses small nucleic acid molecules, DNA molecules, proteins and applications for preventing and/or treating Ebola viral hemorrhagic fever, and those skilled in the art can learn from the contents of the present invention and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • nucleic acid molecules, DNA molecules, proteins, and materials and reagents used in the present invention for preventing and/or treating Ebola viral hemorrhagic fever are commercially available.
  • a small nucleic acid molecule for preventing and/or treating Ebola viral hemorrhagic fever, which is differentially expressed by Ebola virus-infected cells.
  • the small nucleic acid molecule provided by the present invention is a substance completely different from the mechanism of action of the prior art for treating and/or preventing Ebola viral hemorrhagic fever. When the cells are infected with Ebola viral hemorrhagic fever, the expression level of microRNAs in the cells is significantly changed.
  • the small nucleic acid molecules provided by the present invention can directly inhibit the microRNAs expressed in large quantities by complementing the above microRNA sequences.
  • the inhibitor of the microRNA of the present invention By using the inhibitor of the microRNA of the present invention and the inhibitor of the precursor thereof, the expression of the microRNA in the cell infected with Ebola hemorrhagic fever can be effectively suppressed, and the phenomenon of rounding and falling off of the cell is significantly weakened.
  • the cell survival rate is significantly improved, so that Ebola can be effectively treated and/or prevented Viral hemorrhagic fever.
  • microRNA dimer having a double-stranded structure
  • an inhibitor that affects the formation of the precursor of the mature microRNA capable of actually exerting the function of suppressing the target gene is also applicable to the above-described small nucleic acid molecule of the present invention.
  • the above small nucleic acid molecule provided by the present invention refers to any inhibitor of microRNA which has a markedly increased expression amount after the cell is infected with Ebola hemorrhagic fever.
  • the Ebola envelope glycoprotein GP can significantly up-regulate the expression of human umbilical vein endothelial cells by at least 2-fold.
  • the sequence is hsa-miR1246 of SEQ ID NO. 1, and hsa-miR196b of SEQ ID NO. 5p, hsa-miR320a of SEQ ID NO. 3, hsa-miR151a-5p of SEQ ID NO. 4, hsa-miR34a-5p of SEQ ID NO.
  • the above microRNA are all the above-mentioned small nucleic acid molecules of the present invention.
  • the above small nucleic acid molecules can effectively inhibit the expression of corresponding microRNAs in cells infected with Ebola virus, inhibit the phenomenon of rounding off of cells, and improve the survival rate of cells.
  • differential expression is Refers to the expression level up or down by at least 2 times.
  • hsa-miR-152 of SEQ ID NO. 53 hsa-miR-22-3p of SEQ ID NO. 54
  • hsa-miR-424-5p of SEQ ID NO. 55 hsa of SEQ ID NO. -miR-374a-5p
  • the differential expression of these microRNAs is also related to Ebola virus-infected cells, and the expression level is up-regulated by inhibitors of related microRNAs, by inhibiting related microRNAs
  • the expression confirms its relationship with the Ebola virus-infected phenotype; the expression of the associated microRNA is down-regulated, and the relationship between it and Ebola virus infection is confirmed by overexpression of microRNA. Therefore, the above inhibitors or analogs which differentially express microRNAs are suitable for use in the present invention.
  • the small nucleic acid molecule is SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO.
  • the inhibitor of microRNA of 6 is more effective in inhibiting the expression of the corresponding microRNA in the Ebola virus-infected cells, and the cell rounding phenomenon is significantly improved, thereby further improving the cell survival rate.
  • the inhibitor of the microRNA can inhibit the microRNA to complement the sequence of the target gene by complementing the microRNA sequence, thereby inhibiting the inhibition of the target gene expression by the microRNA, thereby increasing the expression of the target gene, thereby improving the expression of the target gene.
  • the survival rate of infected cells An inhibitor of microRNA can be used in the present invention as long as the partial sequence thereof is complementary to the sequence of the microRNA to inhibit the expression of the microRNA.
  • the sequence of the small nucleic acid molecule is SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 has more than 80% homology, preferably has more than 95% homology, more preferably, the sequence of the small nucleic acid molecule is SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO.
  • SEQ ID NO. 7 is an inhibitor of hsa-miR1246, SEQ ID NO. 8 is an inhibitor of hsa-miR196b-5p, SEQ ID NO.
  • SEQ ID NO. 9 is an inhibitor of hsa-miR320a
  • SEQ ID NO. 10 is hsa An inhibitor of -miR151a-5p
  • SEQ ID NO. 11 is an inhibitor of hsa-miR34a-5p
  • SEQ ID NO. 12 is an inhibitor of hsa-miR744a.
  • the sequence of the above small nucleic acid molecule has 80% or more homology to SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. Sexually, it can inhibit the expression of microRNAs that are partially complementary to its sequence, thereby inhibiting the phenotype of rounded cells and improving Ebola virus infection. Cell survival rate.
  • the sequence of the above small nucleic acid molecule has 95% or more homology to SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO.
  • the effect of inhibiting the expression of microRNAs complementary to its sequence is stronger, so that the expression of microRNAs is decreased more, and the effect of improving the survival rate of Ebola virus-infected cells is more obvious.
  • the sequence of the small nucleic acid molecule is SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12, the sequence thereof is related to the microRNA. The sequence is completely complementary, and the effect of inhibiting the expression of microRNA is stronger, and the effect of increasing the survival rate of cells infected with Ebola virus is also more obvious.
  • the small nucleic acid molecule of the present invention can effectively inhibit the rounding and detachment of vascular endothelial cells, thereby causing less damage, thereby protecting the blood vessels and increasing the cell survival rate. Therefore, the above small nucleic acid molecules provided by the present invention are provided.
  • Omsk hemorrhagic fever Kisana forest disease, Crimean hemorrhagic fever, Xinjiang hemorrhagic fever, dengue hemorrhagic fever, Chikungunya fever, yellow fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, Lassa fever, epidemic hemorrhagic fever, Marburg disease or Ebola viral hemorrhagic fever.
  • Another aspect of the present invention is to provide a medicament for preventing and/or treating Ebola viral hemorrhagic fever comprising a therapeutically effective amount of the above small nucleic acid molecule.
  • a medicament comprising a therapeutically effective amount of the above small nucleic acid molecule is effective for inhibiting the expression of microRNA in cells infected with Ebola virus, thereby increasing the survival rate of cells infected with Ebola virus.
  • the medicament further comprises one or more effective substances for preventing and/or treating Ebola viral hemorrhagic fever.
  • a medicament comprising one or more effective substances for preventing and/or treating Ebola viral hemorrhagic fever is effective for preventing and/or treating viral hemorrhagic fever.
  • the above-mentioned effective substance for preventing and/or treating Ebola viral hemorrhagic fever is selected from the group consisting of an antiviral drug, an immunostimulating drug, and a corticosteroid.
  • the invention selects the above effective substance to significantly improve the damage and the degree of shedding of the cells infected with Ebola virus, and at the same time, can significantly improve the cell survival rate.
  • a further aspect of the present invention is to provide the use of the above small nucleic acid molecule to inhibit Ebola virus infection in vitro to cause rounding and detachment of cells, which is to transfect small nucleic acid molecules before or after the cells are infected with Ebola virus.
  • the endogenous expression of GP protein leads to the most prominent phenotype of cell growth and rounding off. Therefore, GP protein is usually used to simulate the growth of Ebola virus in vitro. Shedding experiments.
  • the above small nucleic acid molecule inhibits Ebola virus glycoprotein GP-infected cells in vitro to cause rounding of cell growth. Transfecting the above small nucleic acid molecule into the cell before the cell is infected with the glycoprotein GP of Ebola virus can inhibit the expression of microRNA in time when the expression of microRNA in the cell is up-regulated by the infection, thereby releasing the target gene of the microRNA.
  • the target gene can be expressed in a large amount, thereby reducing the probability of cell detachment caused by Ebola virus glycoprotein GP, and improving the ability of the cell to resist cytotoxicity of Ebola virus glycoprotein GP, thereby enabling Promptly prevent Ebola virus infection.
  • transfection of small nucleic acid molecules into cells after infection with Ebola virus glycoprotein GP inhibits further expression of microRNAs in cells and further inhibition of target gene expression by microRNAs. Furthermore, the cell's resistance to Ebola virus cytotoxicity after infection is increased, and the degree of cell damage is reduced, thereby increasing cell survival rate.
  • the present invention further provides the use of the above small nucleic acid molecule for screening a drug for preventing and/or treating Ebola viral hemorrhagic fever in vitro, the application of which is to homologize a small nucleic acid molecule to a drug to be tested. Sexual comparison. Due to this issue
  • the above small nucleic acid molecule provided by the invention can well inhibit the phenotype of the rounded off cell caused by the Ebola virus envelope glycoprotein GP, and improve the anti-infective ability of the infected cell, and the mechanism of action is that the small nucleic acid molecule can The sequence of the microRNA which is up-regulated by the Ebola virus envelope glycoprotein GP is partially complementary, thereby inhibiting the expression of microRNA in the infected cell.
  • nucleic acid homology alignment between the small nucleic acid molecule and the drug to be tested, according to the principle of higher homology, it can be screened in vitro for prevention and/or treatment of Ebola hemorrhagic fever. Drug.
  • Yet another aspect of the invention provides a DNA molecule whose expression level in Ebola virus infected cells is upregulated by one or more of the above small nucleic acid molecules.
  • a DNA molecule that is up-regulated by one or more of the above small nucleic acid molecules is a target gene that the corresponding microRNA can inhibit.
  • the up-regulated expression of the target genes of these microRNAs can effectively reduce the degree of cell damage caused by Ebola virus, increase the resistance of cells to cytotoxicity caused by Ebola virus, and thereby improve cell survival rate.
  • DNA molecules provided by the present invention, there are many types which can be up-regulated by one or several small nucleic acid molecules, as long as they can be up-regulated by one or several small nucleic acid molecules mentioned above, and can reduce Ebo.
  • DNA molecules which cause damage to cells caused by virus pull DNA molecules which increase the resistance of cells to Ebola virus infection, and thereby increase cell viability are suitable for use in the present invention.
  • the sequence of the above DNA molecule is SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. .18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO.
  • SEQ ID NO. 24 SEQ ID NO. 25, SEQ ID NO. SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32 has 80% or more homology, preferably 95% or more Homology, more preferably, the sequence of the small nucleic acid molecule is SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO.
  • the DNA molecule sequences corresponding to SEQ ID NO. 13 to SEQ ID NO. 32 are: HLTF, DAG1, LAMA4, UBE2H, MCTP1, KIAA1249, SDCBP, CPD, FRMD6, SKIL, TFPI, CFLAR, DPP3, RCAN1 TRIM24, RPAIN, IP6K1, ESM1, SLC9A6, ZCCHC6.
  • the sequence of the above DNA molecule is SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ. ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO .26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32 has greater than 80% homology.
  • DNA molecules with higher homology are more likely to have the same or similar functions, and the possibility of belonging to functionally conserved homologous genes is greater. Therefore, a DNA molecule having 80% or more homology with the DNA molecule of the above sequence can have the same or similar phenotype as the DNA molecule having the above sequence, inhibiting the rounding off of the cell caused by the Ebola virus, and increasing the cell. The possibility of anti-Ebola virus infection is greater.
  • DNA molecule provided by the present invention has the sequence of SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27.
  • the DNA molecule is capable of The sequence of DNA molecules with the same or similar phenotype that inhibits the rounding of the cells caused by the Ebola virus envelope glycoprotein GP is more likely to increase the ability of the cells to resist Ebola virus infection.
  • DNA molecule provided by the present invention has the sequence of SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23. SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. In the case of SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23. SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. In the case of SEQ ID NO.
  • the up-regulated expression can more effectively inhibit the phenotype of the rounded off of the infected cell, thereby more effectively enhancing the cell anti-Ebo The ability to pull a virus.
  • the expression of the above DNA molecules provided by the present invention can be significantly up-regulated by the above small nucleic acid molecules by at least 2-fold, both of which have the ability to enhance the anti-cytotoxic ability of Ebola virus-infected cells.
  • the DNA molecule is SEQ ID NO. 14, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO .26, SEQ ID NO. 27 or SEQ ID NO. 32, preferably the sequence is SEQ ID NO. 14, SEQ ID NO. 23 or SEQ ID NO.
  • RNA is clearly up-regulated; and it is also significantly up-regulated at the level of protein expression.
  • the DNA molecule having the sequence of SEQ ID NO. 14, SEQ ID NO. 23 or SEQ ID NO. 24 is more up-regulated at the level of messenger RNA expression and protein expression, and is damaged by the Ebola-infected cells. Has a more significant improvement.
  • the above DNA molecule for screening a drug for preventing and/or treating Ebola viral hemorrhagic fever in vitro, which is capable of using the above DNA molecule
  • drugs capable of up-regulating the expression of the above DNA molecules are suitable for use in the present invention.
  • the invention also provides a protein encoded by the above DNA molecule, the sequence of the protein is SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO 46. SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51 or SEQ ID NO.
  • the sequence of the protein is SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38. SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51 or SEQ ID NO.
  • the protein encoded by the above DNA molecule provided by the present invention when the sequence thereof is SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38. SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO.
  • SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51 or SEQ ID NO. 52 has more than 80% homology, the protein having the above sequence The homology is relatively high and the possibility of having the same biological function is high.
  • the sequence of the protein encoded by the above DNA molecule has 95% or more homology with the above sequence, it is more likely to have the same biological function as the protein having the above sequence.
  • the expression of the protein having the above sequence is upregulated by the above small nucleic acid molecule, and the up-regulation of the expression can more significantly reduce the degree of damage of the Ebola virus-infected cells and increase the survival rate of the cells.
  • the above sequences are encoded by the DNA molecules of SEQ ID NO. 13 to SEQ ID NO. 32, respectively, from the proteins of SEQ ID NO. 33 to SEQ ID NO.
  • the molecular sequence of the above protein is SEQ ID NO. 34, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. .46, SEQ ID NO. 47 or SEQ ID NO.
  • the molecular sequence is the DAG1 protein of SEQ ID NO. 34, the KIAA1249 protein of SEQ ID NO. 38, the CPD protein of SEQ ID NO. 40, the TFPI protein of SEQ ID NO. 43, the CFLAR protein of SEQ ID NO. 44, SEQ ID. NO.46
  • Overexpression of the RCAN1 protein, the TRIM24 protein of SEQ ID NO. 47, or the ZCCHC6 protein of SEQ ID NO. 52 has a more pronounced effect on the ability of cells to be resistant to Ebola virus infection.
  • a protein encoded by the above DNA molecule for screening a drug for preventing and/or treating Ebola viral hemorrhagic fever in vitro, the application being capable of The protein is a drug screening for preventing and/or treating Ebola viral hemorrhagic fever in vitro, and the drug capable of up-regulating the expression of the above protein can also be used for treating or preventing Ebola viral hemorrhagic fever.
  • the present invention further provides a kit for detecting Ebola viral hemorrhagic fever, the kit comprising a primer capable of specifically amplifying a microRNA inhibited by the small nucleic acid molecule.
  • the primer sequences for specifically amplifying the above-described microRNA molecules are also different depending on the microRNA to be detected.
  • the reverse transcription primer sequence is SEQ ID NO. 140: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCTGCT
  • the primer used for real-time quantitative PCR FP sequence is SEQ ID NO. 146: CCGCGCAATGGATTTTT
  • RP sequence is SEQ ID NO.147: GTGCACGCTCCGAGGT
  • the reverse transcription primer sequence is SEQ ID NO.
  • FP sequence is SEQ ID NO. :CCGCGCTAGGTAGTTTCCT
  • RP sequence is SEQ ID NO. 149: GGTCACCCCCGAGGT
  • the reverse transcription primer sequence is SEQ ID NO. 142: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTCGCCC
  • primer used for real-time quantitative PCR FP
  • the sequence is SEQ ID NO. 150: TCGCGCAAAAGCTGG
  • the RP sequence is SEQ ID NO.
  • the reverse transcription primer sequence is SEQ ID NO. 143: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACTAGA, real-time fluorescence quantification Used by PCR Primer: FP sequence is SEQ ID NO. 152: TCGCGCTCGAGGAGC, RP sequence is SEQ ID NO. 153: GTGCACGCTCCGAGGT; when the sequence of microRNA is SEQ ID NO. 5, the reverse transcription primer sequence is SEQ ID NO. 144: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACAACC.
  • FP sequence is SEQ ID NO. 154: TGCCGCTGGCAGTGT
  • RP sequence is SEQ ID NO. 155: GTGCACGCTCCGAGGT
  • the reverse transcription primer sequence is SEQ ID NO. 145: GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTGCTGT
  • primer used for fluorescent real-time quantitative PCR FP sequence is SEQ ID NO. 156: TCGCGCTGCGGG
  • RP sequence is SEQ ID NO. 157: GTGCACGCTCCGAGGT.
  • the present invention further provides a kit for detecting Ebola viral hemorrhagic fever, the kit comprising a primer which specifically amplifies the above DNA molecule.
  • the DNA molecules which can be significantly up-regulated by the above small nucleic acid molecules in Ebola-infected cells can be efficiently amplified by using the primers provided in the kit of the present invention.
  • the primer sequences for specifically amplifying the above DNA molecules are also different depending on the sequence of the DNA molecule to be detected.
  • sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 102: GTCACTTGCTTCCTTACTTAG
  • the RP sequence is SEQ ID NO. 103: TGAGCCATAACCACAGAG
  • the FP sequence of the primer is SEQ ID NO. 104: ACCAATCAGTGCTCAGTAT
  • the RP sequence is SEQ ID NO. 105: TCAACCCCACATCTCTATC; when the sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 106: TGGTCATCATCTGCTTGTG, and the RP sequence is SEQ ID NO. 107: TTCCTATTCGGTCACTCTCT;
  • SEQ ID NO. 110 CGACGAACAGTAGACAGTATT
  • RP sequence is SEQ ID NO. 111: CAGCAATTCCATCTTCATCACT
  • the FP sequence of the primer is SEQ ID NO. 112: CTAATTCTCCAGCCTCATTG
  • the RP sequence is SEQ ID NO. 113: ATGGTAATCGTCCGTTCA; Where the sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 116: GATTCTCTTCCACAGACTATATG
  • the RP sequence is SEQ ID NO. 117: CCTTCCTCATCCAGTTCATG
  • the FP sequence of the primer is SEQ ID NO. 118: TTCATCTCCGCTTCTTGTG
  • the RP sequence is SEQ ID NO. 119: TGTTCTTCAGTGAGTTCCTT; when the sequence of the DNA molecule is SEQ ID NO. 23, the FP sequence of the primer is SEQ ID NO. 120: GCCTGCTGCTTAATCTTG, and the RP sequence is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 122: AGGCAAGATAAGCAAGGAGAAG, and the RP sequence is SEQ ID NO. 123: AGGTC TATTCTGTGGATGTTCT; when the sequence of the DNA molecule is SEQ ID NO. 25, the FP sequence of the primer is SEQ ID NO. 124: GAACCTCAGTGCCTACAACAC, the RP sequence is SEQ ID NO. 125: GGAAGTCACCTCAGAGTCCAG; when the sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO.
  • the RP sequence is SEQ ID NO. 127: GCTTGGAGATGGCATATAAGA; when the sequence of the DNA molecule is SEQ ID NO. 27, the FP sequence of the primer is SEQ ID NO. ⁇ / RTI> SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 132: GGCGTGGTATCTGTCTGT
  • the RP sequence is SEQ ID NO. 133: CTGTTGTGTCATCCTGTTC; when the sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO.
  • the RP sequence is SEQ ID NO. 135: TTTCTCTGAGGTGGCATAC; when the sequence of the DNA molecule is SEQ ID NO.
  • the FP sequence of the primer is SEQ ID NO. 136: GGTTGGTGTTGATTCAGAC, the RP sequence is SEQ ID NO. 137: GCAGAGGCTTCAGATAGT; when the sequence of the DNA molecule is SEQ ID NO. 32, the FP sequence of the primer is SEQ ID NO. 138: ATTAGCAAGAGTCCTCAGA
  • the RP sequence is SEQ ID NO. 139: ATGGCGGAATAAGCAGATA.
  • the small nucleic acid molecule, the DNA molecule, and the protein encoded by the DNA molecule provided by the present invention are related to the pathogenesis of hemorrhagic fever, various methods for diagnosing and/or treating viral hemorrhagic fever can be developed around the above molecule.
  • Human umbilical vein endothelial cells (HUVEC; Science II); 293A cells;
  • ECM Endothelial Cell Medium
  • FBS fetal bovine serum
  • ECGS endothelial cell growth supplement
  • P/S Science II Cat. No. 0503
  • CMV cytomegalovirus
  • Adenovirus shuttle vector pAdTrack-CMV adenoviral skeleton vector pAdEasy-1;
  • Ad-GP pAdTrack-CMV-GP/pAdEasy-1 recombinant plasmid
  • Ad- ⁇ E1 CMV/pAdEasy-1 recombinant plasmid
  • the adenoviral skeleton plasmid is an adenovirus replication-deficient plasmid, ie, the E1 gene region is deleted, abbreviated as ⁇ E1)
  • HUVEC was resuspended to a single cell solution with ECM, and seeded into a 12-well plate at 105 cells/mL per well at a volume of 1 ml per well; after 24 h, equal amounts of Ad-GP and Ad- ⁇ E1 recombinant glands were added.
  • Virus, multiplicity of infection (MOI) is 500;
  • Example 2 Differentially expressed microRNAs in HUVECs infected with Ad-GP and Ad- ⁇ E1 recombinant adenovirus
  • the HUVEC was resuspended into a single cell solution by ECM, and divided into an experimental group and a control group, and seeded into two 10 cm plates, each cell 10 5 /ml cells, each plate volume 10 ml;
  • the cells were lysed with 1 ml Trizol (invitrogen) after infection for 3h, 6h, 12h, 24h, 36h, 48h, respectively, and total RNA was extracted (for details, see Trizol Instructions for Use).
  • Example 3 Using MTS assay to detect the preventive and/or therapeutic effect of candidate microRNAs on cell shedding
  • the inhibitor of the candidate microRNA is a single-stranded RNA in which all bases synthesized by the methylation are methylated.
  • the inhibitor of the microRNA used in this experiment is: the inhibitor sequence of hsa-miR1246 is SEQ ID NO. 7; hsa The inhibitor sequence of -miR196b-5p is SEQ ID NO. 8; the inhibitor sequence of hsa-miR320a is SEQ ID NO. 9; the inhibitor sequence of hsa-miR151a-5p is SEQ ID NO. 10; hsa-miR34a-5p
  • Transfection reagent lipofect lipofectamine RNAiMAX (Invitrogen);
  • Inoculate cells HUVEC was resuspended into single cell solution with ECM, seeded into 96-well plates, 10 5 cells/ml cells per well, 100 ⁇ l per well; transfected cells were cultured at 37 ° C, 5% CO 2 incubator for 24 h.
  • HUVEC was resuspended into a single cell solution with ECM, seeded into 96-well plates, 10 5 cells/ml per well, 100 ⁇ l per well; 37 ° C, 5% CO 2 incubator cultured cells for 24 h, then added equal amount Ad-GP and Ad- ⁇ E1; transfected with inhibitors of candidate microRNAs after 3 hours of infection, another set of transfection control inhibitors, the final concentration of 100nM; transfection 48h, MTS assay to detect cell viability.
  • phosphate buffer solution PBS, 1 g of water, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 and 0.24 g of KH 2 PO 4 , pH 7.4
  • PBS phosphate buffer solution
  • the absorbance value background value.
  • Fig. 3 The results of the experiment are shown in Fig. 3 and Fig. 4.
  • the results in Fig. 3 are the results of the four experiments after the four experiments were repeated using the inhibitors of the microRNAs listed in Table 1.
  • the control sequence number is SEQ ID NO. 1, SEQ ID NO. 2, compared to the control microRNA inhibitor of SEQ ID NO. 66, which is unable to inhibit microRNA function.
  • Inhibitors of the microRNAs of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 can prevent cell detachment to varying degrees and increase cell survival rate.
  • the sequence numbers are SEQ ID NO. 1, SEQ ID NO.
  • the inhibitors can inhibit cell detachment to varying degrees and increase cell survival rate. Therefore, the inhibitor of the above-mentioned microRNA provided by the present invention can suppress the degree of exfoliation of Ebola virus-infected cells and significantly improve the cell survival rate.
  • Example 4 Determination of corresponding target genes of microRNAs
  • the target gene of the microRNA found in Example 2 was predicted using the microRNA target gene prediction software miRDB.
  • the predicted target genes were searched, and the target genes of the six microRNAs which were significantly inhibited in the phenotype of Ebola virus infection confirmed in Example 3 were mainly searched.
  • the specific selection principle is: compared with the control gene expression level of the recombinant adenovirus after infection, log2 is calculated at 3h, 6h, 12h, 24h after infection, respectively (Ad-GP target gene expression level / Ad- ⁇ E1
  • the result value of the control target gene expression amount in the control group (hereinafter referred to as log2), and the target gene whose absolute value of the result value at any one of the above four time points is greater than or equal to 1 is picked, that is, compared with Ad- ⁇
  • target genes whose expression levels were at least half decreased in the Ad-GP infection group were selected.
  • the target genes and expression changes of the other five small RNAs are shown in Table 2.
  • M-MLV reverse transcription kit Invitrogen, Cat. No. c28025
  • fluorescent dye LightCycler 480Master Green I (Roche, Roche); real-time quantitative PCR instrument (Roche, Roche); internal reference and target gene primer sequences are shown in Table 3 (Guangzhou Ruibo) The company synthesized), the sequences of the primers from top to bottom in Table 3 are SEQ ID NO. 98 to SEQ ID NO.
  • HUVEC was resuspended into a single cell solution with ECM, and seeded into 6-well plates, 10 5 cells/ml per well, 2 ml per well;
  • RNA was reverse transcribed to generate cDNA according to the instructions of the M-MLV Reverse Transcription Kit (Invitrogen).
  • RT-PCR reaction system was prepared as follows: 2 ⁇ SYBR Green I master, 10 ⁇ l; ddH 2 O, 7 ⁇ l; forward primer (10 uM), 1 ⁇ l; reverse primer (10 uM), 1 ⁇ l; cDNA template, 1 ⁇ l.
  • the reaction procedure was: 195 ° C, pre-denaturation for 5 min; 295 ° C, denaturation for 10 s, 355 ° C, renaturation for 10 s, 472 ° C, extension for 20 s, cycle 2 to 435 times.
  • the upregulation of the target gene was detected by the inhibitor of the six microRNAs selected in Example 3, and each microRNA was detected in addition to the target gene corresponding to Table 2, and the other five microRNAs were simultaneously detected.
  • Serial number Gene name Serial number Gene name 1 HLTF 11 TFPI 2 DAG1 12 CFLAR 3 LAMA4 13 DPP3 4 UBE2H 14 RCAN1 5 MCTP1 15 TRIM24 6 KIAA1249 16 RPAIN 7 SDCBP 17 IP6K1 8 CPD 18 ESM1 9 FRMD6 19 SLC9A6 10 SKIL 20 ZCCHC6
  • anti-HLTF rabbit polyclonal antibody (abcam, number ab155031, dilution ratio 1/500);
  • Anti-DAG1 rabbit polyclonal antibody (abcam, number ab105504, dilution ratio 1/1000);
  • Anti-Laminin alpha 4 rabbit polyclonal antibody (abcam, numbered ab69634, dilution ratio 1/200);
  • Anti-Ube2H mouse monoclonal antibody (abcam, number ab58261, dilution ratio 1/1000);
  • Anti-MCTP1 rabbit polyclonal antibody (abcam, number ab83673, dilution ratio 1/500);
  • Anti-KIAA1429 rabbit polyclonal antibody (abcam, numbered ab11011, dilution ratio 1/1500);
  • Anti-CPD rabbit polyclonal antibody (abcam, number ab153874, dilution ratio 1/500);
  • FRMD6 rabbit polyclonal antibody (abcam, numbered ab121133, dilution ratio 1/500);
  • Anti-TRIM24 rabbit polyclonal antibody (abcam, numbered ab70560, dilution ratio 1/1000);
  • Anti-RPAIN mouse polyclonal antibody (abcam, number ab88660, dilution ratio 1/500);
  • Anti-IP6K1 rabbit polyclonal antibody (abcam, number ab96210, dilution ratio 1/500);
  • Anti-ESM1 rabbit polyclonal antibody (abcam, number ab103590, dilution ratio 1/200);
  • Anti-SLC9A6 mouse monoclonal antibody (abcam, number ab77110, dilution ratio / 200);
  • Anti-ZCCHC6 mouse polyclonal antibody (abcam, number ab76901, dilution ratio 1/200);
  • Anti-SDCBP rabbit monoclonal antibody (pitomics, numbered RabMAb 5913-1, dilution ratio 1/2000);
  • Anti-TFPI rabbit monoclonal antibody (epitomics, numbered RabMAb5853-1, dilution ratio 1/1000);
  • Anti-DPP3 rabbit monoclonal antibody (epitomics, numbered RabMAb6523-1, dilution ratio 1/2000);
  • Anti-RCAN1 rabbit monoclonal antibody (epitomics, numbered RabMAb6846-1, dilution ratio 1/1000);
  • Anti-SKIL rabbit polyclonal antibody (CST company, number cst4973, dilution ratio 1/200);
  • Anti-CFLAR rabbit monoclonal antibody (epitomics, number cst8510, dilution ratio 1/500).
  • HUVEC was resuspended to a single cell solution with ECM, seeded into a 12-well plate, 10 5 cells/ml per well, 1 ml volume per well;
  • the protein sample is subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis;
  • the microRNA inhibitor can inhibit the expression of the associated microRNA such that the protein expression encoded by its corresponding target gene is up-regulated.
  • the CFLAR protein of SEQ ID NO. 44, the RCAN1 protein of SEQ ID NO. 46, the TRIM24 protein of SEQ ID NO. 47, and the ZCCHC6 protein of SEQ ID NO. 52 were significantly up-regulated.
  • these genes are regulated by their corresponding microRNAs, and are also involved in GP-induced rounding and shedding of cells. Therefore, in the screening application of these proteins for the prevention and/or treatment of Ebola virus-infected drugs in vitro, any drug capable of up-regulating the expression of the above proteins can effectively attenuate the rounding of cells after Ebola virus infection. Phenotype, increasing cell survival rate.
  • Dual luciferae reporter assay system promega
  • human embryonic kidney 293T cells Dulbecco's modified Eagle's medium (DMEM, Gibco, 10% (v/v) fetal calf Serum, penicillin (100 U/ml) and streptomycin (100 mg/ml); transfection reagent: lipofect lipofectamine 2000 (invitrogen); dual luciferase reporter plasmid psiCHEC-2 (Shanghai Shenggong)
  • a) seed cells 293T cells were prepared into single cell solution in DMEM, seeded into 24-well plates, 10 5 /mL cells per well, 500 ⁇ l per well;
  • the microRNA analogs and the control analogs are double-stranded RNA sequences in which the sense strands of the microRNAs hsa-miR1246, hsa-miR320a, hsa-miR196b-5p, and hsa-miR34a-5p are sense strands.
  • the sequences are SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69 and SEQ ID NO. 70, respectively, and the sense strand sequence of the control analog is SEQ ID NO. 71, the antisense strand sequence of all analogs Each pair is complementary to its corresponding sense strand sequence base pair.
  • the 3'UTR region of the target gene can be constructed into the vector after the reporter gene Renilla luciferase, and after the interference with the microRNA, the report is reported.
  • the change of gene expression (monitoring the change of luciferase activity) can quantitatively reflect the inhibitory effect of microRNA on the target gene; combined with site-directed mutagenesis, the site of action of microRNA and target gene 3'UTR can be further determined.
  • FIG. 8a and 8b An analog of the double-stranded hsa-miR-1246 having the sense strand sequence of SEQ ID NO. 67 is shown in Figures 8a and 8b to be able to significantly inhibit the CFLAR of the target gene sequence of SEQ ID NO. 24 and the sequence is SEQ ID.
  • the sense strand sequence is the double-stranded hsa-miR-320a analog of SEQ ID NO. 68, which can significantly inhibit the TFPI of SEQ ID NO. 23, DAG1, SEQ ID NO.
  • the sense strand sequence is the double-stranded hsa-miR-34a analog of SEQ ID NO. 70, which can significantly inhibit the expression of the RCAN1 gene of SEQ ID NO. 26, and when the RCAN1 gene 3'-UTR is mutated, hsa- The analog of miR-34a does not clearly exhibit inhibition of the RCAN1 gene.
  • Example 5 determining the relationship between the target gene and the cell detachment phenotype
  • HUVEC was resuspended to a single cell solution with ECM, and seeded into a 96-well plate, 10 5 cells/ml per well, 100 ⁇ l per well;
  • Double-stranded oligonucleotide interfering RNA of target genes DAG1, TFPI and CFLAR was transfected at 37 ° C, 5% CO 2 incubator for 24 h, and the control group was transfected into control double-stranded oligonucleotide interfering RNA to a final concentration of 100 nM. , the medium was changed after transfection for 4 hours;
  • the target sequence of RNA interference in the target gene DAG1 is SEQ ID NO.88
  • the sequence of the sense strand of the double-stranded oligonucleotide interfering RNA is SEQ ID NO.89
  • the sequence of the antisense strand from the 3' end to the 5' end is SEQ ID NO. 90 (Guangzhou Ruibo Synthesis, product number: siG000001605B).
  • the target sequence of RNA interference in the target gene TFPI is SEQ ID NO. 91
  • the sequence of the sense strand of the double-stranded oligonucleotide interfering RNA is SEQ ID NO. 92
  • the sequence of the antisense strand from the 3' end to the 5' end is SEQ ID NO. 93 (Guangzhou Ruibo Synthesis, product number: siG000007035C).
  • the target sequence of RNA interference in the target gene CFLAR is SEQ ID NO.94
  • the sequence of the sense strand of the double-stranded oligonucleotide interfering RNA is SEQ ID NO.95
  • the sequence of the antisense strand from the 3' end to the 5' end is SEQ ID NO. 96 (Guangzhou Ruibo Synthesis, product number: siG000008837A).
  • the target sequence of the control double-stranded oligonucleotide interfering RNA is SEQ ID NO. 158: TTCTCCGAACGTGTCACGT.
  • the two bases at the end of the sense strand sequence are dTdT, the remaining base sequence SEQ ID NO.159 is UUCUCCGAACGUGUCACGU, the first two bases of the antisense strand 3' to 5' are dTdT, and the sequence of the remaining bases is SEQ ID NO.
  • AAGAGGCUUGCACAGUGCA Guangzhou Ruibo synthesis.
  • double-stranded oligonucleotide interferes with the dTdT sequence at the 3' end of the sense strand and the antisense strand of the antisense strand to reduce intracellular degradation.
  • small interfering RNAs of some microRNAs are easily due to For the reason of design, the effect of reducing the target gene is not obvious, which is not shown in FIG. 9. Only the small interfering RNA which can significantly reduce the expression of the target gene and the corresponding target gene are detected in the MTS test. The survival rate of the cells.
  • the double-stranded oligonucleotide interfering RNA corresponding to TFPI, DAG1, and CFLAR can significantly reduce the expression level of the corresponding target gene, and when the expression of the above three proteins is decreased, the corresponding cells are The survival rate decreased significantly, indicating that the expression of at least three of the above six microRNA target genes was significantly decreased, and the degree of cell shedding after infection in the corresponding Ad-GP group was increased, and the cell survival rate was decreased.
  • DMEM Dulbecco's modified Eagle's medium
  • the target gene overexpressing plasmids were: Peak13-TFPI-flag, Peak13-DAG1-flag, Peak13-CFLAR-flag; GP and GP deletion plasmids which are the key toxic regions causing cell shedding, ie, GP- ⁇ mucin expression plasmid, and Example 1 Transfection reagent liposome lipofectamine2000; blood cell counting plate.
  • DMEM DMEM is used to prepare 293T cells into a single cell solution, and seeded into a 12-well plate, 10 5 /mL cells per well, 1 ml per well;
  • the control group is co-transfected with the peak13-GFP expression plasmid and the GP expression plasmid or the GP-SEQ ID NO.97. ⁇ mucin expression plasmid, and the medium was changed after 4 hours;
  • Figure 10 after the target genes TFPI, DAG1 and CFLAR were over-expressed, the degree of shedding of the cells in the over-expressed group was significantly alleviated, indicating that the expression of these three target genes can significantly inhibit the Ebola envelope glycoprotein.
  • the GP causes the cells to become rounded off the phenotype, thereby increasing cell survival.
  • Figure 11 also shows the relief of the degree of shedding of cells after over-expression of different target genes, indicating that different combinations of these three target genes can also inhibit Ebola envelope glycoprotein GP. The cells become rounded off the phenotype, thereby increasing cell survival.

Abstract

提供一种用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及其应用。该小核酸分子为埃博拉病毒感染细胞差异化表达的微小RNA的抑制物和/或微小RNA前体的抑制物，能够上调微小RNA的靶基因的表达，从而提高细胞对埃博拉病毒感染的抵抗力。

Description

用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及应用
本申请要求于2014年03月25日提交中国专利局、申请号为201410114552.5、发明名称为“用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，具体而言，涉及一种用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白以及应用。
背景技术
病毒性出血热泛指由数类病毒引起的、常伴以出血症状的一种严重疾病。按照病原分属四科，即沙粒病毒科(拉沙热、胡宁和普马拉)、布尼亚病毒科(克里米亚-刚果出血热、裂谷热、汉坦出血热)、线状病毒科(埃博拉和马尔堡)以及黄病毒科(黄热病、登革出血热、鄂木斯克出血热、基萨那森林病)。
以上各种病毒性出血热虽然在病原体、寄生宿主和传播途径上各有差异，但都具有发热、出血倾向及肾脏损害的主要临床特征，都属于急性病毒性传染病。目前均无特效疗法，采用对症和支持疗法，纠正水和电解质平衡，必要时补液、输血和抗休克治疗。
其中，埃博拉出血热因其病原体是埃博拉病毒，也叫埃博拉病毒病，是目前已知的毒性最大的病毒性疾病之一，病死率高达50－90％。目前已确定了埃博拉病毒的5个亚型：埃博拉-扎伊尔型(EBO-Zaire，ZEBO)、埃博拉-苏丹型(EBO-Sudan，SEBO)、埃博拉-莱斯顿型(EBO-R，REBO)和埃博拉-科特迪瓦型(EBO-CI，ICEBO)，埃博拉-本迪布乌干达型(EBO-Bundibugyo Uganda，BEBO)，其中ZEBO致病性最强。
埃博拉病毒可以通过接触传播，传播速度很快。患者一旦发病，可在24小时内死亡。主要症状可表现为高热、头痛、恶心、呕吐、腹泻、体内外大出血、全身酸痛等。它属于生物安全等级四级(BSL-4)的病毒，具有极高的生物危险性。
埃博拉病毒的宿主尚未确定，最初被认为是啮齿类动物，但实验后很快被否认。实验人员发现，感染病毒后的蝙蝠一般不会死亡，因此推断其传染源为带有病毒的蝙蝠及蝙蝠的排泄物。但这只是推测，是否为蝙蝠还有待确定。
从遗传角度来看，埃博拉病毒属于单股负链RNA病毒，其基因组约19kb，具有7个基因：NP、vp35、vp40、gp、vp30、vp24和L，其中NP，VP30，VP35和RNA依赖的RNA聚合酶构成病毒体的中心，即核糖核蛋白复合物，该复合物负责转录和复制。VP40和VP24负责核壳体的形成，病毒的出芽和包装以及决定宿主范围。在这些编码的蛋白质中，GP表面糖蛋白可能介导埃博拉病毒和靶细胞的结合和融合，在埃博拉出血热的发病机理中起非常重要的作用。将GP单独转到人源化细胞或其他几种非人源化细胞中，会导致细胞变圆、脱落，细胞表面粘附因子下调；将GP转入到人或猪血管中，会增加血管的通透性，从而导致血管出血。
但由于从血样中检测特异的抗原或病毒的基因，或通过细胞培养分离或者检测病毒的IgM和IgG抗体的实验都有严重的生物危害，必须在最大限度地保证生物安全的条件下才能进行，所以，到目前为止，商业上也没有针对病毒感染建立起特异的诊断方法。
在埃博拉出血热的治疗方面，一般的抗病毒药物包括利巴韦林和干扰素都对埃博拉病毒无效。尽管有实验显示，凝固干扰素在感染埃博拉病毒的猴群中似乎可以起一些作用，但凝固干扰素在人体的效果如何尚未确定。此外，目前也尚无对人类有效的疫苗，埃博拉出血热的康复者的血清也没有太大的作用，甚至有可能带来更坏的影响。目前的治疗仍以支持治疗为主，如使病毒的侵入最小化，或及时补充损失的血小板，平衡电解质，保持血液中氧元素含量以及对并发症的治疗等。现今唯一的治疗方法为注射NPC1阻碍剂，因埃博拉病毒需透过NPC1进入细胞核进行自我复制，NPC1蛋白于细胞间进行运输胆固醇，但阻碍剂会阻挡胆固醇的运输路线造成尼曼匹克症。因此，还需要通过更多更广泛的研究思路来寻找预防和治疗埃博拉出血热疾病方面的药物。
发明内容
有鉴于此，本发明提供一种用于预防和/或治疗出血热的小核酸分子、DNA分子、蛋白以及它们在药物筛选中的应用，并提供了一种预防和/或治疗出血热的药物研发的新思路。
需要说明的是，本发明是基于背景技术部分所提到的各种病毒性出血热所引起的血管结构和功能异常而导致的渗血、出血这一共同症状的前提下，通过大量研究而得到的结果。因此，本发明下面所提到的以埃博拉病毒为主要研究对象得到的能够抑制或缓解上述症状的各种物质也适用于上述其他病毒性出血热。
由于血管内皮细胞是埃博拉病毒的主要攻击的对象之一，血管内皮细胞在维持血管通透性和体液平衡方面发挥着重要作用，在埃博拉病毒感染血管内皮细胞后，通常引起血管结构和功能的异常，进而导致埃博拉出血热弥漫性血管内凝血(DIC)。由于人脐静脉(human umbilical vein，HUV)是血管内皮细胞的常用来源，其供源丰富，取材方便，血管条件优越，混杂其他细胞较少，可获得细胞免疫原性较低的血管内皮细胞，可使实验条件和所获结果更符合人体情况。因此，本发明在进行血管内皮细胞实验时，选用人脐静脉血管内皮细胞作为细胞模型来进行研究。
为了实现上述发明目的，本发明提供以下技术方案：
根据本发明的一个方面，提供了一种用于预防和/或治疗埃博拉病毒性出血热的小核酸分子，该小核酸分子为埃博拉病毒感染细胞差异化表达的微小RNA的抑制物和/或该微小RNA前体的抑制物。
进一步地，上述小核酸分子是序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.6的微小RNA及其前体的抑制物。
进一步地，上述小核酸分子的序列与SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12具有80％以上的同源性，优选具有95％以上的同源性，更优选地，上述小核酸分子的序列为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、或者SEQ ID NO.12。
根据本发明的另一个方面，提供了一种上述小核酸分子在制备用于预防和/或治疗鄂木斯克出血热、基萨那森林病、克里米亚出血热、新疆出血热、登革出血热、基孔肯雅热、黄热病、阿根廷出血热、玻利维亚出血热、拉沙热、流行性出血热或者马尔堡病的药物中的应用。
根据本发明的又一个方面，提供了一种用于预防和/或治疗埃博拉病毒性出血热的药物，该药物包括治疗有效量的上述小核酸分子。
进一步地，上述药物进一步包括一种或多种用于预防和/或治疗埃博拉病毒性出血热有效物质，优选地，上述用于预防和/或治疗埃博拉病毒性出血热有效物质选自抗病毒类药物、免疫提升类药物及皮质激素类药物。
根据本发明的一个方面，提供了一种上述小核酸分子在体外抑制埃博拉病毒性出血热感染引起细胞生长脱落的应用，在细胞感染埃博拉病毒之前或之后，将上述小核酸分子转染至细胞中。
根据本发明的一个方面，提供了一种上述小核酸分子在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用，将上述小核酸分子与待测药物进行核酸同源性比对。
根据本发明的又一个方面，提供了一种DNA分子，上述DNA分子在埃博拉病毒感染细胞中的表达水平被上述一种或多种小核酸分子上调。
进一步地，上述DNA分子的序列与SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32具有80％以上的同源性，优选具有95％以上的同源性，更优选地，上述DNA分子的序列为SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32。
进一步地，上述DNA分子为序列为SEQ ID NO.14、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.26、SEQ ID NO.27或者SEQ ID NO.32，优选序列为SEQ ID NO.14、SEQ ID NO.23或者SEQ ID NO.24。
根据本发明的又一个方面，提供了一种上述DNA分子在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用。
根据本发明的又一个方面，提供了一种上述DNA分子编码的蛋白，上述蛋白的序列与SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ ID NO.52具有80％以上的同源性，优选具有95％以上的同源性，更优选地，上述蛋白的序列为SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ ID NO.52。
进一步地，上述蛋白的序列为SEQ ID NO.34、SEQ ID NO.38、SEQ ID NO.40、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.46、SEQ ID NO.47或者SEQ ID NO.52，优选序列为SEQ ID NO.34、SEQ ID NO.43或者SEQ ID NO.44。
根据本发明的又一个方面，提供了一种上述蛋白在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用。
根据本发明的再一个方面，提供了一种用于检测埃博拉病毒性出血热的试剂盒，该试剂盒包括可特异性扩增上述小核酸分子所抑制的微小RNA的引物。
进一步地，当微小RNA的序列为SEQ ID NO.1时，逆转录引物序列为SEQ ID NO.140：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCTGCT，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.146：CCGCGCAATGGATTTTT，RP序列为SEQ ID NO.147：GTGCACGCTCCGAGGT；
当微小RNA的序列为SEQ ID NO.2时，逆转录引物序列为SEQ ID NO.141：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCCAAC，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.148：CCGCGCTAGGTAGTTTCCT，RP序列为SEQ ID NO.149：GTGCACGCTCCGAGGT；
当微小RNA的序列为SEQ ID NO.3时，逆转录引物序列为SEQ ID NO.142：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTCGCCC，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.150：TCGCGCAAAAGCTGG，RP序列为SEQ ID NO.151：GTGCACGCTCCGAGGT；
当微小RNA的序列为SEQ ID NO.4时，逆转录引物序列为SEQ ID NO.143：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACTAGA，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.152：TCGCGCTCGAGGAGC，RP序列为SEQ ID NO.153：GTGCACGCTCCGAGGT；
当微小RNA的序列为SEQ ID NO.5时，逆转录引物序列为SEQ ID NO.144：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACAACC.，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.154：TCGCGCTGGCAGTGT，RP序列为SEQ ID NO.155：GTGCACGCTCCGAGGT；
当微小RNA的序列为SEQ ID NO.6时，逆转录引物序列为SEQ ID NO.145：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTGCTGT，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.156：TCGCGCTGCGGG，RP序列为SEQ ID NO.157：GTGCACGCTCCGAGGT。
根据本发明的再一个方面，提供了一种用于检测埃博拉病毒性出血热的试剂盒，该试剂盒包括可特异性扩增上述DNA分子的引物。
进一步地，当DNA分子的序列为SEQ ID NO.13时，引物的FP序列为SEQ ID NO.100：AAGCAGGATCTTCTAAGGTT，RP序列为SEQ ID NO.101：AGTGTTGTTCTTGGTCTCTC；当DNA分子的序列为SEQ ID NO.14时，引物的FP序列为SEQ ID NO.102：GTCACTTGCTTCCTTACTTAG，RP序列为SEQ ID NO.103：TGAGCCATAACCACAGAG；当DNA分子的序列为SEQ ID NO.15时，引物的FP序列为SEQ ID NO.104：ACCAATCAGTGCTCAGTAT，RP序列为SEQ ID NO.105：TCAACCTCCACATCTCTATC；当DNA分子的序列为SEQ ID NO.16时，引物的FP序列为SEQ ID NO.106：TGGTCATCATCTGCTTGTG，RP序列为SEQ ID NO.107：TTCCTATTCGGTCACTCTCT；当DNA分子的序列为SEQ ID NO.17时，引物的FP序列为SEQ ID NO.108：GGTAGAACTGAACAACGATAG，RP序列为SEQ ID NO.109：CAGCAATGGTATAGCAACTT；当DNA分子的序列为SEQ ID NO.18时，引物的FP序列为SEQ ID NO.110：CGACGAACAGTAGACAGTATT，RP序列为SEQ ID NO.111：CAGCAATTCCATCTTCATCACT；当DNA分子的序列为SEQ ID NO.19时，引物的FP序列为SEQ ID NO.112：CTAATTCTCCAGCCTCATTG，RP序列为SEQ ID NO.113：ATGGTAATCGTCCGTTCA；当DNA  分子的序列为SEQ ID NO.20时，引物的FP序列为SEQ ID NO.114：ACCATTAGTGTTGCTGAGA，RP序列为SEQ ID NO.115：GTGTGAAGTTGACCTGAATAG；当DNA分子的序列为SEQ ID NO.21时，引物的FP序列为SEQ ID NO.116：GATTCTCTTCCACAGACTATATG，RP序列为SEQ ID NO.117：CCTTCCTCATCCAGTTCATG；当DNA分子的序列为SEQ ID NO.22时，引物的FP序列为SEQ ID NO.118：TTCATCTCCGCTTCTTGTG，RP序列为SEQ ID NO.119：TGTTCTTCAGTGAGTTCCTT；当DNA分子的序列为SEQ ID NO.23时，引物的FP序列为SEQ ID NO.120：GCCTGCTGCTTAATCTTG，RP序列为SEQ ID NO.121：CATCATCCGCCTTGAATG；当DNA分子的序列为SEQ ID NO.24时，引物的FP序列为SEQ ID NO.122：AGGCAAGATAAGCAAGGAGAAG，RP序列为SEQ ID NO.123：AGGTCTATTCTGTGGATGTTCT；当DNA分子的序列为SEQ ID NO.25时，引物的FP序列为SEQ ID NO.124：GAACCTCAGTGCCTACAACAC，RP序列为SEQ ID NO.125：GGAAGTCACCTCAGAGTCCAG；当DNA分子的序列为SEQ ID NO.26时，引物的FP序列为SEQ ID NO.126：
TTGCTCAGACCTTACACATAG，RP序列为SEQ ID NO.127：GCTTGGAGATGGCATATAAGA；当DNA分子的序列为SEQ ID NO.27时，引物的FP序列为SEQ ID NO.128：CAGAACATACCACGACAAG，RP序列为SEQ ID NO.129：CTGGAAGGAGTAGAGGATGT；当DNA分子的序列为SEQ ID NO.28时，引物的FP序列为SEQ ID NO.130：GAGCCTGAGACTTGTGTATC，RP序列为SEQ ID NO.131：CGTATTCTGGGAAGTGTTG；当DNA分子的序列为SEQ ID NO.29时，引物的FP序列为SEQ ID NO.132：GGCGTGGTATCTGTCTGT，RP序列为SEQ ID NO.133：CTGTTGTGTCATCCTGTTC；当DNA分子的序列为SEQ ID NO.30时，引物的FP序列为SEQ ID NO.134：TATCTGAAGGACGGTTCTGG，RP序列为SEQ ID NO.135：TTATCTCTGAGGTGGCATAC；当DNA分子的序列为SEQ ID NO.31时，引物的FP序列为SEQ ID NO.136：GGTTGGTGTTGATTCAGAC，RP序列为SEQ ID NO.137：GCAGAGGCTTCAGATAGT；当DNA分子的序列为SEQ ID NO.32时，引物的FP序列为SEQ ID NO.138：ATTAGCAAGAGTCCTCAGA，RP序列为SEQ ID NO.139：ATGGCGGAATAAGCAGATA。
应用本发明的技术方案，通过利用本发明的小核酸分子，能够抑制埃博拉病毒感染细胞差异化表达的微小RNA和/或微小RNA前体的表达，进而能够上调微小RNA的靶基因的表达，从而提高细胞对埃博拉病毒性出血热感染的抵抗力。通过本发明所提供的用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及它们的应用，不但提供了预防和/或治疗埃博拉病毒性出血热新的药物分子，而且为相关病毒性出血热药物的开发提供了新的研究方向。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示Ad-GP重组腺病毒上清及Ad-△E1重组腺病毒上清(对照病毒上清)感染人脐静脉血管内皮细胞不同时间点细胞存活率的情况；
图2示Ad-GP重组腺病毒上清及Ad-△E1重组腺病毒上清(对照病毒上清)感染人脐静脉血管内皮细胞不同时间点后提取的总RNA电泳图谱；
图3示Ad-GP重组腺病毒上清及Ad-△E1重组腺病毒上清(对照病毒上清)感染人脐静脉血管内皮细胞，分别在感染前24h转染微小RNA抑制剂，并在感染后48h进行MTS实验检测细胞的存活率；
图4示Ad-GP重组腺病毒上清及Ad-△E1重组腺病毒上清(对照病毒上清)感染人脐静脉血管内皮细胞，分别在感染后3h转染微小RNA抑制剂，并在感染后48h进行MTS实验 检测细胞的存活率；
图5a、5b、5c示荧光定量PCR法验证微小RNA的抑制物能够上调靶基因的表达情况；
图6和图7a、7b示Ad-GP重组腺病毒上清及Ad-△E1重组腺病毒上清(对照病毒上清)感染人脐静脉血管内皮细胞，分别在感染前24h转染微小RNA抑制剂，并在感染后24h检测微小RNA靶基因蛋白的表达情况；其中，图6是蛋白杂交印迹图；图7a、7b是对图6中的统计学分析图；
图8a和图8b示微小RNA的类似物对海肾荧光素标记的野生型靶基因和突变靶基因的mRNA表达的影响；
图9示采用双链寡核苷酸干扰RNA方法降低微小RNA靶基因的表达会降低Ad-GP感染HUVEC细胞的存活率；
图10示采用过量表达的方法使微小RNA靶基因过量表达会显著降低GP过表达所致的细胞脱落漂浮的比率；
图11示采用过量表达的方法同时使多个不同微小RNA的靶基因过量表达会显著降低GP过表达所致的细胞脱落漂浮的比率。
具体实施方式
本发明公开了用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及应用中所用原料及试剂均可由市场购得。
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
首先，需要声明的是，在本发明中，除有特殊说明外，所有涉及到DNA或RNA序列的，均是指从5’端到3’端的碱基序列。此外，在本发明中，FP表示正向引物，RP表示反向引物。
在本发明一种典型的实施方式中，提供了一种用于预防和/或治疗埃博拉病毒性出血热的小核酸分子，该小核酸分子为埃博拉病毒感染细胞差异化表达出的微小RNA的抑制物和/或其前体的抑制物。本发明所提供的小核酸分子是与现有技术中治疗和/或预防埃博拉病毒性出血热的药物作用机理完全不同的物质。细胞感染埃博拉病毒性出血热后引起细胞内微小RNA的表达量发生明显改变，通过本发明所提供的小核酸分子，能够直接与上述微小RNA序列互补而显著抑制大量表达的微小RNA。通过利用本发明的微小RNA的抑制物及其前体的抑制物，可以有效抑制感染埃博拉病毒性出血热的细胞中的微小RNA的表达，使得细胞变圆、脱落的现象明显减弱，进而使细胞的存活率显著提高，从而能够有效地治疗和/或预防埃博拉 病毒性出血热。
由于微小RNA的成熟过程需要经过形成茎环结构的前体形式，然后经过剪切加工形成具有双链结构的微小RNA二聚体，但在发挥功能阶段还需要去除其中的正义链形成真正起到抑制靶基因功能的成熟微小RNA。因此，影响形成上述能够真正发挥抑制靶基因功能的成熟微小RNA的前体的抑制物也适用于本发明的上述小核酸分子。
本发明所提供的上述小核酸分子是指任何一个在细胞感染埃博拉病毒性出血热后表达量明显上升的微小RNA的抑制物。例如，埃博拉病毒包膜糖蛋白GP感染人脐静脉血管内皮细胞后表达量能够明显上调至少2倍的序列为SEQ ID NO.1的hsa-miR1246、SEQ ID NO.2的hsa-miR196b-5p、SEQ ID NO.3的hsa-miR320a、SEQ ID NO.4的hsa-miR151a-5p、SEQ ID NO.5的hsa-miR34a-5p或SEQ ID NO.6的hsa-miR744a，上述微小RNA的抑制物均属于本发明的上述小核酸分子。上述小核酸分子均能有效地抑制埃博拉病毒感染的细胞中相应微小RNA的表达，抑制细胞变圆脱落的现象，提高细胞的存活率。
此外，除了上述的表达量上调至少2倍的微小RNA外，在埃博拉病毒感染不同时间长度的细胞中还存在一些与细胞变圆脱落相关的差异表达的微小RNA，此处的差异表达是指表达量上调或下调至少2倍。比如序列为SEQ ID NO.53的hsa-miR-152、SEQ ID NO.54的hsa-miR-22-3p、SEQ ID NO.55的hsa-miR-424-5p、SEQ ID NO.56的hsa-miR-374a-5p、SEQ ID NO.57的hsa-miR-24-3p、SEQ ID NO.58的hsa-miR-222-3p、SEQ ID NO.59的hsa-miR-4521、SEQ ID NO.60的hsa-miR-15b-5p、SEQ ID NO.61的hsa-miR-93-5p、SEQ ID NO.62的hsa-miR-193a-5p、SEQ ID NO.63的hsa-miR-29a-3p或者SEQ ID NO.64的hsa-miR-29c-3p，这些微小RNA的差异表达也与埃博拉病毒感染细胞有关，表达量上调的用相关微小RNA的抑制物，通过抑制相关微小RNA的表达来证实其与埃博拉病毒感染表型之间的关系；表达量下调的用相关微小RNA的类似物，通过微小RNA的过量表达来证实其与埃博拉病毒感染之间的关系。因此，不论是上述差异表达微小RNA的抑制剂还是类似物，均适用于本发明。
在本发明一种优选的实施例中，上述小核酸分子为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.6的微小RNA的抑制物，在埃博拉病毒感染的细胞中抑制相应微小RNA的表达的作用更明显，细胞变圆脱落现象明显改善，进而更有效地提高了细胞的存活率。
上述微小RNA的抑制物能够通过与微小RNA序列互补从而抑制微小RNA与靶基因的序列进行互补，从而抑制了微小RNA对靶基因表达的抑制作用，使得靶基因的表达相对升高，进而提高了被感染细胞的存活率。对于微小RNA的抑制物，只要其部分序列能够与微小RNA的序列进行互补起到抑制微小RNA表达的功能的抑制物均可用于本发明。
在本发明一种优选的实施例中，上述小核酸分子的序列与SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12具有80％以上的同源性，优选具有95％以上的同源性，更优选地，小核酸分子的序列为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12。其中，SEQ ID NO.7为hsa-miR1246的抑制剂、SEQ ID NO.8为hsa-miR196b-5p的抑制剂、SEQ ID NO.9为hsa-miR320a的抑制剂、SEQ ID NO.10为hsa-miR151a-5p的抑制剂、SEQ ID NO.11为hsa-miR34a-5p的抑制剂以及SEQ ID NO.12为hsa-miR744a的抑制剂。
上述小核酸分子的序列具有与SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12具有80％以上的同源性时能够很好地抑制与其序列部分互补的微小RNA的表达，从而抑制了细胞变圆脱落的表型，提高了埃博拉病毒感染 细胞的存活率。上述小核酸分子的序列具有与SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12具有95％以上的同源性时，抑制与其序列互补的微小RNA的表达的作用较强，使得微小RNA的表达下降的更多，进而提高埃博拉病毒感染细胞的存活率的作用较明显。当小核酸分子的序列为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12时，其序列与微小RNA的序列完全互补，抑制微小RNA表达的作用更强，对提高埃博拉病毒感染的细胞的存活率的作用也更明显。
本领域技术人员熟知的是，引起病毒性出血热的病毒有13种，共同的特点是都能引起血管内皮细胞的出血。由于本发明的上述小核酸分子能够有效地抑制血管内皮细胞变圆脱落，使其受到的损伤更小，进而起到保护血管提高细胞存活率的功能，因此，本发明所提供的上述小核酸分子也适用于预防和/或治疗鄂木斯克出血热、基萨那森林病、克里米亚出血热、新疆出血热、登革出血热、基孔肯雅热、黄热病、阿根廷出血热、玻利维亚出血热、拉沙热、流行性出血热、马尔堡病或埃博拉病毒性出血热。
本发明的另一方面在于提供了一种用于预防和/或治疗埃博拉病毒性出血热的药物，该药物包括治疗有效量的上述小核酸分子。包含治疗有效量的上述小核酸分子的药物能够有效地抑制被埃博拉病毒感染的细胞中微小RNA的表达，从而提高被埃博拉病毒感染的细胞的存活率。
在本发明所提供的上述药物中，只要包括治疗有效量的上述小核酸分子的药物均可用于本发明，对于药物中的有效物质的种类并无特殊要求。在本发明一种优选的实施例中，上述药物进一步包括一种或多种用于预防和/或治疗埃博拉病毒性出血热有效物质。包含一种或多种用于预防和/或治疗埃博拉病毒性出血热有效物质的药物均能有效地预防和/或治疗病毒性出血热。在本发明又一种优选的实施例中，上述用于预防和/或治疗埃博拉病毒性出血热有效物质选自抗病毒类药物、免疫提升类药物及皮质激素类药物。本发明选择上述有效物质对于受埃博拉病毒感染的细胞的损伤及脱落程度有明显的改善，同时能够更明显地提高细胞的存活率。
本发明的再一方面在于提供了上述小核酸分子在体外抑制埃博拉病毒感染导致细胞生长变圆脱落的应用，该应用是在细胞感染埃博拉病毒之前或之后将小核酸分子转染至细胞中。在埃博拉病毒基因组编码的7个蛋白中，GP蛋白的内源性表达导致细胞生长变圆脱落的表型最显著，因此，通常用GP蛋白来模拟埃博拉病毒体外感染细胞生长变圆脱落的实验。
在本发明上述应用中，优选上述小核酸分子在体外抑制埃博拉病毒糖蛋白GP感染细胞导致细胞生长变圆脱落的应用。将上述小核酸分子在细胞感染埃博拉病毒的糖蛋白GP之前转染至细胞中，能够在感染引起细胞中微小RNA表达上调时及时抑制微小RNA的表达，进而解除了微小RNA对其靶基因的抑制作用，使得靶基因能够大量表达，从而降低了埃博拉病毒糖蛋白GP导致的细胞变圆脱落的几率，同时提高了细胞抗埃博拉病毒糖蛋白GP的细胞毒性的能力，从而能够及时地防治埃博拉病毒的感染。
同理，在感染埃博拉病毒糖蛋白GP之后将小核酸分子转染至细胞中，是在引起细胞中微小RNA大量表达之后及时抑制其进一步的表达和微小RNA进一步对靶基因表达的抑制，进而提高了细胞在感染后对埃博拉病毒的细胞毒性的抵抗力，降低了细胞损伤程度，从而提高了细胞的存活率。
本发明另外还提供了一种上述小核酸分子在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用，该应用是将小核酸分子与待测药物进行核酸同源性比对。由于本发 明所提供的上述小核酸分子能够很好地抑制被埃博拉病毒包膜糖蛋白GP引起的细胞变圆脱落的表型，提高感染细胞的抗感染能力，其作用机理在于该小核酸分子能够与被埃博拉病毒包膜糖蛋白GP所引起的表达上调的微小RNA的序列进行部分互补，进而抑制感染细胞内微小RNA的表达。因此，通过将该小核酸分子与待测药物进行核酸同源性比对，根据同源性越高越好的原则，便可在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物。
本发明的又一个方面提供了一种DNA分子，该DNA分子在埃博拉病毒感染细胞中的表达水平可被上述一种或几种小核酸分子上调。被上述一种或几种小核酸分子上调表达的DNA分子，即为相应微小RNA能够抑制的靶基因。这些微小RNA的靶基因的上调表达能够有效降低埃博拉病毒所致的细胞受损伤的程度，提高细胞对埃博拉病毒引起的细胞毒性的抵抗力，进而提高细胞的存活率。
在本发明所提供的上述DNA分子中，能够被上述一种或几种小核酸分子上调表达的有很多种，只要能够被上述的一种或几种小核酸分子上调表达同时又能降低埃博拉病毒导致的细胞受损伤的程度，提高细胞对埃博拉病毒感染的抵抗力，进而提高细胞的存活率的DNA分子均适用于本发明。在本发明一种优选的实施例中，上述DNA分子的序列与SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32具有80％以上的同源性，优选具有95％以上的同源性，更优选地，小核酸分子的序列为SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32。其中，DNA分子序列SEQ ID NO.13至SEQ ID NO.32对应的基因分别为：HLTF、DAG1、LAMA4、UBE2H、MCTP1、KIAA1249、SDCBP、CPD、FRMD6、SKIL、TFPI、CFLAR、DPP3、RCAN1、TRIM24、RPAIN、IP6K1、ESM1、SLC9A6、ZCCHC6。
在本发明所提供的上述优选的实施例中，上述DNA分子的序列与SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32具有80％以上的同源性。从生物进化的角度看，同源性越高的DNA分子之间具有相同或相似功能的可能性就比较高，属于功能保守的同源基因的可能性就比较大。因此，具有与上述序列的DNA分子具有80％以上同源性的DNA分子能够起到与具有上述序列的DNA分子相同或相似的抑制埃博拉病毒导致的细胞变圆脱落的表型，提高细胞抗埃博拉病毒感染的能力的可能性就比较大。
当本发明所提供的上述DNA分子的序列与SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32具有95％以上的同源性时，该DNA分子能够起到与具有上述序列的DNA分子相同或相似的抑制埃博拉病毒包膜糖蛋白GP导致的细胞变圆脱落的表型，提高细胞抗埃博拉病毒感染的能力的可能性就更大。
当本发明所提供的上述DNA分子的序列为SEQ ID NO.13、SEQ ID NO.14、SEQ ID  NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32时，由于具有上述序列的DNA分子的表达受到上述小核酸分子的上调，其上调表达能够更加有效地抑制感染细胞变圆脱落的表型，进而更有效地提高细胞抗埃博拉病毒感染的能力。
本发明所提供的上述DNA分子的表达均能被上述小核酸分子明显上调至少2倍，都具有提高埃博拉病毒感染细胞的抗细胞毒性能力。在本发明一种典型的实施例中，上述DNA分子为序列为SEQ ID NO.14、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.26、SEQ ID NO.27或者SEQ ID NO.32，优选序列为SEQ ID NO.14、SEQ ID NO.23或者SEQ ID NO.24。序列为SEQ ID NO.14、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.27或者SEQ ID NO.32的DNA分子，不仅在信使RNA表达水平上明显表现为上调；而且在蛋白表达水平上也明显被上调。序列为SEQ ID NO.14、SEQ ID NO.23或者SEQ ID NO.24的DNA分子在信使RNA表达水平和蛋白表达水平上上调的程度更加明显，对于埃博拉感染细胞受损伤、脱落变圆具有更加显著的改善效果。
在本发明另一种典型的实施方式中，还提供了一种上述DNA分子在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用，该应用能够以上述DNA分子为对象在体外开展用于预防和/或治疗埃博拉病毒性出血热的药物筛选，能够使上述DNA分子表达上调的药物均适用于本发明。
本发明还提供了一种上述DNA分子编码的蛋白，该蛋白的序列与SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ ID NO.52具有80％以上的同源性，优选具有95％以上的同源性，更有选地，蛋白的序列为SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ ID NO.52。
本发明所提供的上述DNA分子编码的蛋白，当其序列与SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ IDNO.52具有80％以上的同源性时，与具有上述序列的蛋白的同源性比较高，具有相同生物学功能的可能性就高。当上述DNA分子编码的蛋白的序列与上述序列具有95％以上的同源性时，其与具有上述序列的蛋白起到相同生物学功能的可能性就更高。具有上述序列的蛋白的表达被上述小核酸分子上调，其表达的上调能够更加明显地降低埃博拉病毒感染细胞受损伤的程度，提高细胞的存活率。其中，上述序列从SEQ ID NO.33至SEQ ID NO.52的蛋白分别由序列从SEQ ID NO.13至SEQ ID NO.32的DNA分子所编码。
在本发明一种优选的实施例中，上述蛋白的分子序列为SEQ ID NO.34、SEQ ID NO.38、SEQ ID NO.40、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.46、SEQ ID NO.47或者SEQ ID NO.52。分子序列为SEQ ID NO.34的DAG1蛋白、SEQ ID NO.38的KIAA1249蛋白、SEQ ID NO.40的CPD蛋白、SEQ ID NO.43的TFPI蛋白、SEQ ID NO.44的CFLAR蛋白、SEQ ID NO.46 的RCAN1蛋白、SEQ ID NO.47的TRIM24蛋白或者SEQ ID NO.52的ZCCHC6蛋白过量表达之后对细胞抗埃博拉病毒感染的能力的提高有更显著的效果。进一步地优选序列为SEQ ID NO.34、SEQ ID NO.43或者SEQ ID NO.44的蛋白。
在本发明一种典型的实施方式中，还提供了一种上述DNA分子编码的蛋白在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用，该应用能够以上述蛋白为对象在体外开展用于预防和/或治疗埃博拉病毒性出血热的药物筛选，能够上调上述蛋白表达的药物亦可应用于治疗或预防埃博拉病毒性出血热。
本发明进一步提供了一种用于检测埃博拉病毒性出血热的试剂盒，该试剂盒包括可特异性扩增上述小核酸分子所抑制的微小RNA的引物。
在本发明所提供的上述试剂盒中，根据所要检测的微小RNA的不同，其所提供的用于特异扩增上述微小RNA分子的引物序列也不同。当微小RNA的序列为SEQ ID NO.1时，逆转录引物序列为SEQ ID NO.140：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCTGCT，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.146：CCGCGCAATGGATTTTT，RP序列为SEQ ID NO.147：GTGCACGCTCCGAGGT；当微小RNA的序列为SEQ ID NO.2时，逆转录引物序列为SEQ ID NO.141：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCCAAC，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.148：CCGCGCTAGGTAGTTTCCT，RP序列为SEQ ID NO.149：GTGCACGCTCCGAGGT；当微小RNA的序列为SEQ ID NO.3时，逆转录引物序列为SEQ ID NO.142：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTCGCCC，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.150：TCGCGCAAAAGCTGG，RP序列为SEQ ID NO.151：GTGCACGCTCCGAGGT；当微小RNA的序列为SEQ ID NO.4时，逆转录引物序列为SEQ ID NO.143：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACTAGA，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.152：TCGCGCTCGAGGAGC，RP序列为SEQ ID NO.153：GTGCACGCTCCGAGGT；当微小RNA的序列为SEQ ID NO.5时，逆转录引物序列为SEQ ID NO.144：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACAACC.，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.154：TCGCGCTGGCAGTGT，RP序列为SEQ ID NO.155：GTGCACGCTCCGAGGT；当微小RNA的序列为SEQ ID NO.6时，逆转录引物序列为SEQ ID NO.145：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTGCTGT，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.156：TCGCGCTGCGGG，RP序列为SEQ ID NO.157：GTGCACGCTCCGAGGT。
本发明进一步提供了一种用于检测埃博拉病毒性出血热的试剂盒，该试剂盒包括可特异性扩增上述DNA分子的引物。利用本发明的试剂盒中提供的引物能够有效地扩增出埃博拉感染细胞中能被上述小核酸分子明显上调表达的DNA分子。
在本发明所提供的上述试剂盒中，根据所要检测的DNA分子的序列不同，其所提供的用于特异扩增上述DNA分子的引物序列也不同。当DNA分子的序列为SEQ ID NO.13时，引物的FP序列为SEQ ID NO.100：AAGCAGGATCTTCTAAGGTT，RP序列为SEQ ID NO.101：AGTGTTGTTCTTGGTCTCTC；当DNA分子的序列为SEQ ID NO.14时，引物的FP序列为SEQ ID NO.102：GTCACTTGCTTCCTTACTTAG，RP序列为SEQ ID NO.103：TGAGCCATAACCACAGAG；当DNA分子的序列为SEQ ID NO.15时，引物的FP序列为SEQ ID NO.104：ACCAATCAGTGCTCAGTAT，RP序列为SEQ ID NO.105：TCAACCTCCACATCTCTATC；当DNA分子的序列为SEQ ID NO.16时，引物的FP序列为SEQ ID NO.106：TGGTCATCATCTGCTTGTG，RP序列为SEQ ID NO.107：TTCCTATTCGGTCACTCTCT；当DNA分子的序列为SEQ ID NO.17时，引物的FP序列为SEQ ID NO.108：GGTAGAACTGAACAACGATAG，RP序列为SEQ ID NO.109：CAGCAATGGTATAGCAACTT；当DNA分子的序列为SEQ ID NO.18时，引物的FP序列为SEQ ID NO.110： CGACGAACAGTAGACAGTATT，RP序列为SEQ ID NO.111：CAGCAATTCCATCTTCATCACT；当DNA分子的序列为SEQ ID NO.19时，引物的FP序列为SEQ ID NO.112：CTAATTCTCCAGCCTCATTG，RP序列为SEQ ID NO.113：ATGGTAATCGTCCGTTCA；当DNA分子的序列为SEQ ID NO.20时，引物的FP序列为SEQ ID NO.114：ACCATTAGTGTTGCTGAGA，RP序列为SEQ ID NO.115：GTGTGAAGTTGACCTGAATAG；当DNA分子的序列为SEQ ID NO.21时，引物的FP序列为SEQ ID NO.116：GATTCTCTTCCACAGACTATATG，RP序列为SEQ ID NO.117：CCTTCCTCATCCAGTTCATG；当DNA分子的序列为SEQ ID NO.22时，引物的FP序列为SEQ ID NO.118：TTCATCTCCGCTTCTTGTG，RP序列为SEQ ID NO.119：TGTTCTTCAGTGAGTTCCTT；当DNA分子的序列为SEQ ID NO.23时，引物的FP序列为SEQ ID NO.120：GCCTGCTGCTTAATCTTG，RP序列为SEQ ID NO.121：CATCATCCGCCTTGAATG；当DNA分子的序列为SEQ ID NO.24时，引物的FP序列为SEQ ID NO.122：AGGCAAGATAAGCAAGGAGAAG，RP序列为SEQ ID NO.123：AGGTCTATTCTGTGGATGTTCT；当DNA分子的序列为SEQ ID NO.25时，引物的FP序列为SEQ ID NO.124：GAACCTCAGTGCCTACAACAC，RP序列为SEQ ID NO.125：GGAAGTCACCTCAGAGTCCAG；当DNA分子的序列为SEQ ID NO.26时，引物的FP序列为SEQ ID NO.126：TTGCTCAGACCTTACACATAG，RP序列为SEQ ID NO.127：GCTTGGAGATGGCATATAAGA；当DNA分子的序列为SEQ ID NO.27时，引物的FP序列为SEQ ID NO.128：CAGAACATACCACGACAAG，RP序列为SEQ ID NO.129：CTGGAAGGAGTAGAGGATGT；当DNA分子的序列为SEQ ID NO.28时，引物的FP序列为SEQ ID NO.130：GAGCCTGAGACTTGTGTATC，RP序列为SEQ ID NO.131：CGTATTCTGGGAAGTGTTG；当DNA分子的序列为SEQ ID NO.29时，引物的FP序列为SEQ ID NO.132：GGCGTGGTATCTGTCTGT，RP序列为SEQ ID NO.133：CTGTTGTGTCATCCTGTTC；当DNA分子的序列为SEQ ID NO.30时，引物的FP序列为SEQ ID NO.134：TATCTGAAGGACGGTTCTGG，RP序列为SEQ ID NO.135：TTATCTCTGAGGTGGCATAC；当DNA分子的序列为SEQ ID NO.31时，引物的FP序列为SEQ ID NO.136：GGTTGGTGTTGATTCAGAC，RP序列为SEQ ID NO.137：GCAGAGGCTTCAGATAGT；当DNA分子的序列为SEQ ID NO.32时，引物的FP序列为SEQ ID NO.138：ATTAGCAAGAGTCCTCAGA，RP序列为SEQ ID NO.139：ATGGCGGAATAAGCAGATA。
由于本发明所提供的小核酸分子、DNA分子以及DNA分子所编码的蛋白的变化与出血热的发病机制有关，因此，围绕上述分子可以展开各种诊断和/或治疗病毒性出血热的方法。
下面将结合具体的实施例来说明本发明的有益效果。
实施例一、构建细胞脱落模型
1.实验材料
人脐静脉血管内皮细胞Human umbilical vein endothelial cells(HUVEC；Science II)；293A细胞；
内皮细胞培养基(Endothelial Cell Medium，ECM)(Science II)：含5％胎牛血清(FBS，Science II,货号为0025)，1％内皮细胞生长添加剂(endothelial cell growth supplement，ECGS,Science II,货号为1052)和1％青霉素链霉素溶液(penicillin/streptomycin solution，P/S Science II,货号为0503)；
加入了巨细胞病毒(cytomegalovirus，CMV)强启动子的经过序列改造的pEAK13质粒，该质粒上带有有氨卡青霉素和嘌呤霉素抗性基因，分别可用于原核细胞和真核细胞的筛选，含有NheⅠ、NotⅠ、BspHⅠ、EcoRⅠ和HindⅢ多克隆酶切位点。
腺病毒穿梭载体pAdTrack-CMV；腺病毒骨架载体pAdEasy-1；
细胞培养皿；荧光显微镜。
2.实验方法
2.1)将序列为SEQ ID NO.65的埃博拉(扎伊尔型)病毒糖蛋白GP的基因序列插入pAdTrack-CMV质粒中，构建pAdTrack-CMV-GP质粒。
2.2)pAdTrack-CMV-GP质粒及不含GP的对照空载体与与腺病毒骨架质粒pAdEasy-1重组，分别构建pAdTrack-CMV-GP/pAdEasy-1重组质粒(以下简称Ad-GP)和pAdTrack-CMV/pAdEasy-1重组质粒(以下简称Ad-△E1)。重组腺病毒构建具体步骤参照：Jinyong Luo,Zhong-Liang Deng,Xiaoji Luo，etal.A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.Nature.17May 2007；doi:10.1038/nprot.2007.135。(注：腺病毒骨架质粒是腺病毒复制缺陷型质粒，即缺失E1基因区，简写为△E1)
2.3)细胞脱落模型的建立
a)将HUVEC用ECM重悬成单细胞溶液，种到12孔板，每孔105个/mL细胞，每孔体积1ml；24h后分别加入等量的Ad-GP和Ad-△E1重组的腺病毒，感染复数(MOI)为500；
b)用荧光显微镜观察细胞形态的变化，从附图1中可以看出，在感染48h后，Ad-GP重组腺病毒感染的细胞组中近45％的细胞明显变圆、脱落，而Ad-△E1重组腺病毒感染的细胞组中并未受到明显的影响，长势良好。因此，GP蛋白所致的细胞脱落模型构建成功。
实施例二、寻找Ad-GP和Ad-△E1重组的腺病毒感染的HUVEC中差异表达的微小RNA
2.1.实验材料：同实施例一
2.2.实验方法
将HUVEC用ECM重悬成单细胞溶液，分为实验组和对照组，分别种到两个10cm板上，每孔105个/ml细胞，每板体积10ml；
24小时后分别向实验组和对照组中加入等量的Ad-GP和Ad-△E1重组的腺病毒，感染复数(MOI)为500；
分别在感染3h，6h，12h，24h，36h，48h后用1ml Trizol(invitrogen)裂解细胞，提取总RNA(具体步骤参见Trizol使用操作指南)；
利用北京贝瑞和康生物技术有限公司的Illumina Hiseq2500对所提的总RNA进行高通量测序。为了分析Ad-GP和Ad-△E1重组的腺病毒感染的细胞中差异表达的基因，利用miRDeep软件分析测得的数据中每个转录本的RPM值，比较两组感染细胞的数据，筛选出在感染3h，6h，12h，24h，36h，48h中只要满足一个时间点上差异表达的倍数大于等于2的微小RNA，利用miRDB分析相应的靶基因，通过STRING数据库构建微小RNA-靶基因相互作用网络，结合基因功能注释，挑选处于网络关键节点的微小RNA。
2.3.实验结果
2.3.1总RNA的提取质量见图2，从图2中可以看出，实验组和对照组中总RNA提取的质量均较好，没有明显的RNA降解。
2.3.2利用生物信息学分析具有差异表达的微小RNA及其差异表达数据，具体见表1。
表1
Figure PCTCN2015072624-appb-000001
实施例三、利用MTS实验检测候选微小RNA对细胞脱落的预防和/或治疗效果
3.1实验材料：除下列试剂外，其余同实施例一。
微小RNA的抑制物(广州锐博合成)
候选微小RNA的抑制物是人工合成的所有碱基被甲基化修饰的单链RNA，此实验使用的微小RNA的抑制物分别是：hsa-miR1246的抑制物序列为SEQ ID NO.7；hsa-miR196b-5p的抑制物序列为SEQ ID NO.8；hsa-miR320a的抑制物序列为SEQ ID NO.9；hsa-miR151a-5p的抑制物序列为SEQ ID NO.10；hsa-miR34a-5p的抑制物序列为SEQ ID NO.11；hsa-miR744a的抑制物序列为SEQ ID NO.12；对照抑制物序列为SEQ ID NO.66。
转染试剂：脂质体lipofectamine RNAiMAX(Invitrogen)；
细胞增殖与毒性检测试剂盒(MTS)(Promega)
3.2实验方法
3.2.1分组检测：
a.预防组
接种细胞：将HUVEC用ECM重悬成单细胞溶液，种到96孔板，每孔105个/ml细胞，每孔体积100μl；37℃，5％CO2孵箱培养细胞24h后转染候选微小RNA的抑制物，另设一组转染对照抑制物，培养基中抑制物的终浓度为100nM。转染4h后更换培养基；转染24h后分别加入等量的Ad-GP和Ad-△E1；48h后进行MTS实验检测细胞存活率。
b.治疗组
接种细胞：将HUVEC用ECM重悬成单细胞溶液，种到96孔板，每孔105个/ml，每孔体积100μl；37℃，5％CO2孵箱培养细胞24h后加入等量的Ad-GP和Ad-△E1；加毒感染3h后转染候选微小RNA的抑制物，另设一组转染对照抑制物，终浓度100nM；转染48h后进行MTS实验检测细胞存活率。
3.2.2细胞增殖与毒性检测试剂盒(MTS)实验检测
弃去96孔板中的ECM培养基；每孔加入100μl磷酸盐缓冲溶液(PBS，1升水中溶解8g NaCl、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4，pH值7.4)轻轻洗一遍细胞；弃去PBS，每孔加入100μl细胞培养基和20μl MTS混合液；37℃避光孵育1-2h，用酶标仪检测在490nm处的吸光值，同时检测在630nm处的吸光值(背景值)。
3.3实验结果
实验结果见附图3和附图4，附图3中的结果是利用表1中所列的微小RNA的抑制物重复四次实验后，挑出的效果比较明显的6个。从图3中可以看出，在预防组中，相比不能抑制微小RNA功能的序列号为SEQ ID NO.66的对照微小RNA抑制物，序列号为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6的微小RNA的抑制物均能不同程度地预防细胞脱落，提高细胞的存活率。相应地，从图4中可以看出，在治疗组中，相比不能抑制微小RNA功能的对照微小RNA抑制物，序列号为SEQ ID NO.1、SEQ ID NO.3和SEQ ID NO.6的抑制物均能不同程度地抑制细胞脱落，提高细胞的存活率。由此说明，本发明所提供的上述微小RNA的抑制物都能够抑制埃博拉病毒感染细胞的脱落程度，显著提高细胞的存活率。
实施例四、微小RNA相应靶基因的确定
4.1.生物信息学方法预测靶基因
利用微小RNA靶基因预测软件miRDB预测实施例二中所找到的微小RNA的靶基因。在实施例二的高通量测序结果中寻找预测的靶基因，且重点寻找了实施例三中验证的对埃博拉病毒感染表型抑制比较明显的6个微小RNA的靶基因。具体挑选原则是：相比对照的重组腺病毒感染后靶基因的表达量，分别在加毒感染3h，6h，12h，24h时，计算log2(Ad-GP组靶基因表达量/Ad-△E1对照组靶基因表达量)的结果值(下述以log2表示)，挑取上述4个时间点中任一时间点所取结果值的绝对值大于等于1的靶基因，即相比Ad-△E1对照组，Ad-GP感染组的表达量至少下降一半的靶基因挑选出来。其中，除了hsa-miR744没有显著性差异表达的靶基因外(即log2的绝对值小于1)，其余5个小RNA的靶基因及表达量变化见表2。
表2
Figure PCTCN2015072624-appb-000002
Figure PCTCN2015072624-appb-000003
4.2实时定量PCR法验证微小RNA的抑制物能够上调靶基因的表达
4.2.1实验材料：除下列试剂外，其余同实施例一。
M-MLV逆转录试剂盒(Invitrogen，货号c28025)；荧光染料：LightCycler 480Master Green Ⅰ(Roche，罗氏)；实时定量PCR仪(Roche，罗氏)；内参及靶基因引物序列见表3(广州瑞博公司合成)，表3中从上到下的引物的序列为SEQ ID NO.98至SEQ ID NO.139。
表3
Figure PCTCN2015072624-appb-000004
Figure PCTCN2015072624-appb-000005
4.2.2实验方法：
a)种细胞：将HUVEC用ECM重悬成单细胞溶液，种到6孔板中，每孔105个/ml细胞，每孔体积2ml；
b)在37℃，5％的CO2孵箱中培养24小时后，转染微小RNA抑制物和对照抑制物，终浓度100nM；
c)转染4小时后更换培养基；转染24小时后加入Ad-GP重组腺病毒；
d)分别在感染3h，6h，12h，24h用trizol裂解细胞收取细胞样品提取RNA；
e)逆转录：按照M-MLV逆转录试剂盒(Invitrogen)说明书，将1.0ug总RNA逆转录生成cDNA。
f)用Real-time PCR仪进行靶基因mRNA水平的检测，用看家基因GAPDH作为内参， 每个基因设三个复孔，内参也为三个复孔，配制RT-PCR反应体系如下：2×SYBR Green Ⅰ master，10μl；ddH2O，7μl；正向引物(10uM)，1μl；反向引物(10uM)，1μl；cDNA模板，1μl。反应程序为：①95℃，预变性5min；②95℃，变性10s，③55℃，复性10s，④72℃，延伸20s，循环②至④35次。
g)数据处理：在熔点曲线为单峰的情况下，数据处理即可开始。根据每次扩增DNA量约增加两倍来计算。在反应的前期，DNA量的扩增是呈对数扩增的，越早达到定量PCR仪的阈值Ct，说明模板量越大，通过内参较正后，即可比较目的基因的丰度。目的基因相对内参丰度＝2-(Ct目的-Ct内参)
h)利用GraphPad Prism作图软件做出柱状图并作t检验。
4.2.3实施定量PCR的检测结果
对实施例3中筛选出的6个微小RNA的抑制物对靶基因的上调情况进行了检测，每个微小RNA除了检测表2中自身对应的靶基因外，同时检测其余5个微小RNA所对应的靶基因，以检测单一靶基因是否被多个微小RNA抑制剂所上调，并对靶基因能够被微小RNA上调表达的程度进行显著性检验，*表示P<0.05时有显著性差异；**表示P<0.01时有显著性差异，具体每个基因上调表达的情况见附图5a、5b和5c。然后，将四个时间点中任一时间点能够被上述至少一种微小RNA抑制剂显著上调的靶基因挑选出来，总结在表4中。
表4
序号 基因名称 序号 基因名称
1 HLTF 11 TFPI
2 DAG1 12 CFLAR
3 LAMA4 13 DPP3
4 UBE2H 14 RCAN1
5 MCTP1 15 TRIM24
6 KIAA1249 16 RPAIN
7 SDCBP 17 IP6K1
8 CPD 18 ESM1
9 FRMD6 19 SLC9A6
10 SKIL 20 ZCCHC6
4.3蛋白水平上验证微小RNA的靶基因
4.3.1实验材料：除下列试剂外，其余材料同实施例一和实施例三。
一抗：抗HLTF兔多克隆抗体(abcam公司，编号为ab155031,稀释比例1/500)；
抗DAG1兔多克隆抗体(abcam公司,编号为ab105504，稀释比例1/1000)；
抗Laminin alpha 4兔多克隆抗体(abcam公司，编号为ab69634，稀释比例1/200)；
抗Ube2H小鼠单克隆抗体(abcam公司，编号为ab58261，稀释比例1/1000)；
抗MCTP1兔多克隆抗体(abcam公司，编号为ab83673，稀释比例1/500)；
抗KIAA1429兔多克隆抗体(abcam公司，编号为ab11011，稀释比例1/1500)；
抗CPD兔多克隆抗体(abcam公司，编号为ab153874，稀释比例1/500)；
FRMD6兔多克隆抗体(abcam公司，编号为ab121133，稀释比例1/500)；
抗TRIM24兔多克隆抗体(abcam公司，编号为ab70560，稀释比例1/1000)；
抗RPAIN小鼠多克隆抗体(abcam公司，编号为ab88660，稀释比例1/500)；
抗IP6K1兔多克隆抗体(abcam公司，编号为ab96210，稀释比例1/500)；
抗ESM1兔多克隆抗体(abcam公司，编号为ab103590，稀释比例1/200)；
抗SLC9A6小鼠单克隆抗体(abcam公司，编号为ab77110，稀释比例/200)；
抗ZCCHC6小鼠多克隆抗体(abcam公司，编号为ab76901，稀释比例1/200)；
抗SDCBP兔单克隆抗体(pitomics公司，编号为RabMAb 5913-1，稀释比例1/2000)；
抗TFPI兔单克隆抗体(epitomics公司，编号为RabMAb5853-1，稀释比例1/1000)；
抗DPP3兔单克隆抗体(epitomics公司，编号为RabMAb6523-1，稀释比例1/2000)；
抗RCAN1兔单克隆抗体(epitomics公司，编号为RabMAb6846-1，稀释比例1/1000)；
抗SKIL兔多克隆抗体(CST公司，编号为cst4973，稀释比例1/200)；
抗CFLAR兔单克隆抗体(epitomics公司，编号为cst8510，稀释比例1/500)。
4.3.2实验方法
a)种细胞：将HUVEC用ECM重悬成单细胞溶液，种到12孔板中，每孔105个/ml细胞，每孔体积1ml；
b)37℃，5％的CO2孵箱中培养24小时后，转染微小RNA抑制物和对照抑制物，抑制物的终浓度为100nM；
c)转染4小时后更换培养基；转染24小时后加入Ad-GP和Ad-△E1；
d)感染后24h用细胞裂解液裂解细胞收取细胞蛋白样品；
e)利用BCA蛋白质定量试剂盒(天根，PA115)将样品蛋白浓度调成一致；
f))加入等体积的2×SDS加样缓冲液，95℃变性10分钟，放回冰上；
g)将蛋白样品进行十二烷基硫酸钠聚丙烯酰胺(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)凝胶电泳；
h)显影检测蛋白表达情况，用密理博(millipore)ECL显色液，发光时间约为10分钟。
4.3.3实验结果
从附图6及附图7a、7b上可以看出，微小RNA抑制物可以抑制相关微小RNA的表达，使得其对应的靶基因编码的蛋白表达被上调。其中，序列号为SEQ ID NO.34的DAG1蛋白、序列号为SEQ ID NO.38的KIAA1429蛋白、序列号为SEQ ID NO.40的CPD蛋白、序列号为SEQ ID NO.43的TFPI、序列号为SEQ ID NO.44的CFLAR蛋白、序列号为SEQ ID NO.46的RCAN1蛋白、序列号为SEQ ID NO.47的TRIM24蛋白以及序列号为SEQ ID NO.52的ZCCHC6蛋白被明显上调。由此可进一步确定，这些基因是受其相应的微小RNA调控的，并且也参与GP导致细胞变圆、脱落过程的。因此，以这些蛋白为对象在体外进行预防和/或治疗埃博拉病毒感染药物的筛选应用中，只要能够上调上述蛋白表达的药物均可以有效地减弱埃博拉病毒感染后细胞变圆脱落的表型，提高细胞的存活率。
4.4体内实验验证微小RNA所调控的靶基因
4.4.1实验材料：除下列试剂外，其余材料同实施例一。
双荧光素酶报告系统(dual luciferae reporter assay system，promega)；人胚肾293T细胞；良伊格尔培养基培养基(Dulbecco’s modified Eagle’s medium，DMEM，Gibco，加入10％(v/v)胎牛血清，青霉素(100U/ml)和链霉素(100mg/ml)；转染试剂：脂质体lipofectamine2000(invitrogen)；双荧光素酶报告质粒psiCHEC-2(上海生工)
4.4.2实验方法
a)种细胞：用DMEM将293T细胞配制成单细胞溶液，种到24孔板中，每孔105/mL个细胞，每孔体积500μl；
b)37℃，5％CO2孵箱培养24小时后转染微小RNA的类似物，对照组转入对照类似物，转染6小时后更换培养基。此处，微小RNA的类似物及对照类似物均为双链RNA序列，其中微小RNA hsa-miR1246、hsa-miR320a、hsa-miR196b-5p和hsa-miR34a-5p的类似物的正义链(sense strand)序列分别为SEQ ID NO.67、SEQ ID NO.68、SEQ ID NO.69以及SEQ ID NO.70，对照类似物的正义链序列为SEQ ID NO.71，所有类似物的反义链序列均与其对应的正义链序列碱基互补配对。
c)转染24h后转染对应的双荧光素酶报告质粒，该质粒同时包含靶基因3’-UTR，海肾荧光报告基因和萤火虫荧光报告基因，其中萤火虫荧光报告基因作为转染效率的内部参考。对照组转入3’-UTR突变质粒，实验组转入3’-UTR野生型质粒，48h后检测海肾荧光素酶活性。微小RNA对应的靶基因的野生型3’-UTR序列与对应的突变型3’-UTR序列信息见表5。
表5
序列号 微小RNA调控的靶基因的3'UTR类型
SEQ ID NO.72 hsa-miR1246-ZCCHC6-野生型
SEQ ID NO.73 hsa-miR1246-ZCCHC6-突变型
SEQ ID NO.74 hsa-miR1246-CFLAR-野生型
SEQ ID NO.75 hsa-miR1246-CFLAR-突变型
SEQ ID NO.76 hsa-miR320-TFPI-野生型
SEQ ID NO.77 hsa-miR320-TFPI-突变型
SEQ ID NO.78 hsa-miR320-CPD-野生型
SEQ ID NO.79 hsa-miR320-CPD-突变型
SEQ ID NO.80 hsa-miR320a-KIAA1429-野生型
SEQ ID NO.81 hsa-miR320a-KIAA1429-突变型
SEQ ID NO.82 hsa-miR320a-DAG1-野生型
SEQ ID NO.83 hsa-miR320a-DAG1-突变型
SEQ ID NO.84 hsa-miR196b-TFPI-野生型
SEQ ID NO.85 hsa-miR196b-TFPI-突变型
SEQ ID NO.86 hsa-miR34-RCAN1-野生型
SEQ ID NO.87 hsa-miR34-RCAN1-突变型
4.4.3实验结果
由于微小RNA主要通过作用于靶基因的3’UTR发挥抑制靶基因的功能，可以将目的基因3’UTR区域构建至载体中报告基因海肾荧光素酶的后面，通过比较干扰微小RNA后，报告基因表达的改变(监测萤光素酶的活性变化)可以定量反映微小RNA对目的基因的抑制作用；结合定点突变等方法可以进一步确定微小RNA与靶基因3’UTR的作用位点。
附图8a和图8b中显示了正义链序列为SEQ ID NO.67的双链hsa-miR-1246的类似物能够很显著地抑制靶基因序列为SEQ ID NO.24的CFLAR和序列为SEQ ID NO.32的ZCCHC6的表达，而当这两个基因的3’-UTR发生突变时，hsa-miR-1246的类似物就不再能抑制这两个基因的表达。同理，正义链序列为SEQ ID NO.68的双链hsa-miR-320a的类似物能够明显抑制序列分别为SEQ ID NO.23的TFPI、SEQ ID NO.14的DAG1、SEQ ID NO.20的CPD以及EQ ID NO.18的KIAA-1429四个基因的表达，而当这四个基因的3’-UTR发生突变时，hsa-miR-320a的类似物对这四个基因的抑制作用就表现的不明显。正义链序列为SEQ ID NO.69的双链hsa-miR196b-5p的类似物能够明显抑制序列为SEQ ID NO.23的TFPI的基因表达，而当TFPI基因的3’-UTR发生突变时，hsa-miR-196b-5p的类似物对这个基因的抑制作用就表现的不明显。正义链序列为SEQ ID NO.70的双链hsa-miR-34a的类似物能明显抑制序列为SEQ ID NO.26的RCAN1基因的表达，而当RCAN1基因3’-UTR发生突变时，hsa-miR-34a的类似物就不能明显表现出对RCAN1基因的抑制作用。
实施例五、确定靶基因与细胞脱落表型之间的关系
5.1用双链寡核苷酸RNA降低靶基因的表达验证靶基因促进细胞脱落方面的作用
5.1.1实验方法
a)将HUVEC用ECM重悬成单细胞溶液，种到96孔板中，每孔105个/ml细胞，每孔体积100μl；
b)37℃，5％CO2孵箱培养24h后转染靶基因DAG1、TFPI以及CFLAR的双链寡核苷酸干扰RNA，对照组转入对照双链寡核苷酸干扰RNA，终浓度100nM，转染4h后更换培养基；
靶基因DAG1中RNA干扰的靶序列为SEQ ID NO.88，其双链寡核苷酸干扰RNA的正义链的序列为SEQ ID NO.89，反义链从3’端到5’端的序列为SEQ ID NO.90(广州锐博合成，产品编号为：siG000001605B)。
靶基因TFPI中RNA干扰的靶序列为SEQ ID NO.91，其双链寡核苷酸干扰RNA的正义链的序列为SEQ ID NO.92，反义链从3’端到5’端的序列为SEQ ID NO.93(广州锐博合成，产品编号为：siG000007035C)。
靶基因CFLAR中RNA干扰的靶序列为SEQ ID NO.94，其双链寡核苷酸干扰RNA的正义链的序列为SEQ ID NO.95，反义链从3’端到5’端的序列为SEQ ID NO.96(广州锐博合成，产品编号为：siG000008837A)。
对照双链寡核苷酸干扰RNA的靶序列为SEQ ID NO.158：TTCTCCGAACGTGTCACGT。正义链序列的末尾两位碱基为dTdT，其余碱基序列SEQ ID NO.159为UUCUCCGAACGUGUCACGU，反义链3’至5’的前两位碱基为dTdT，其余碱基的序列SEQID NO.160为AAGAGGCUUGCACAGUGCA(广州锐博合成)。
其中，上述双链寡核苷酸干扰RNA的正义链和反义链的3’末端的最后两位核苷酸为dTdT序列，以减少细胞内降解。
c)转染24h后加入等量的Ad-GP和Ad-△E1，48h后进行MTS实验检测细胞存活率。
5.1.2实验结果
由于小干扰RNA设计的有效性不能完全预知，部分微小RNA的小干扰RNA容易由于 设计的原因，对靶基因的降低效果不明显，在附图9中没有示出，附图9中仅显示了能明显降低靶基因表达的小干扰RNA及对应的靶基因经过MTS试验后检测到的细胞的存活率。从附图9中可以看出，TFPI、DAG1、CFLAR对应的双链寡核苷酸干扰RNA均能显著降低相应靶基因的表达量，而当上述三个蛋白表达被降低后，相应的细胞的存活率就明显下降了，说明上述6个微小RNA的靶基因中，至少这三个基因的表达显著降低后，相应的Ad-GP组感染后的细胞脱落程度加剧，细胞的存活率下降。
5.2用过量表达靶基因的方法验证靶基因在抑制细胞脱落方面的作用
5.2.1实验材料：除下列材料外，其余同其他实施例
人胚肾293T细胞；良伊格尔培养基(Dulbecco’s modified Eagle’s medium，DMEM，Gibco，加入10％(v/v)胎牛血清，青霉素(100U/ml)和链霉素(100mg/ml)；靶基因过表达质粒分别为：Peak13-TFPI-flag，Peak13-DAG1-flag，Peak13-CFLAR-flag；GP以及GP缺失导致细胞脱落的关键毒性区域的质粒即GP-△mucin表达质粒同实施例一；转染试剂脂质体lipofectamine2000；血球计数板。
5.2.2实验方法
a)用DMEM将293T细胞配成单细胞溶液，种到12孔板，每孔105/mL个细胞，每孔体积1ml；
b)培养24h后共同转染序列为SEQ ID NO.23、SEQ ID NO.14和SEQ ID NO.24的靶基因表达质粒和GP表达质粒或序列为SEQ ID NO.97的GP-△mucin(即GP缺失mucin区域，文献研究报道此区域是GP导致细胞脱落的关键毒性区域)的表达质粒，对照组共同转染peak13-GFP表达质粒和GP表达质粒或序列为SEQ ID NO.97的GP-△mucin表达质粒，4小时后更换培养基；
c)转染48h后对漂浮细胞进行计数分析。
5.2.3实验结果
附图10中显示了靶基因TFPI、DAG1和CFLAR被过量表达后，过表达组的细胞的脱落程度被明显缓解，说明这三个靶基因表达上调后可以明显抑制埃博拉病毒包膜糖蛋白GP导致的细胞变圆脱落的表型，从而提高细胞的存活率。此外，附图11中还显示了不同靶基因相互组合过表达后对细胞的脱落程度的缓解情况，表明这三个靶基因之间的不同组合也能够抑制埃博拉病毒包膜糖蛋白GP导致的细胞变圆脱落的表型，从而提高细胞的存活率。
以上对本发明所提供的用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、DNA分子、蛋白及应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
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Claims (19)

  1. 一种用于预防和/或治疗埃博拉病毒性出血热的小核酸分子，其特征在于，所述小核酸分子为埃博拉病毒感染细胞差异化表达的微小RNA的抑制物和/或所述微小RNA前体的抑制物。
  2. 根据权利要求1所述的小核酸分子，其特征在于，所述小核酸分子是序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.6的微小RNA及其前体的抑制物。
  3. 根据权利要求1或2所述的小核酸分子，其特征在于，所述小核酸分子的序列与SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或者SEQ ID NO.12具有80％以上的同源性，优选具有95％以上的同源性，更优选地，所述小核酸分子的序列为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、或者SEQ ID NO.12。
  4. 一种权利要求1至3中任一项所述的小核酸分子在制备用于预防和/或治疗鄂木斯克出血热、基萨那森林病、克里米亚出血热、新疆出血热、登革出血热、基孔肯雅热、黄热病、阿根廷出血热、玻利维亚出血热、拉沙热、流行性出血热或者马尔堡病的药物中的应用。
  5. 一种用于预防和/或治疗埃博拉病毒性出血热的药物，其特征在于，所述药物包括治疗有效量的权利要求1至3中任一项所述的小核酸分子。
  6. 根据权利要求5所述的药物，其特征在于，所述药物进一步包括一种或多种用于预防和/或治疗埃博拉病毒性出血热有效物质，优选地，所述用于预防和/或治疗埃博拉病毒性出血热有效物质选自抗病毒类药物、免疫提升类药物及皮质激素类药物。
  7. 一种权利要求1至3中任一项所述的小核酸分子在体外抑制埃博拉病毒感染细胞生长脱落的应用，其特征在于，在细胞感染埃博拉病毒之前或之后，将所述小核酸分子转染至所述细胞中。
  8. 一种权利要求1至3中任一项所述的小核酸分子在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用，其特征在于，将所述小核酸分子与待测药物进行核酸同源性比对。
  9. 一种DNA分子，其特征在于，所述DNA分子在埃博拉病毒感染细胞中的表达水平能被权利要求1至3中任一项所述的一种或多种小核酸分子上调。
  10. 根据权利要求9所述的DNA分子，其特征在于，所述DNA分子的序列与SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32具有80％以上的同源性，优选具有95％以上 的同源性，更优选地，所述DNA分子的序列为SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31或者SEQ ID NO.32。
  11. 根据权利要求10所述的DNA分子，其特征在于，所述DNA分子为序列为SEQ ID NO.14、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.26、SEQ ID NO.27或者SEQ ID NO.32，优选序列为SEQ ID NO.14、SEQ ID NO.23或者SEQ ID NO.24。
  12. 一种权利要求9至11中任一项所述的DNA分子在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用。
  13. 一种权利要求9至11中任一项所述DNA分子编码的蛋白，其特征在于，所述蛋白的序列与SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ ID NO.52具有80％以上的同源性，优选具有95％以上的同源性，更优选地，所述蛋白的序列为SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51或者SEQ ID NO.52。
  14. 根据权利要求13所述的蛋白，其特征在于，所述蛋白的分子序列为SEQ ID NO.34、SEQ ID NO.38、SEQ ID NO.40、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.46、SEQ ID NO.47或者SEQ ID NO.52，优选序列为SEQ ID NO.34、SEQ ID NO.43或者SEQ ID NO.44。
  15. 一种权利要求13或14所述的蛋白在体外筛选用于预防和/或治疗埃博拉病毒性出血热的药物中的应用。
  16. 一种用于检测埃博拉病毒性出血热的试剂盒，其特征在于，所述试剂盒包括可特异性扩增权利要求1至3中任一项所述的小核酸分子所抑制的微小RNA的引物。
  17. 根据权利要求16所述的试剂盒，其特征在于，
    当所述微小RNA的序列为SEQ ID NO.1时，逆转录引物序列为SEQ ID NO.140：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCTGCT，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.146：CCGCGCAATGGATTTTT，RP序列为SEQ ID NO.147：GTGCACGCTCCGAGGT；
    当所述微小RNA的序列为SEQ ID NO.2时，逆转录引物序列为SEQ ID NO.141：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACCCCAAC，荧光实时定量PCR所用到的 引物：FP序列为SEQ ID NO.148：CCGCGCTAGGTAGTTTCCT，RP序列为SEQ ID NO.149：GTGCACGCTCCGAGGT；
    当所述微小RNA的序列为SEQ ID NO.3时，逆转录引物序列为SEQ ID NO.142：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTCGCCC，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.150：TCGCGCAAAAGCTGG，RP序列为SEQ ID NO.151：GTGCACGCTCCGAGGT；
    当所述微小RNA的序列为SEQ ID NO.4时，逆转录引物序列为SEQ ID NO.143：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACTAGA，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.152：TCGCGCTCGAGGAGC，RP序列为SEQ ID NO.153：GTGCACGCTCCGAGGT；
    当所述微小RNA的序列为SEQ ID NO.5时，逆转录引物序列为SEQ ID NO.144：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACAACC，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.154：TCGCGCTGGCAGTGT，RP序列为SEQ ID NO.155：GTGCACGCTCCGAGGT；
    当所述微小RNA的序列为SEQ ID NO.6时，逆转录引物序列为SEQ ID NO.145：GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACTGCTGT，荧光实时定量PCR所用到的引物：FP序列为SEQ ID NO.156：TCGCGCTGCGGG，RP序列为SEQ ID NO.157：GTGCACGCTCCGAGGT。
  18. 一种用于检测埃博拉病毒性出血热的试剂盒，其特征在于，所述试剂盒包括可特异性扩增权利要求9至11中任一项所述DNA分子的引物。
  19. 根据权利要求18所述的试剂盒，其特征在于，
    当所述DNA分子的序列为SEQ ID NO.13时，所述引物的FP序列为SEQ ID NO.100：AAGCAGGATCTTCTAAGGTT，RP序列为SEQ ID NO.101：AGTGTTGTTCTTGGTCTCTC；
    当所述DNA分子的序列为SEQ ID NO.14时，所述引物的FP序列为SEQ ID NO.102：GTCACTTGCTTCCTTACTTAG，RP序列为SEQ ID NO.103：TGAGCCATAACCACAGAG；
    当所述DNA分子的序列为SEQ ID NO.15时，所述引物的FP序列为SEQ ID NO.104：ACCAATCAGTGCTCAGTAT，RP序列为SEQ ID NO.105：TCAACCTCCACATCTCTATC；
    当所述DNA分子的序列为SEQ ID NO.16时，所述引物的FP序列为SEQ ID NO.106：TGGTCATCATCTGCTTGTG，RP序列为SEQ ID NO.107：TTCCTATTCGGTCACTCTCT；
    当所述DNA分子的序列为SEQ ID NO.17时，所述引物的FP序列为SEQ ID NO.108：GGTAGAACTGAACAACGATAG，RP序列为SEQ ID NO.109：CAGCAATGGTATAGCAACTT；
    当所述DNA分子的序列为SEQ ID NO.18时，所述引物的FP序列为SEQ ID NO.110：CGACGAACAGTAGACAGTATT，RP序列为SEQ ID NO.111：CAGCAATTCCATCTTCATCACT；
    当所述DNA分子的序列为SEQ ID NO.19时，所述引物的FP序列为SEQ ID NO.112：CTAATTCTCCAGCCTCATTG，RP序列为SEQ ID NO.113：ATGGTAATCGTCCGTTCA；
    当所述DNA分子的序列为SEQ ID NO.20时，所述引物的FP序列为SEQ ID NO.114：ACCATTAGTGTTGCTGAGA，RP序列为SEQ ID NO.115：GTGTGAAGTTGACCTGAATAG；
    当所述DNA分子的序列为SEQ ID NO.21时，所述引物的FP序列为SEQ ID NO.116：GATTCTCTTCCACAGACTATATG，RP序列为SEQ ID NO.117：CCTTCCTCATCCAGTTCATG；
    当所述DNA分子的序列为SEQ ID NO.22时，所述引物的FP序列为SEQ ID NO.118：TTCATCTCCGCTTCTTGTG，RP序列为SEQ ID NO.119：TGTTCTTCAGTGAGTTCCTT；
    当所述DNA分子的序列为SEQ ID NO.23时，所述引物的FP序列为SEQ ID NO.120：GCCTGCTGCTTAATCTTG，RP序列为SEQ ID NO.121：CATCATCCGCCTTGAATG；
    当所述DNA分子的序列为SEQ ID NO.24时，所述引物的FP序列为SEQ ID NO.122：AGGCAAGATAAGCAAGGAGAAG，RP序列为SEQ ID NO.123：AGGTCTATTCTGTGGATGTTCT；
    当所述DNA分子的序列为SEQ ID NO.25时，所述引物的FP序列为SEQ ID NO.124：GAACCTCAGTGCCTACAACAC，RP序列为SEQ ID NO.125：GGAAGTCACCTCAGAGTCCAG；
    当所述DNA分子的序列为SEQ ID NO.26时，所述引物的FP序列为SEQ ID NO.126：TTGCTCAGACCTTACACATAG，RP序列为SEQ ID NO.127：GCTTGGAGATGGCATATAAGA；
    当所述DNA分子的序列为SEQ ID NO.27时，所述引物的FP序列为SEQ ID NO.128：CAGAACATACCACGACAAG，RP序列为SEQ ID NO.129：CTGGAAGGAGTAGAGGATGT；
    当所述DNA分子的序列为SEQ ID NO.28时，所述引物的FP序列为SEQ ID NO.130：GAGCCTGAGACTTGTGTATC，RP序列为SEQ ID NO.131：CGTATTCTGGGAAGTGTTG；
    当所述DNA分子的序列为SEQ ID NO.29时，所述引物的FP序列为SEQ ID NO.132：GGCGTGGTATCTGTCTGT，RP序列为SEQ ID NO.133：CTGTTGTGTCATCCTGTTC；
    当所述DNA分子的序列为SEQ ID NO.30时，所述引物的FP序列为SEQ ID NO.134：TATCTGAAGGACGGTTCTGG，RP序列为SEQ ID NO.135：TTATCTCTGAGGTGGCATAC；
    当所述DNA分子的序列为SEQ ID NO.31时，所述引物的FP序列为SEQ ID NO.136：GGTTGGTGTTGATTCAGAC，RP序列为SEQ ID NO.137：GCAGAGGCTTCAGATAGT；
    当所述DNA分子的序列为SEQ ID NO.32时，所述引物的FP序列为SEQ ID NO.138：ATTAGCAAGAGTCCTCAGA，RP序列为SEQ ID NO.139：ATGGCGGAATAAGCAGATA。
PCT/CN2015/072624 2014-03-25 2015-02-10 用于预防和/或治疗埃博拉病毒性出血热的小核酸分子、dna分子、蛋白及应用 WO2015143958A1 (zh)

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CN106589079B (zh) * 2016-11-25 2020-03-10 武汉大学 一种多肽s1及其在制备治疗脂肪肝和/或ⅱ型糖尿病药物中的应用
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102834513A (zh) * 2008-03-05 2012-12-19 拜奥克里斯特制药公司 抗病毒治疗剂
WO2013036993A1 (en) * 2011-09-13 2013-03-21 Commonwealth Scientific And Industrial Research Organisation Detection of viral infection
CN103068835A (zh) * 2010-06-06 2013-04-24 西奈山医学院 重组rna 病毒及其用途
CN103940998A (zh) * 2014-05-04 2014-07-23 山东大学 血清microRNA作为肝细胞癌转移的早期诊断标志物的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2666481T3 (en) * 2003-12-26 2019-02-25 Masatoshi Hagiwara A method for regulating the phosphorylation of SR protein and antiviral agents comprising an SR protein activity regulator as the active ingredient
WO2007084488A2 (en) * 2006-01-13 2007-07-26 Vanderbilt University Adam10 and its uses related to infection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102834513A (zh) * 2008-03-05 2012-12-19 拜奥克里斯特制药公司 抗病毒治疗剂
CN103068835A (zh) * 2010-06-06 2013-04-24 西奈山医学院 重组rna 病毒及其用途
WO2013036993A1 (en) * 2011-09-13 2013-03-21 Commonwealth Scientific And Industrial Research Organisation Detection of viral infection
CN103940998A (zh) * 2014-05-04 2014-07-23 山东大学 血清microRNA作为肝细胞癌转移的早期诊断标志物的应用

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