CN104789641A - Animal response type gene under microbial infection, identification and applications thereof - Google Patents
Animal response type gene under microbial infection, identification and applications thereof Download PDFInfo
- Publication number
- CN104789641A CN104789641A CN201410020196.0A CN201410020196A CN104789641A CN 104789641 A CN104789641 A CN 104789641A CN 201410020196 A CN201410020196 A CN 201410020196A CN 104789641 A CN104789641 A CN 104789641A
- Authority
- CN
- China
- Prior art keywords
- mirna
- animal
- infected
- response type
- microbes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 60
- 230000004044 response Effects 0.000 title claims abstract description 54
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 33
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 30
- 230000000813 microbial effect Effects 0.000 title abstract description 7
- 239000002679 microRNA Substances 0.000 claims abstract description 208
- 108091070501 miRNA Proteins 0.000 claims abstract description 200
- 238000000034 method Methods 0.000 claims abstract description 60
- 238000012360 testing method Methods 0.000 claims abstract description 38
- 238000002474 experimental method Methods 0.000 claims abstract description 11
- 239000012805 animal sample Substances 0.000 claims abstract description 10
- 108700005443 Microbial Genes Proteins 0.000 claims abstract description 4
- 244000005700 microbiome Species 0.000 claims description 26
- 230000000295 complement effect Effects 0.000 claims description 22
- 239000002773 nucleotide Substances 0.000 claims description 21
- 125000003729 nucleotide group Chemical group 0.000 claims description 21
- 201000010099 disease Diseases 0.000 claims description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 19
- 239000003112 inhibitor Substances 0.000 claims description 15
- 230000033228 biological regulation Effects 0.000 claims description 8
- 238000001228 spectrum Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000000556 agonist Substances 0.000 claims description 4
- 208000024891 symptom Diseases 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 210000004102 animal cell Anatomy 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 46
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 35
- 239000000523 sample Substances 0.000 description 30
- 108700011259 MicroRNAs Proteins 0.000 description 19
- 108091028066 Mir-126 Proteins 0.000 description 17
- 108091053257 miR-99b stem-loop Proteins 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 16
- 108091034117 Oligonucleotide Proteins 0.000 description 15
- 210000004443 dendritic cell Anatomy 0.000 description 15
- 239000000074 antisense oligonucleotide Substances 0.000 description 13
- 238000012230 antisense oligonucleotides Methods 0.000 description 13
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 230000008569 process Effects 0.000 description 12
- 241000709675 Coxsackievirus B3 Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 108020004999 messenger RNA Proteins 0.000 description 11
- 239000002243 precursor Substances 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 238000012986 modification Methods 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 108020004707 nucleic acids Proteins 0.000 description 10
- 102000040430 polynucleotide Human genes 0.000 description 10
- 108091033319 polynucleotide Proteins 0.000 description 10
- 239000002157 polynucleotide Substances 0.000 description 10
- 238000003753 real-time PCR Methods 0.000 description 10
- 241000700605 Viruses Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 108091027963 non-coding RNA Proteins 0.000 description 9
- 102000042567 non-coding RNA Human genes 0.000 description 9
- 239000008194 pharmaceutical composition Substances 0.000 description 9
- 102000039634 Untranslated RNA Human genes 0.000 description 8
- 108020004417 Untranslated RNA Proteins 0.000 description 8
- 108010067390 Viral Proteins Proteins 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 238000010276 construction Methods 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 230000001717 pathogenic effect Effects 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 102000042773 Small Nucleolar RNA Human genes 0.000 description 7
- 108020003224 Small Nucleolar RNA Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 108091044988 miR-125a stem-loop Proteins 0.000 description 6
- 108091032320 miR-146 stem-loop Proteins 0.000 description 6
- 108091024530 miR-146a stem-loop Proteins 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 5
- 102000039471 Small Nuclear RNA Human genes 0.000 description 5
- 108020004566 Transfer RNA Proteins 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 201000008827 tuberculosis Diseases 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- -1 comprising rRNA Proteins 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 208000030507 AIDS Diseases 0.000 description 3
- 108091032955 Bacterial small RNA Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 108091092195 Intron Proteins 0.000 description 3
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- 108020004688 Small Nuclear RNA Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000003278 mimic effect Effects 0.000 description 3
- 238000002966 oligonucleotide array Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000712461 unidentified influenza virus Species 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241001646725 Mycobacterium tuberculosis H37Rv Species 0.000 description 2
- 108700035964 Mycobacterium tuberculosis HsaD Proteins 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 102000004598 Small Nuclear Ribonucleoproteins Human genes 0.000 description 2
- 108010003165 Small Nuclear Ribonucleoproteins Proteins 0.000 description 2
- 108091032917 Transfer-messenger RNA Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 230000000721 bacterilogical effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000007621 cluster analysis Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 210000003606 umbilical vein Anatomy 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 235000012571 Ficus glomerata Nutrition 0.000 description 1
- 244000153665 Ficus glomerata Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108091092238 Homo sapiens miR-146b stem-loop Proteins 0.000 description 1
- 108091069088 Homo sapiens miR-150 stem-loop Proteins 0.000 description 1
- 108091065981 Homo sapiens miR-155 stem-loop Proteins 0.000 description 1
- 108091070395 Homo sapiens miR-31 stem-loop Proteins 0.000 description 1
- 108091067008 Homo sapiens miR-342 stem-loop Proteins 0.000 description 1
- 241000589248 Legionella Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091028049 Mir-221 microRNA Proteins 0.000 description 1
- 108091060585 Mir-31 Proteins 0.000 description 1
- 206010062207 Mycobacterial infection Diseases 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 1
- 101150034459 Parpbp gene Proteins 0.000 description 1
- 108091007412 Piwi-interacting RNA Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002350 accommodative effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000011278 co-treatment Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- YQYJSBFKSSDGFO-FWAVGLHBSA-N hygromycin A Chemical compound O[C@H]1[C@H](O)[C@H](C(=O)C)O[C@@H]1Oc1ccc(\C=C(/C)C(=O)N[C@@H]2[C@@H]([C@H]3OCO[C@H]3[C@@H](O)[C@@H]2O)O)cc1O YQYJSBFKSSDGFO-FWAVGLHBSA-N 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- XBCXJKGHPABGSD-UHFFFAOYSA-N methyluracil Natural products CN1C=CC(=O)NC1=O XBCXJKGHPABGSD-UHFFFAOYSA-N 0.000 description 1
- 108091027034 miR-148a stem-loop Proteins 0.000 description 1
- 108091080321 miR-222 stem-loop Proteins 0.000 description 1
- 108091088477 miR-29a stem-loop Proteins 0.000 description 1
- 108091029716 miR-29a-1 stem-loop Proteins 0.000 description 1
- 108091092089 miR-29a-2 stem-loop Proteins 0.000 description 1
- 108091066559 miR-29a-3 stem-loop Proteins 0.000 description 1
- 108091046889 miR-710 stem-loop Proteins 0.000 description 1
- 108091058491 miR-877 stem-loop Proteins 0.000 description 1
- 108091032373 miR-881 stem-loop Proteins 0.000 description 1
- 108091086591 miR-882 stem-loop Proteins 0.000 description 1
- 108091007426 microRNA precursor Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 210000004332 phalangeal cell Anatomy 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 206010047470 viral myocarditis Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to an animal response type gene under microbial infection, identification and applications thereof, and particularly provides a method for non-diagnostic identification of animal body miRNA. The method comprises: (a) providing a test group and a control group, wherein the test group is the animal individual, the animal sample or the animal cells being subjected to any one microbial infection; (b) comparing the expression profiles of the miRNA derived from the animals in the test group and the control group so as to identify the response type miRNA; (c) comparing the identified response type miRNA and the microbial gene database so as to determine the type of the response type miRNA having the target relationship with the microbial; and (d) verifying the regulating relation on the type of the response type miRNA having the target relationship through experiments.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to the response type gene of animal under infected by microbes and qualification thereof and application.
Background technology
Microorganism (Microorganism) is that to be extensively present in a group of occurring in nature invisible, must amplify hundreds of times, thousands of times even general name of the tens thousand of times of tiny organisms just can observed by opticmicroscope or electron microscope.They have that the bodily form is small, structure is simple, breeding rapidly, easily variation and the feature such as accommodative ability of environment is strong.
Microbe species is various, very extensive in the distribution of occurring in nature.In the body surface of the mankind, animal and plant and the cavity that communicates with the external world thereof, also there is multiple-microorganism to exist.
Microorganism mainly comprises: bacterium, archeobacteria, protobiont, virus etc.
Microorganism is cause some disease on one of most important impact of the mankind, especially transmissible disease.Although in prevention and treatment of diseases, the mankind make significant progress, and new infected by microbes that is existing and that reproduce still constantly occurs, and numerous disease lacks effective medicine always.In addition, the abuse of a large amount of Broad spectrum antibioticss causes powerful selective pressure, and many bacterial strains are morphed, and causes the generation endangering larger endurance strain.
Infected by microbes (Microbial infection) is the disease that clinical common microorganism causes.Microorganism enters in human or animal body by oral cavity, skin or respiratory tract, utilizes the breeding of nourishing and growing of human or animal body.If these bacteriums produce toxin, ill will be made, as cholera, typhoid fever, pneumonia etc.The disease such as acquired immune deficiency syndrome (AIDS), flu is then caused by virus.The diagnosis of bacteriosis, the overwhelming majority all needs to carry out bacteriodiagnosis with the clear and definite cause of disease.But in sample, be separated to bacterium might not mean that this bacterium is the cause of disease of disease, therefore comprehensively should analyze according to the bacterial species of the position of the clinical setting of patient, collect specimen, acquisition.Sometimes tests such as doing toxicity, cell and animal is still needed to determine the pathogenic of this bacterial strain.
At present, it is generally acknowledged, the process of common infection is as follows: activate pathogen-associated molecular path, as cytolemma is correlated with Toll-like receptor and tenuigenin Nod sample acceptor, activating cells Nuclear Factor kappa B subsequently, and then activate congenital and acquired immunity, finally eliminate the cause of disease be detected, form the long-term immunizing power for future infection.
Infectious diseases, particularly Chronic persistent infect, and are one of global persistent ailments of harm humans health and life.The mankind are had to the intra-cellular pathogens of material impact, comprise mycobacterium tuberculosis, Salmonellas, brucella, legionella pneumophilia, listeria bacteria and leprosy bacillus etc.Seven serious diseases such as tuberculosis, AIDS, hepatitis form the disease of global threat, and born of the same parents' endoparasitism of the reason that its treatment is difficult and pathogenic agent is closely related.
A difficult problem for the treatment of infected by microbes in born of the same parents.Major cause comprises: invade when these pathogenic agent and hide in cell, and conventional microbiotic is difficult to be transported in the cell that bacterium hides, even and if be transported to, concentration is also difficult to reach effective sterilization effect; Antibody can not enter in cell and play a role; When in born of the same parents bacterium success parasitic cell after, immunity of can not only escaping is engulfed, kills and wounds and is removed, and can in cell long-term existence, in due course machine (as immunity of organisms decline) cause body to be fallen ill.
Therefore, for infected by microbes disease, this area is in the urgent need to developing new Diagnosis and Treat technology.
Summary of the invention
Object of the present invention is just to provide a kind of therapeutic substance that can be used for treating infected by microbes, and differentiates the method for described therapeutic substance.
In a first aspect of the present invention, provide a kind of method that nondiagnostic differentiates animal body miRNA, comprise step:
A () provides a test group and a control group, wherein said test group is be subject to the animal individual of a certain infected by microbes, animal sample or zooblast; A described control group is that experiment condition is identical but not by the animal individual of described infected by microbes, animal sample or zooblast;
The express spectra of the miRNA of described animal is derived from (b) more described test group and control group, thus identify compared with described control group, there is the miRNA kind of significant difference in expression level in described test group, be decided to be response type miRNA (responding miRNA), wherein, described response type miRNA comprises: express the rise type miRNA and/or the remarkable downward type miRNA lowered of expression that significantly raise.
In another preference, in step (c), described response type miRNA kind expresses the rise type miRNA significantly raised.
In another preference, in step (d), described regulation relationship refers to the downward of described miRNA to described microorganism target gene.
In another preference, in step (c), described response type miRNA kind expresses the downward type miRNA significantly lowered.
In another preference, in step (d), described regulation relationship refers to the downward of described miRNA to described microorganism target gene.
At another preference, described remarkable rise refers to for a concrete miRNA, E1/E0 >=1.5, preferably >=2, more preferably >=2.5, best >=4, wherein E1 is the expression amount of described miRNA kind in described test group (level), and E0 is the expression amount of identical miRNA kind in described control group (level); And/or
Described remarkable downward refers to for a concrete miRNA, E1/E0≤0.75, preferably≤0.67, more preferably≤0.5, best≤0.25, wherein E1 is the expression amount of described miRNA kind in described test group (level), and E0 is the expression amount of identical miRNA kind in described control group (level).
In another preference, described method also comprises step:
C response type miRNA kind that () will identify, compare with the gene database of described microorganism and/or the gene database of described animal, thus determine the response type miRNA kind that there is target relation with described microbial gene and/or described animal gene, wherein said target relation represents for a certain specific miRNA kind X, it is complete or substantially the same with one section of nucleotide sequence section of the gene Y of described microorganism or the gene Ya of described animal, or completely or substantially complementary;
D () is for the described response type miRNA kind that there is target relation, verify the regulation relationship of described miRNA kind X and described gene Y or Ya (target gene) by experiment, if wherein the result is positive, then differentiate that described animal body miRNA is the response type miRNA participating in infected by microbes; If the result is negative, then differentiate that described animal body miRNA is the response type miRNA not participating in infected by microbes.
In another preference, described test group comprises the first test subgroup and/or the second test subgroup, wherein the first test subgroup is subject to infected by microbes and shows the animal individual of infection symptoms, animal sample or zooblast, and the second test subgroup is subject to infected by microbes and does not show the animal individual of infection symptoms, animal sample or zooblast.
In another preference, described method also comprises step:
E (), to the miRNA identified, tests the ability that it resists described infected by microbes.
In a second aspect of the present invention, provide the miRNA that described in a kind of first aspect present invention, method identifies.
In another preference, described miRNA comprises the rise type miRNA and/or the remarkable downward type miRNA lowered of expression that express and significantly raise.
In 3rd of the present invention, provide a kind of composition, described composition contains (a) antisense sequences of acceptable carrier and the miRNA described in (a) second aspect present invention or its agonist or described miRNA or inhibitor pharmaceutically or on food.
In another preference, described composition is pharmaceutical composition.
In another preference, when described miRNA is rise type miRNA, described pharmaceutical composition contains antisense sequences or the inhibitor of described miRNA.
In another preference, when described miRNA is downward type miRNA, described pharmaceutical composition contains described miRNA or its agonist.
In another preference, whether or the reagent of infected by microbes relative disease, chip or test kit the purposes of miRNA as described in respect of the second aspect of the invention, for the preparation of detecting infected by microbes.
In another preference, described reagent comprises primer, probe or its combination.
In another preference, described microorganism comprises pathogenic microorganism, as bacterium, virus, chlamydozoan, mycoplasma etc.; Preferably, described microorganism comprises (but being not limited to): influenza virus, parainfluenza virus, Salmonellas, gonococcus, avian influenza virus etc.
In another preference, described avian influenza virus is H5N1.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the detected result of miRNA in the dendritic cell of m tuberculosis infection.
Fig. 2 shows miR-146a, miR-125a-5p and miR-99b expression of results in the dendritic cell be infected by bacterial.
Fig. 3 shows miR-99b inhibitor and the expression of results of miR-99b process LAN in the dendritic cell be infected by bacterial.
Fig. 4 shows and suppresses miR-99b can significantly reduce H37Rv level.
Fig. 5 shows the check result of embodiment 2, and 41 kinds of miRNAs expression in infection sample and normal healthy controls have significant difference.
Fig. 6 shows the cluster analysis result in embodiment 2, utilizes the multiple response miRNAs shown in figure infection sample and normal healthy controls can be distinguished completely.
Fig. 7 shows microspur battle array and Real-time PCR records Comparative result to miRNA expression amount.
Fig. 8 shows the ROC tracing analysis situation to response miRNA checking in embodiment 2.
Fig. 9 shows the detected result of miR-126 in HeLa cell, HL-1 cell and HUVEC cell.
Figure 10 shows and uses CVB3 cells infected after 7 hours, uses western blot method method to detect the result of viral protein (VP-1) content.
Figure 11 shows response miRNA inhibitor can reduce virus activity.
In above-mentioned each figure, " * " or " * * " represents the difference (p<0.05) of the remarkable or highly significant existed statistically.
Embodiment
The present inventor, through extensive and deep research, is surprised to find that first, and animal body is when being subject to pathogenic infection, and animal body can respond to infected by microbes, causes the miRNA of animal body self to raise or lowers (being generally response type miRNA).When the non-coding RNA (non-coding RNA, ncRNA) (comprising miRNA) of animal release, these specific ncRNA can be used as the main or complementary biomarker detecting infected by microbes.In addition, some response type miRNA also participates in the antagonistic process of animal body to infected by microbes, contributes to zooblast and resists the infection that microorganism causes.Complete the present invention on this basis.
Term
As used herein, term " microorganism " comprises the various tiny organisms such as virus, bacterium, chlamydozoan.In the present invention, " internal microorganism " refers to be present in the various microorganisms in host (people or other animals) body, comprises microbial infection, parasitic microbe and symbiotic microorganism.The internal microorganism of one quasi-representative is the pathogenic agent that can cause disease.
In the present invention, according to the relation of pathogenic agent and host cell, pathogenic agent can be divided into bacterium two class in extracellular bacteria and born of the same parents.Extracellular bacteria refers to the bacterium of growth and breeding in the body fluid such as intercellular substance, blood, lymph liquid, tissue juice outside host cell.In born of the same parents, bacterium is divided into again two kinds: in facultative born of the same parents, bacterium refers in host, mainly to reside in Intracellular growth breeding, externally also can grow without in the substratum of viable cell.No matter obligate intracellular bacterial is then external in vivo, all must in viable cell growth and breeding.
As used herein, term " milRNA " i.e. microRNA like, refers to the RNA sequence being similar to microRNA.
As used herein, term " sRNA " i.e. small RNA, refers to small noncoding RNA sequence.In the present invention, sRNA comprises microRNA and milRNA.
Non-coding RNA
Non-coding RNA (Non-coding RNA, ncRNA), refer to the RNA not being translated into protein, comprising rRNA, tRNA, snRNA, the various different RNA such as snoRNA and microRNA, these RNA transcribe from genome, do not translate into albumen, but participate in protein translation process, rna level just can exercise respective biological function.Such as, snRNA, snoRNA participate in RNA montage and RNA modification.
Non-coding RNA divides from length can be divided into 3 classes: be less than 50nt, comprise microRNA, siRNA, piRNA; 50nt to 500nt, comprises rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA etc.; Be greater than 500nt, comprise the non-coding RNA of long mRNA-like, the long non-coding RNA etc. not with polyA tail.
SnRNA is the abbreviation of small nuclear RNA, also referred to as small nuclear rna.Its function is combined with protein factor to form small nut nucleoglucoprotein particle (small nuclear ribonucleo-protein partcle is called for short snRNPs), exercises the function of montage mRNA.
SnoRNA is the tiny RNA found as far back as kernel, is called little nucleolar RNA, and initial their biological function of discovery is used to modify rRNA's.Most of little nucleolar RNA can be divided into two classes.One class is C Dbox snoRNA, this class snoRNA is methylate to the base of RNA to modify.Another kind of is H/ACA box, and this class snoRNA carries out methyluracil modification to the base of RNA.Be characterized in the two stem of formation one, centre adds a loop district, has a boxH in middle loop district.And have individual boxACA at the tail of afterbody.Due to boxH and boxACA primary sequence feature define more loose.
MiRNA is that little RNA molecule and open gene are complementary, mediated gene silencing.MicroRNA is the tiny RNA of a class 21-23nt, its precursor the chances are about 70-100nt, forms stem (stem) structure of standard, becomes the single stranded RNA of 21-23nt after processing.The mechanism of action of microRNA be and mRNA complementary, allow the reticent or degraded of mRNA.Based on the RNAi technology that miRNA mechanism is developed, the small RNA of similar microRNA is utilized to carry out the mRNA of reticent correspondence exactly.
GRNA, also known as guiding RNA, refers to the RNA with mRNA complementary sequence that has participating in rna editing in eukaryote.
ERNA, from the RNA molecule that intron (introns) or noncoding DNA are transcribed, transcribing and translation efficiency of finely regulating gene.
Srp rna, identifies in phalangeal cell matter with containing signal peptide mRNA, determines the RNA functional molecular of secretion.
PRNA, refers to phage rna.Such as, research shows, utilizes ATP to participate in packing of DNA in fi29 phage with 6 same small RNA moleculars.
TmRNA, refers to the RNA with tRNA sample and mRNA sample compound.TmRNA extensively exists in bacterium, the rrna that identification is translated or reading code is wrong, also identifies that those postpone the rrna of stalls, mediates these problematic ribosomal disintegrations.
In addition, the non-translational region in mRNA, comprises intron region and rrna recognition component as 5'-UTR, 3'-UTR etc., also can regard non-coding RNA as.
Mechanism
For the ease of understanding the present invention, the present inventor provides following mechanism.However, it should be understood that these mechanism and be not used in and any restriction is produced to the present invention.
Research of the present invention discloses, and after infected by microbes animal body (as people), animal body can respond or reply, and causes the rise of some miRNA kind or downward (being commonly referred to as " response type miRNA ").Such as, in many cases, animal body can be secreted or raise some self miRNA and enter the recycle system, thus the infection of response (comprise opposing or assist (comprising passive assistance)) specified microorganisms, therefore, these responses type miRNA can be used as the diagnosis marker of infected by microbes disease.
In addition, for the response type miRNA that can resist or resist infected by microbes, also can be used as active constituents of medicine.
MiRNA and precursor thereof
The invention provides a class new, the miRNA that comes from animal body.As used herein, described " miRNA " refers to a kind of RNA molecule, from the transcript processing that can form miRNA precursor.Ripe miRNA has 18-26 Nucleotide (nt) (more particularly about 19-22nt) usually, does not also get rid of the miRNA molecule with other number Nucleotide.MiRNA can be detected by Northern trace usually.
As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.
MiRNA can process from precursor miRNA (Precursor miRNA, Pre-miRNA), and described precursor miRNA can be folded into a kind of stable stem ring (hair clip) structure, and described loop-stem structure length is generally between 50-100bp.Described precursor miRNA can be folded into stable loop-stem structure, and the stem both sides of loop-stem structure comprise substantially complementary two sequences.Described precursor miRNA can be natural or synthetic.
Precursor miRNA can be sheared generation miRNA, described miRNA can be substantially complementary with the sequence at least partially of the mRNA of encoding gene.As used herein, " substantially complementary " refers to that the sequence of Nucleotide is enough complementary, can interact, as formed secondary structure (as loop-stem structure) in the foreseeable mode of one.Usually, the nucleotide sequence of two " substantially complementary " mutually between have at least the Nucleotide of 70% to be complementary; Preferably, the Nucleotide of 80% is had at least to be complementary; Preferred, have at least the Nucleotide of 90% to be complementary; Preferred further, have at least the Nucleotide of 95% to be complementary; As 98%, 99% or 100%.Usually, maximum 40 unmatched Nucleotide can be had between two enough complementary molecules; Preferably, there are maximum 30 unmatched Nucleotide; Preferred, there are maximum 20 unmatched Nucleotide; Preferred further, there are maximum 10 unmatched Nucleotide, as having 1,2,3,4,5,8 unmatched Nucleotide.
As used herein, " stem ring " structure is also referred to as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form the secondary structure that one comprises double-stranded region (stem), described double-stranded region is formed by two regions (being positioned on same a part) of this nucleic acid molecule, the both sides of two region apportion double stranded section; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. single-stranded regions.Even if two of this nucleic acid molecule regions are not complete complementaries, the double stranded section of Nucleotide also can keep double-stranded state.Such as, insertion, disappearance, replacement etc. can cause the not complementary of a zonule or this zonule self to form the secondary structure of loop-stem structure or other form, but these two regions still can be substantially complementary, and interact in foreseeable mode, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, usually obtain one there is the nucleic acid of the nucleotide sequence of primary structure after, those skilled in the art can determine whether this nucleic acid can form loop-stem structure.
Antisense oligonucleotide
According to miRNA sequence provided by the present invention, can have devised its antisense oligonucleotide, described antisense oligonucleotide can lower the expression of corresponding miRNA in vivo.As used herein, " antisense oligonucleotide (antisense-oligonucleotides; AS-Ons or ASO) " is also called " antisense nucleotide ", refers to that length is about the DNA molecular of 18-26nt (more particularly about 19-22nt) or RNA molecule or its analogue.
In the present invention, described " antisense oligonucleotide " also comprises the modified antisense nucleotide adopted as obtained based on means such as nucleic acid lock or nucleic acid chains backbone modification technology, described modification does not change the activity of antisense oligonucleotide substantially, more preferably, described modification can improve the stability of antisense oligonucleotide, activity or result for the treatment of.Nucleic acid lock (locked nucleic acid, LNA) typically refers to the modification technique 2' Sauerstoffatom of ribose and 4' carbon atom coupled together by a methylene bridge.LNA can extend the serum half-life of miRNA, improves target affinity, reduces scope and the degree of the effect of missing the target.The antisense drug developed based on the modification technique of nucleic acid chains skeleton is in solubility, and the aspects such as nuclease-resistant degraded are improved greatly, and are easy to a large amount of synthesis.The backbone modification method of oligonucleotide has multiple, comprises sulfo-method, such as, be sulfo-deoxynucleotide chain by deoxynucleotide chain thio-modification.The method is substituted by the Sauerstoffatom sulphur atom of the phosphate bond on DNA skeleton, can resist nuclease degradation.Should be understood that and anyly the major part of described antisense oligonucleotide or all active modification can be kept to be included in the present invention.
As optimal way of the present invention, nucleic acid lock is carried out to antisense oligonucleotide and modifies; More preferably also carry out thio-modification.
Transferred to after in animal body by antisense oligonucleotide of the present invention, they obviously can lower the expression of relevant miRNA.
Polynucleotide construction
According to miRNA sequence provided by the present invention, can design the polynucleotide construction that can be processed to the miRNA that can affect corresponding mrna expression after being imported into, also namely described polynucleotide construction can raise the amount of corresponding miRNA in vivo.Therefore, the invention provides a kind of polynucleotide (construction) of separation, described polynucleotide (construction) can become precursor miRNA by people's cell transcription, and described precursor miRNA can be expressed as described miRNA by people's cell shearing.
As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown in formula I:
Seq
forward-X-Seq
oppositely
Formula I
In formula I,
Seq
forwardfor the nucleotide sequence of miRNA described in can becoming at cells, Seq
oppositelyfor with Seq
forwardsubstantially complementary nucleotide sequence; Or, Seq
oppositelyfor the nucleotide sequence of miRNA described in can becoming at cells, Seq
forwardfor with Seq
forwardsubstantially complementary nucleotide sequence;
X is for being positioned at Seq
forwardand Seq
oppositelybetween intervening sequence, and described intervening sequence and Seq
forwardand Seq
oppositelynot complementary;
Structure shown in formula I, after proceeding to cell, forms the secondary structure shown in formula II:
Formula II
In formula II, Seq
forward, Seq
oppositelywith the definition of X as above-mentioned;
|| represent at Seq
forwardand Seq
oppositelybetween formed base pair complementarity relation.
Usually, described polynucleotide construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described miRNA, or described polynucleotide construction.Described expression vector is usually also containing promotor, replication orgin and/or marker gene etc.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.
Described expression vector can preferably comprise one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as kalamycin, gentamicin, Totomycin, amicillin resistance.
Detection reagent, detection chip and detection kit
Present invention also offers a kind of test kit for detecting, in described test kit, containing the detection reagent for ncRNA of the present invention (as miRNA) or detection chip.Described test kit can be used for the express spectra detecting miRNA of the present invention.Preferably, also containing the marker for labeled rna sample in described test kit, and the substrate corresponding with described marker.
In addition, also can comprising in described test kit for extracting the required all ingredients such as RNA, PCR, hybridization, colour developing, including but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, nitrite ion, washing lotion, antibody etc.
In addition, working instructions and/or chip image analysis software can also be comprised in described test kit.
Chip
MicroRNA chip of expression spectrum containing how up to a hundred, thousands of or more probes, contains multiple microRNA usually, utilizes the principle of double-strand homologous complementary to detect the content of contained various microRNA in sample.Therefore, can detect the transcriptional level of the microRNA in sample to be tested at one time.
Utilize miRNA sequence of the present invention, corresponding miRNA chip can also be prepared, and then study the regulative mode of its express spectra and miRNAs.
The present invention also provides a kind of chip for analyzing miRNA express spectra, and described chip can be used for detecting in infected by microbes situation, the response type miRNA of animal body.
The oligonucleotide probe that described miRNA chip of the present invention comprises solid phase carrier and is fixed in order on described solid phase carrier.
Particularly, according to miRNA of the present invention, applicable probe can be designed, be fixed on solid phase carrier, be formed " oligonucleotide arrays ".Described " oligonucleotide arrays " refers to the array with addressable point (namely with distinctive, addressable address is the position of feature), and each addressable point is all containing a coupled characteristic oligonucleotide.As required, oligonucleotide arrays can be divided into multiple sub-battle array.
Described solid phase carrier can adopt the various common used materials in gene chip field, such as but not limited to nylon membrane, and the slide, plastic sheet etc. of the slide modified through active group (as aldehyde radical, amino etc.) or silicon chip, unmodified.
The preparation of described miRNA chip can adopt the common manufacturing method of biochip known in the art.Such as, if what solid phase carrier adopted is modify slide or silicon chip, the 5' end of probe is containing amido modified poly-dT string, oligonucleotide probe can be mixed with solution, then point sample instrument is adopted to modify on slide or silicon chip by its point, be arranged in predetermined sequence or array, then being spent the night by placement is fixed, and just can obtain miRNA chip of the present invention.
Solid-phase hybridization between RNA and miRNA chip of the present invention carries out according to the classical way of this area, and the general personnel in this area empirically easily determine the optimum condition of related buffers, probe and concentration of specimens, prehybridization temperature, hybridization temperature and time etc.Or also can with reference to described in " Molecular Cloning: A Laboratory guide ".
Then measurement information is treated according to acquisition of informations such as the position of marking signal on miRNA chip, intensity.If amplified production fluorophor marks, also directly can obtain with fluorescence detection device (as laser confocal scanner Scanarray3000 etc.) and treat measurement information.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, comprise the response type miRNA of the present invention of pharmaceutically acceptable carrier or significant quantity, or its promotor or agonist.
As used herein, term " significant quantity " or " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or animal (as Mammals and bird) and without excessive bad side reaction (as toxicity, stimulation and transformation reactions), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various vehicle and thinner.
Pharmaceutical composition of the present invention contains the activeconstituents of the present invention of safe and effective amount and pharmaceutically acceptable carrier.This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Usual pharmaceutical preparation should match with administering mode, and the formulation of pharmaceutical composition of the present invention is injection, oral preparations (tablet, capsule, oral liquid), transdermal agent, sustained release dosage.Such as be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.Described pharmaceutical composition should aseptically manufacture.
The significant quantity of activeconstituents of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio, metabolism, transformation period etc. of described activeconstituents; The severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.Usually, when activeconstituents of the present invention gives with the dosage of about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals) every day, gratifying effect can be obtained.Such as, by an urgent demand for the treatment of situation, the dosage that several times separate can be given every day, or dosage is reduced pari passu.
Pharmaceutically acceptable carrier of the present invention includes, but is not limited to: water, salt solution, liposome, lipid, albumen, Protein-antibody conjugate, peptide matters, Mierocrystalline cellulose, nanogel or its combination.The selection of carrier should match with administering mode, and these are all known by those of ordinary skill in the art.
Present invention also offers the purposes of described pharmaceutical composition, for the preparation of antagonism or the medicine etc. resisting infected by microbes.
Authentication method
Present invention also offers a kind of method of qualification response type miRNA (or other ncRNA).Described method can based on any sample deriving from animal body or zooblast.
Usually, the inventive method comprises step:
A () provides a test group and a control group, wherein said test group is be subject to the animal individual of a certain infected by microbes, animal sample or zooblast;
Derive from the express spectra of the miRNA of described animal in (b) more described test group and control group, thus identify response type miRNA;
C response type miRNA kind that () will identify, compares with the gene database of described microorganism, thus determines the response type miRNA kind that there is target relation with described microbial gene;
D (), for the described response type miRNA kind that there is target relation, verifies described regulation relationship by experiment.
A kind of typical method is analyzed by cell in vitro infection experiment.The method generally includes following steps:
A. with various microorganism (comprising bacterium, virus, mycoplasma, chlamydozoan etc.) infection animal cell, infected cell training liquid is collected;
B. utilize the means that high throughput sequencing technologies (Solexa sequencing technologies) and bioinformatic analysis comparison combine, derive from first sieve cell training liquid animal and after infected by microbes quantity there is the one group of ncRNA (comprising miRNA) significantly raised or significantly lower;
C. use sensitive further, the ncRNA of real-time fluorescence quantitative PCR to primary dcreening operation verifies accurately.
By aforesaid method provided by the present invention, can differentiate and evaluate after infected by microbes animal, one or more ncRNA (comprising miRNA) that animal body raises as response or lowers.
Major advantage of the present invention comprises:
(1) disclose in animal body the miRNA that there is a class and respond to infected by microbes first, this kind of response type miRNA contributes to animal body antagonism or opposing infected by microbes.
(2) a kind of novel method detecting infected by microbes is provided.
(3) new way that a kind of combating microorganisms infects and treats microbial diseases is provided.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.
Embodiment 1
To discriminating and the application of the response miRNA of mycobacterium tuberculosis
(1) to the discriminating of replying miRNA in m tuberculosis infection cell
1.1 cell cultures
At the RPMI substratum of antibiotic-free, use 24 porose discs, use mycobacterium tuberculosis (deriving from Nanjing University's Life Science College) to infect C57BL/6 mouse (deriving from Chinese Academy of Sciences's cell bank) dendritic cell (DC) with the ratio of 1:10; At 5%CO
2in incubator, 37 DEG C of cultivations.With respectively without any process (U), through 1 μ g/ml sugar greasiness (LPS), through H37Rv infect and with the dendritic cell of LPS process (LPS+H37RV) in contrast.
MiRNA is replied in 1.2 screening m tuberculosis infection cells
First use miRNA Mini Kit (purchased from Yu Bo bio tech ltd, Shanghai) to extract total serum IgE, then adopt EXIQON (purchased from Vedbaek company of Denmark) to detect sample, concrete outcome is shown in Fig. 1.
Fig. 1 shows the detected result of miRNA in the dendritic cell of m tuberculosis infection.
As can be seen from Figure 1, in the dendritic cell of mycobacterial infections, 8 kinds of miRNA, miR-710, miR-881*, miR-882, miR-877, miR-146a, miR-125a-5p, miR-99b and miR-222 up-regulateds.By these 8 kinds of miRNA alternatively express spectra, in order to screening further.
1.3 adopt Real – time PCR to detect the expression of miRNA special in the dendritic cell be infected by bacterial.
For screening the miRNA special to mycobacterium tuberculosis further, Real-time PCR method is used to detect the dendritic cell be infected by bacterial.MiRNA Mini Kit (purchased from Yu Bo bio tech ltd, Shanghai) is used to extract total serum IgE; MiRNA and mRNA being respectively to be detected synthesizes cDNA; Real-time (SYBR Green dye method) (be bio tech ltd purchased from Beijing health) is used to detect sample.Detected result to show in 3-4 group independent experiment one group of data representatively.Each group of experiment is separate, and the result marked in its figure is mean+/-standard error average (SEM).
Detect and find, the expression change of three kinds of miRNA, miR-146a, miR-125a-5p and miR-99b is maximum, therefore continues to test to the dependency that Mycobacterium tuberculosis H37Rv infects to these three kinds of miRNA.
Fig. 2 shows miR-146a, miR-125a-5p and miR-99b expression of results in the dendritic cell be infected by bacterial.
miR-146a：ugagaacugaauuccauggguu(SEQ ID NO.:1)；
miR-125a-5p:ucccugagacccuuuaaccuguga(SEQ ID NO.:2);
miR-99b:cacccguagaaccgaccuugcg(SEQ ID NO.:3)
As can be seen from Figure 2, LPS-H37Rv co-treatment can cause miR-146a and miR-125a-5p to raise, and the rise of other 6 kinds of miRNAs is all infected relevant to H37Rv.Wherein, miR-99b significantly raises after H37Rv infects, but does not raise after LPS process.As can be seen here, miR-99b infects tool specificity to Mycobacterium tuberculosis H37Rv.
(2) inhibitor for miRNA can reduce bacterial population
Use the dendritic cell that anti-miR-99b process is infected through H37Rv, to check miRNA inhibitor to the restraining effect of bacteriological infection.Wherein, knock out miR-99b with antisense oligonucleotide (antisense oligonucleotide), obtain anti-miR-99b (Ambion company).
Cultivate the 4th day at dendritic cell, use antibiotic-free RPMI1640 substratum cleaning cell once, then at CO
2in incubator, at 37 DEG C, cultivate 4 hours in 500 μ l antibiotic-free RPMI1640 substratum.Then, 100nM anti-miR-99b is added in 50 μ l Opti-MEM substratum (purchased from Invitrogen).After one day, replaced medium, uses H37Rv bacteria-infected cells.Bacteriological infection is after 24 hours, and collecting cell, extracts RNA to carry out Real-time PCR inspection.In contrast, random oligonucleotide (scramble) is used to process Opti-MEM substratum.Concrete outcome is shown in Fig. 3.
Fig. 3 shows miR-99b inhibitor and the expression of results of miR-99b process LAN in the dendritic cell be infected by bacterial.Detect from the qRT-PCR of Fig. 3 and find, anti-miR-99b process can miR-99b in effective reticent dendritic cell.
Infect after 48 hours, detect sample H37Rv level (CFU, colony-forming unit), concrete outcome is shown in Fig. 4.
Fig. 4 shows and suppresses miR-99b can significantly reduce H37Rv level.Further, raising miR-99b level by adding mimic, finding that H37Rv level can increase.As can be seen here, the fecundity of mycobacterium tuberculosis can be reduced for the inhibitor of miR-99b.
Embodiment 2
H1N1 is replied to the screening and identification of miRNA
In the present embodiment, infected by influenza H1N1 (available from Nanjing University's Life Science College) studies, and shows after H1N1 infects, and in zooblast, response change can occur miRNA level.And utilize these responses miRNA, can the higher diagnostic value of auxiliary diagnosis infector/tool.
(1) screening of miRNA is replied in virus infected cell
Use Ficoll density gradient centrifugation instrument centrifugation of blood samples, obtain human peripheral blood mononuclear cell (PBMC), be stored in RNA Sample preservation liquid RNAlater (Ambion), at-80 DEG C, in order to extracting RNA; Then miRNA Mini Kit (purchased from Yu Bo bio tech ltd, Shanghai) extracts the total serum IgE of peripheral blood lymphocytes.
The method of biochip is used to detect infection sample and normal healthy controls.Concrete operations are as follows:
1) extract total serum IgE in serum, denaturing formaldehyde gel electrophoresis detects the quality of total serum IgE;
2) separation of miRNA: the miRNA Isolation Kit (Cat#.1560) getting 50-100 μ g total serum IgE Ambion company is separated miRNA;
3) fluorescent mark of miRNA sample: utilize T4RNA ligase enzyme marking method to carry out fluorescent mark, and then by dehydrated alcohol precipitation, for chip hybridization after drying up;
4) hybridization and cleaning: RNA is dissolved in (15% methane amide in 16 μ L hybridization solutions; 0.2%SDS; 3 × SSC; 50 × Denhardt solution), in 42 DEG C of hybridized overnight.After hybridization terminates, first in about the 42 DEG C liquid containing 0.2%SDS, 2 × SSC, wash 4 minutes, then in 0.2 × SSC liquid, room temperature washes 4 minutes, and namely slide can be used for scanning after drying;
5) chip scanning: chip LuxScan10K/A twin-channel laser scanner scans;
6) data are extracted and are analyzed: adopt LuxScan3.0 image analysis software to analyze chip image, picture signal is converted into numerary signal, finally analyze with SAM and select difference expression gene.
Concrete outcome is shown in Fig. 5 and Fig. 6.
Fig. 5 shows Normal group and infects microRNA differential expression result in sample.Adopt SAM software analysis, FDR, lower than 0.05, is considered as normal higher than 1.5; Higher than transverse axis or the column lower than transverse axis, represent the miRNAs raising or lower.
The detected result detected from Fig. 5 finds, 41 kinds of miRNAs expression in infection sample and normal healthy controls have significant difference.
Fig. 6 shows the expression hierarchical cluster tree result of the miRNA of normal healthy controls group and patient.What green or red bar represented is the level reduction of specific microRNA or increases.
Show from the cluster analysis of Fig. 6, utilizing these 41 kinds to reply miRNAs can distinguish completely by infection sample and normal healthy controls.
For screening response miRNAs further, use Real-time PCR method (Taqman probe method) detection by quantitative 9 kinds to express change maximum miRNAs:hsa-miR-146b-5p, hsa-miR-148a, hsa-miR-150, hsa-miR-31, hsa-miR-155, hsa-miR-29a, hsa-miR-29b, hsa-miR-342-5p and hsa-miR-886-3p, use RNU44 as stdn reference.Concrete operation step comprises:
Extract the serum total serum IgE of animal, obtain cDNA sample by RNA reverse transcription reaction;
Primer is designed for microRNA;
Add TaqMan probe and carry out PCR reaction;
Detect the change of the amount of microRNA in animal serum.
All detections all carry out four times; Use 2-Δ Δ Ct method to calculate relative content, use
software is by t method of inspection processing data.When p value≤0.05, think tool significant difference.Concrete outcome is shown in Fig. 7.
Fig. 7 shows micromatrix and Real-time PCR records Comparative result to miRNA expression amount.The detected result of Fig. 7 conforms to microarray result, and namely the expression of these 9 kinds of miRNA really in infection sample and normal healthy controls exists significant difference.
(2) to the checking/qualification of response miRNA
For verifying further the 9 kinds of response miRNA filtered out, setting up ROC curve, evaluating the value that it differentiates to infect sample and normal healthy controls.Checking uses Graphpad Prism V5.01 software; When P value is less than 0.05, namely think tool significant difference.Get and multiplely block (cut-off) value, when specificity and sensitivity the highest time, then think that this cutoff value is optimum.Concrete outcome is shown in Fig. 8.
The ROC tracing analysis of Fig. 8 finds, miR-31, miR-29a and miR-148a are when P value is less than 0.05, and sensitivity and specificity are very high, infection sample and normal healthy controls can be distinguished, the very high diagnostic value of tool.
Embodiment 3
Coxsackie B virus 3 (coxsackie virus B3, CVB3) is replied to the screening and identification of miRNA
The known Coxsackie B virus group positive is often the etiological diagnosis standard of viral myocarditis, and microRNA miR-126 tool heartspecific.In the present embodiment, study Coxsackie B virus 3, in checking zooblast, whether miR-126 is its response gene.Show to utilize miR-126 inhibitor, can obviously reduce CVB3 virus activity.
(1) to the discriminating of replying miRNA in zooblast after CVB3 virus infection
1. cell cultures and virus infection
HeLa cell (deriving from Chinese Academy of Sciences's cell bank) is cultivated in containing the DMEM of 10%FBS, Human Cardiomyocytes HL-1 (deriving from Chinese Academy of Sciences's cell bank) cultivates in containing the Claycomb substratum of 10%FBS, Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) (deriving from Chinese Academy of Sciences's cell bank) is cultivated in EGM BulletKit.After making cell be infected 1 hour by CVB3 (available from Nanjing University's Life Science College), use PBS to rinse, then be placed in new substratum.
2.RNA extracts and real-time RT-PCR
MiRNA Mini Kit (purchased from Yu Bo bio tech ltd, Shanghai) is used to extract cell RNA; Use TaqMan MicroRNA Reverse Transcription box (purchased from Life Technologies) to carry out rna transcription, then use relative quantitation method to detect.Use U6 as internal reference.All real-time RT-PCR detect and all carry out three times.Each group of experiment is separate, and the result marked in its figure is mean+/-standard error average (SEM).Concrete outcome is shown in Fig. 9.
Fig. 9 a-c shows the detected result of miR-126 in HeLa cell;
Fig. 9 d shows the detected result of miR-126 in HL-1 cell;
Fig. 9 e shows the detected result of miR-126 in HUVEC cell.
As can be seen from Figure 9, the expression level of miR-126 in HeLa cell, HL-1 cell, HUVEC cell occurs significantly to raise.
3. adopt western blot method to verify that CVB3 is by the protein regulation miR-126 relevant to miR-126 regulatory pathway further.
Response is produced for checking miR-126 infects CBV3 really, liposome Oligofectamine (purchased from Life Technologies company) is used to be intended by synthetic miRNA entering cell like thing (miRNA mimic, this plan is microRNA stochastic sequence like the sequence of thing) transfection.The random miRNA of transfection intends like thing in contrast (miR-CL).Transfection concentrations adopts two kinds, 0.1nM and 1nM.Transfection, after 48 hours, extracts RNA, uses Real-time PCR to detect miR-126 content, confirms that miRNA intends having raised this miRNA level like thing.
MiRNA intends: caucccuugcaugguggaggg (SEQ ID NO.:4)
After confirmation, use CVB3 cells infected.Infect after 7 hours, use western blot method method to detect viral protein (VP-1) content.Concrete outcome is shown in Figure 10.As can be seen from Figure 10, the miRNA mimic transfection of 0.1nM and 1nM concentration, all makes VP-1 express and obviously raises.As can be seen here, the response miRNA of the CVB3 really of miR-126.
(2) reply miRNA inhibitor and can reduce virus activity
By the response miRNA of CVB3, the inhibitor of miR-126, cell is entered in the transfection of synthetic miR-126inhibitor (126-in) inhibitor.Transfection random synthetic miRNA inhibitor is (CL-in) (purchased from Life Technologies company) in contrast.Transfection, after 48 hours, is extracted RNA and detects miR-126 content, confirms that miR-126 expresses and is effectively lowered.
The sequence of random synthetic miRNA inhibitor: guagggaacguaccaccuccc (SEQ ID NO.:5).
After confirmation, use CVB3 cells infected.Infect after 7 hours, use western blot method method to detect viral protein (VP-1) content.Concrete outcome is shown in Figure 11.As can be seen from Figure 11, suppress miR-126 to express, VP-1 can be made to express and obviously reduce.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. nondiagnostic differentiates a method of animal body miRNA, it is characterized in that, comprises step:
A () provides a test group and a control group, wherein said test group is be subject to the animal individual of a certain infected by microbes, animal sample or zooblast; A described control group is that experiment condition is identical but not by the animal individual of described infected by microbes, animal sample or zooblast;
The express spectra of the miRNA of described animal is derived from (b) more described test group and control group, thus identify compared with described control group, there is the miRNA kind of significant difference in expression level in described test group, be decided to be response type miRNA (responding miRNA), wherein, described response type miRNA comprises: express the rise type miRNA and/or the remarkable downward type miRNA lowered of expression that significantly raise.
2. the method for claim 1, is characterized in that, in step (c), described response type miRNA kind expresses the rise type miRNA significantly raised.
3. method as claimed in claim 2, it is characterized in that, in step (d), described regulation relationship refers to the downward of described miRNA to described microorganism target gene; Or
In step (d), described regulation relationship refers to the downward of described miRNA to described microorganism target gene.
4. the method for claim 1, is characterized in that, in step (c), described response type miRNA kind expresses the downward type miRNA significantly lowered.
5. the method for claim 1, is characterized in that, described method also comprises step:
C response type miRNA kind that () will identify, compare with the gene database of described microorganism and/or the gene database of described animal, thus determine the response type miRNA kind that there is target relation with described microbial gene and/or described animal gene, wherein said target relation represents for a certain specific miRNA kind X, it is complete or substantially the same with one section of nucleotide sequence section of the gene Y of described microorganism or the gene Ya of described animal, or completely or substantially complementary;
D () is for the described response type miRNA kind that there is target relation, verify the regulation relationship of described miRNA kind X and described gene Y or Ya (target gene) by experiment, if wherein the result is positive, then differentiate that described animal body miRNA is the response type miRNA participating in infected by microbes; If the result is negative, then differentiate that described animal body miRNA is the response type miRNA not participating in infected by microbes.
6. the method for claim 1, it is characterized in that, described test group comprises the first test subgroup and/or the second test subgroup, wherein the first test subgroup is subject to infected by microbes and shows the animal individual of infection symptoms, animal sample or zooblast, and the second test subgroup is subject to infected by microbes and does not show the animal individual of infection symptoms, animal sample or zooblast.
7. the method for claim 1, is characterized in that, described method also comprises step:
E (), to the miRNA identified, tests the ability that it resists described infected by microbes.
8. the miRNA identified by method described in claim 1;
Preferably, described miRNA comprises the rise type miRNA and/or the remarkable downward type miRNA lowered of expression that express and significantly raise.
9. a composition, is characterized in that, described composition contains (a) antisense sequences of acceptable carrier and (a) miRNA according to claim 8 or its agonist or described miRNA or inhibitor pharmaceutically or on food.
10. whether or the reagent of infected by microbes relative disease, chip or test kit the purposes of miRNA as claimed in claim 8, is characterized in that, for the preparation of detecting infected by microbes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410020196.0A CN104789641A (en) | 2014-01-16 | 2014-01-16 | Animal response type gene under microbial infection, identification and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410020196.0A CN104789641A (en) | 2014-01-16 | 2014-01-16 | Animal response type gene under microbial infection, identification and applications thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104789641A true CN104789641A (en) | 2015-07-22 |
Family
ID=53554831
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410020196.0A Withdrawn CN104789641A (en) | 2014-01-16 | 2014-01-16 | Animal response type gene under microbial infection, identification and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104789641A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111041001A (en) * | 2018-10-15 | 2020-04-21 | 吴可行 | Safe coxsackie virus for treating KRAS mutant tumors and pharmaceutical composition thereof |
-
2014
- 2014-01-16 CN CN201410020196.0A patent/CN104789641A/en not_active Withdrawn
Non-Patent Citations (3)
| Title |
|---|
| HAO SONG等: "Microarray analysis of MicroRNA expression in peripheral blood mononuclear cells of critically ill patients with influenza A (H1N1)", 《BMC INFECTIOUS DISEASES》 * |
| XIN YE等: "MiR‑126 promotes coxsackievirus replication by mediating cross‑talk of ERK1/2 and Wnt/β‑catenin signal pathways", 《CELL. MOL. LIFE SCI.》 * |
| YOGESH SINGH等: "Mycobacterium tuberculosis Controls MicroRNA-99b (miR-99b) Expression in Infected Murine Dendritic Cells to Modulate Host Immunity", 《JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111041001A (en) * | 2018-10-15 | 2020-04-21 | 吴可行 | Safe coxsackie virus for treating KRAS mutant tumors and pharmaceutical composition thereof |
| CN111041001B (en) * | 2018-10-15 | 2023-02-28 | 上海行深生物科技有限公司 | Safe coxsackie virus for treating KRAS mutant tumor and pharmaceutical composition thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9903002B2 (en) | Non-coding RNA of Salmonella and identification and use thereof | |
| CN110004234B (en) | Chicken anti-salmonella infection related miRNA and application thereof | |
| CN112779329B (en) | Molecular marker for auxiliary diagnosis of viral meningitis and application and kit thereof | |
| Bellehumeur et al. | Propidium monoazide (PMA) and ethidium bromide monoazide (EMA) improve DNA array and high-throughput sequencing of porcine reproductive and respiratory syndrome virus identification | |
| WO2021194603A2 (en) | Methods, oligonucleotides, and kits for detection and treatment of coronavirus | |
| CN106103714B (en) | In vivo infection microorganism, parasitic microbe, the noncoding RNA of symbiotic microorganism and its identification and application | |
| CN105814203B (en) | Avian influenza virus miRNA and its identification, detection and application | |
| US20240254566A1 (en) | Analysis method, kit, and detection device | |
| CN104789641A (en) | Animal response type gene under microbial infection, identification and applications thereof | |
| Han et al. | Comparative analysis of microRNA in schistosomula isolated from non-permissive host and susceptible host | |
| CN110387415A (en) | A circular RNA marker for detecting cerebral ischemia-reperfusion injury and its application | |
| CN109652529A (en) | Osteoporosis specificity miRNA, composition and its diagnostic use | |
| CN112941209A (en) | Molecular marker for detecting chicken infected salmonella enteritidis, method and application thereof | |
| CN105567832B (en) | A method for detecting microbial drug resistance genes for non-disease diagnosis and treatment purposes | |
| CN115873967B (en) | Nucleic acid molecule and application thereof as well as method for detecting genome pollution in trichomonas fowl nucleic acid sample | |
| CN113403403B (en) | Molecular marker, detection method and application of a chicken infected with Campylobacter jejuni | |
| KR102408783B1 (en) | Use of SCARNA13 for Diagnosis or Treatment of Alzheimer's Disease | |
| US10144974B2 (en) | Methods and reagents that specifically detect, distinguish and quantify IFN-LAMBDA2 mRNA from IFN-LAMBDA3 mRNA in humans | |
| CN105219852B (en) | A kind of primer and application and method for detecting gene expression level of phosphorus metabolism-related enzymes in Anabaena sp. | |
| CN116440152A (en) | Application of inhibitor of WTAP in preparation of medicine for treating atopic dermatitis | |
| CN108179197A (en) | Chicken ring RNA Chr9:10814512 | 10838667 genetic test primers, method and application | |
| CN103160507A (en) | MiRNA serum marker capable of detecting liver cirrhosis and application thereof | |
| HK1237372A1 (en) | Methods of detecting trichomonas vaginalis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20150722 |
|
| WW01 | Invention patent application withdrawn after publication |