CN111778253B - Small interfering nucleic acid, composition and application - Google Patents

Abstract

The invention discloses a small interfering RNA, a composition and an application thereof. The invention claims a siRNA, wherein the siRNA is SP-2013, part of nucleotides of a sense strand are 2' -O-methyl ribonucleotides, and part of phosphate groups are phosphorothioate groups. The invention provides novel siRNA and a composition thereof, which can effectively prevent and/or treat novel coronavirus.

Description

Small interfering nucleic acid, composition and application

The invention relates to a small interfering RNA, a composition and a divisional application of the application, wherein the application date is 4/3/2020, the application number is 202010256867.9, and the name of the invention is small interfering RNA.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a small interfering RNA for inhibiting the expression of a novel coronavirus gene and application of the small interfering RNA in preparing a pharmaceutical composition for preventing and/or treating novel coronavirus pneumonia.

Background

At the end of 2019 and early 2020, a novel coronavirus pneumonia (coronas disease2019, COVID-19) is an acute infectious pneumonia, and researchers found that its pathogen is caused by a novel beta coronavirus that was not previously found in humans, which was subsequently named COVID-19 by the World Health Organization (WHO). Patients with COVID-19 mostly have the initial symptoms of fever, hypodynamia and dry cough, and gradually have severe manifestations such as dyspnea and the like. The prognosis is good in most patients and acute respiratory distress syndrome and septic shock can occur in some severe cases and even death. By 1/3 of 2020, COVID-19 has infected 88000 people, causing nearly 3000 deaths. Scientists and clinicians around the world are working rapidly to combat the respiratory disease covi-19 caused by the new coronavirus, but there is still a lack of effective antiviral drugs against the covi-19 virus, and current treatments are mainly isolation treatment and symptomatic support treatment. Therefore, the development of siRNA and pharmaceutical products thereof effective in preventing and/or treating novel coronavirus pneumonia is an urgent problem to be solved.

Disclosure of Invention

The invention aims to provide siRNA targeting a novel coronavirus gene and application thereof in preparation of a novel coronavirus pneumonia medicament.

The invention provides siRNA, which comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence shown as SEQ ID NO: 2, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO: 3; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 28, and the antisense strand contains the nucleotide sequence shown as SEQ ID NO: 29; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 40, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO: 41; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 42, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 43; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 52, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 53; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 54, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 55; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 62, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 63; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 64, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 65; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 66, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 67; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 68, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 69; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 84, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 85; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 92, and the antisense strand comprises the nucleotide sequence shown as SEQ ID NO: 93; alternatively, the sense strand comprises the sequence set forth as SEQ ID NO: 94, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 95;

wherein the content of the first and second substances,

sense strand 5'-CAUCUGAUUUGGCUACUAACA-3' (SEQ ID NO: 2),

antisense strand 5'-UUAGUAGCCAAAUCAGAUGUG-3' (SEQ ID NO: 3);

sense strand 5'-UCUGCAAUUAACAGGCCACAA-3' (SEQ ID NO: 28),

antisense strand 5'-GUGGCCUGUUAAUUGCAGAUG-3' (SEQ ID NO: 29);

sense strand 5'-AAACAAGCUAGUCUUAAUGGA-3' (SEQ ID NO: 40),

antisense strand 5'-CAUUAAGACUAGCUUGUUUGG-3' (SEQ ID NO: 41);

sense strand 5'-GUUGAUGGUGUUGUCCAACAA-3' (SEQ ID NO: 42),

antisense strand 5'-GUUGGACAACACCAUCAACUU-3' (SEQ ID NO: 43);

sense strand 5'-CUAGUCUCUAGUCAGUGUGUU-3' (SEQ ID NO: 52),

antisense strand 5'-CACACUGACUAGAGACUAGUG-3' (SEQ ID NO: 53);

sense strand 5'-AAUGUUACUUGGUUCCAUGCU-3' (SEQ ID NO: 54),

antisense strand 5'-CAUGGAACCAAGUAACAUUGG-3' (SEQ ID NO: 55);

sense strand 5'-CUAGUUAUCAGACUCAGACUA-3' (SEQ ID NO: 62),

antisense strand 5'-GUCUGAGUCUGAUAACUAGCG-3' (SEQ ID NO: 63);

sense strand 5'-CAAGCUUUAAACACGCUUGUU-3' (SEQ ID NO: 64),

antisense strand 5'-CAAGCGUGUUUAAAGCUUGUG-3' (SEQ ID NO: 65);

sense strand 5'-UCAUGGUGUAGUCUUCUUGCA-3' (SEQ ID NO: 66),

antisense strand 5'-CAAGAAGACUACACCAUGAGG-3' (SEQ ID NO: 67);

sense strand 5'-GCCUCAAUGAGGUUGCCAAGA-3' (SEQ ID NO: 68),

antisense strand 5'-UUGGCAACCUCAUUGAGGCGG-3' (SEQ ID NO: 69);

sense strand 5'-CUUACAUGGAUUUGUCUUCUA-3' (SEQ ID NO: 84),

antisense strand 5'-GAAGACAAAUCCAUGUAAGGA-3' (SEQ ID NO: 85);

sense strand 5'-AGGCGUUCCAAUUAACACCAA-3' (SEQ ID NO: 92),

antisense strand 5'-GGUGUUAAUUGGAACGCCUUG-3' (SEQ ID NO: 93);

sense strand 5'-UGACCAAAUUGGCUACUACCG-3' (SEQ ID NO: 94),

antisense strand 5'-GUAGUAGCCAAUUUGGUCAUC-3' (SEQ ID NO: 95).

Preferably, at least one single strand of the sense strand and the antisense strand of the siRNA is deleted or added with one or several nucleotides, thereby obtaining an siRNA derivative having the same function as the siRNA.

Preferably, at least a portion of the phosphate-sugar backbone of at least one single strand of said sense strand and said antisense strand of said siRNA is a phosphorothioate backbone;

or, at least a portion of the nucleotides of at least one single strand of the sense strand and the antisense strand are substituted or modified with nucleotides;

alternatively, at least one single strand of the sense strand and the antisense strand is linked to a signaling molecule and/or an active molecule and/or a functional group.

Preferably, at least a portion of the phosphate-sugar backbone of at least one single strand of said sense strand and said antisense strand of said siRNA is a phosphorothioate backbone;

or, at least a portion of the nucleotides of at least one single strand of the sense strand and the antisense strand are substituted or modified with nucleotides;

alternatively, at least one single strand of the sense strand and the antisense strand is linked to a signaling molecule and/or an active molecule and/or a functional group.

Preferably, the sense strand of the siRNA is SEQ ID NO: 2, the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 28 th nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 40 th nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 st nucleotides of the 52 nd nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 54 th nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 62 nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 st nucleotides of the 64 th nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 st nucleotides of the 66 nd nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 68 th nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 84 nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 st nucleotides of the 92 nd nucleotide sequence are all 2' -O-methyl ribonucleotides;

alternatively, the sense strand of the siRNA SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 st nucleotides of the 94 nucleotide sequence are all 2' -O-methyl ribonucleotides.

Preferably, the phosphate groups between the 1 st, 2 nd and 3 rd nucleotides and the phosphate groups between the 19 th, 20 th and 21 st nucleotides of the nucleotide sequence of the sense strand of the siRNA are all phosphorothioate groups.

Preferably, cholesterol is linked to the 5' -end of the sense strand of the siRNA.

The invention also provides application of the siRNA in preparation of a medicament for preventing and/or treating viral pneumonia.

Preferably, the viral pneumonia is novel coronavirus pneumonia COVID-19.

The invention also provides a kit, which comprises the siRNA and has the function of treating the novel coronavirus COVID-19.

Experiments prove that the siRNA modified by the invention has obvious inhibition effect on the novel coronavirus gene, lays a foundation for clinically treating the novel coronavirus pneumonia, and has great clinical significance and popularization value.

Drawings

FIG. 1 is a map of the dual luciferase reporter plasmid GP-mirGlo plasmid in example 3 of the present invention;

FIG. 2 is a graph showing the results of a dose-dependent experiment using siRNA numbered SP-26 in example 5 of the present invention;

FIG. 3 is a graph showing the results of a dose-dependent experiment using siRNA numbered SP-3552 in example 5 of the present invention;

FIG. 4 is a graph showing the results of a dose-dependent experiment using siRNA numbered SP-2013 in example 5 of the present invention;

FIG. 5 is a graph showing the results of a dose-dependent experiment using siRNA numbered SP-3169 in example 5 of the present invention;

FIG. 6 is a graph showing the results of a dose-dependent experiment using siRNA numbered SP-2867 in example 5 of the present invention;

FIG. 7 is a graph showing the results of a dose-dependent experiment using siRNA numbered SP-179 in example 5 of the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following non-limiting examples will allow those skilled in the art to better understand the present invention.

Any person skilled in the art can substitute or change the technical scheme and concept of the present invention within the careless scope of the present invention.

The 293T cell line used in the examples below was purchased from the classical collection of cells of the Central institute of technology.

Example 1

Synthesis of Small interfering nucleic acids

A novel coronavirus (COVID-19) genome (Genebank accession number; MN908947.3) (SEQ ID NO: 1) with a relatively conserved sequence was selected as a template. Respectively aiming at the conserved regions of the COVID-19 gene, a nucleotide sequence of 21bp is selected to design small interfering nucleic acid (siRNA).

The siRNA designed in this example was synthesized by shanghai gimar pharmaceutical technology ltd, and the sequences of the synthesized siRNA are shown in table 1.

TABLE 1

As shown in table 1, this example also sets up the sense strand nucleotide sequence as set forth in SEQ ID NO: 102, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO: 103, M-siNC as a negative control, which is an unrelated sequence having no target site corresponding to the novel coronavirus (COVID-19) gene.

Example 2

Synthesis of modified Small interfering nucleic acids

The modified siRNA shown in the table 2 is obtained by chemically modifying the sense strand of the sequence in the table 1, namely, the 2' hydroxyl group of the pentose group in the nucleotide residues of the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 st bases of the sense strand is modified by methoxy, the phosphate group between the 1 st, 2 nd and 3 rd bases of the sense strand is phosphorothioate group, and the phosphate group between the 19 th, 20 th and 21 st bases of the sense strand is phosphorothioate group; the antisense strand is not modified, the siRNA designed in this example is synthesized by shanghai gimar pharmaceutical technology ltd, and cholesterol is linked to the 5' end of the sense strand of the siRNA for modification.

TABLE 2

Wherein m represents a 2' -methoxy group as a pentose group in the nucleotide residue on the left side, and s represents a phosphorothioate group between deoxyribonucleotide residues on the left and right sides thereof.

Example 3

Construction of dual-luciferase reporter plasmid

The dual-luciferase reporter plasmid GP-miRGLO vector used in this example (the sequence of this empty vector is shown in the specification), purchased from promega (cat. No. E1330), and the map information is shown in FIG. 1. A dual-luciferase reporter plasmid as shown in Table 3 was prepared by inserting a novel coronavirus (COVID-19) genome sequence into the GP-mirGAL vector.

TABLE 3

Example 4

Cell transfection

1.293T cells were cultured in 10cm dishes to 80-90% confluency, the culture broth was decanted and the cells were washed twice with 2ml PBS.

2. Adding 2ml of Trypsin-EDTAsolution, mixing uniformly, and standing at 37 ℃ for 1 minute.

3. The pancreatin solution was carefully aspirated, 2ml of DMEM medium containing 10% FBS was added, and the cells were pipetted to form a single cell suspension.

4. Counting with a hemocytometer, diluting the cells to 1X 10⁶Cells/ml. By 5X 10⁵Inoculating 12-well plate with cell/well concentration, mixing well at 37 deg.C with 5% CO₂The culture was carried out for 24 hours.

5. Each 1OD₂₆₀120 μ l DEPC-H for siRNA₂O dissolved to a final concentration of about 20. mu.M.

6. Adding 100 μ l serum-free DMEM into 1.5ml EP tube, adding 7ul siRNA, adding 1.2ug of corresponding dual-fluorescence report carrier, and mixing; another 1.5ml EP tube was added with 100. mu.l serum-free DMEM, 4. mu.l Lipofectamine2000, mixed well, left at room temperature for 5 minutes, then mixed and left at room temperature for 20 minutes.

Grouping was performed according to the correspondence between plasmids and siRNA, and the specific correspondence is shown in Table 2.

Each group was done 3 more wells, averaged, and tested at time point 24/48h, once.

The culture medium was aspirated from the 12-well plate, and the transfection mixture was added dropwise to the 12-well plate, mixed well, and incubated in an incubator for 5 hours.

7. The transfection solution was aspirated off, and 500ul of DMEM medium containing 10% FBS was added. 5% CO at 37 ℃₂The culture was continued for 24 and 48 hours, and the samples were collected.

Dual luciferase systemic detection

The Dual-Luciferase assay was performed using the Dual-Luciferase reporter assay kit (promega) (see instructions for specific methods of use).

Preparation of reagents:

1. preparation of passive lysis buffer 1 × PLB: 1 volume of 5XpassiveLysis buffer (PLB) was added to 4 volumes of distilled water. And (4) uniformly mixing. Storing at 4 ℃ for later use.

2. LAR II, dissolving the freeze-dried powder of LuciferaseAssaySubstrate by LuciferaseAssayBuffer II. Store at-20 ℃ until use.

3. Preparation of

Reagent (prepared as used): according to the required dosage of the experiment, a certain amount of the medicine is taken

Added in corresponding amount

In the preparation, the final concentration of the seed is 1 Xconcentration for standby.

A detection step:

1. the culture medium in the 12-well plate was decanted and the cells were washed twice with 500ul PBS.

2. 1 XPLB 300ul was added to the culture wells. Passive lysis of cells: the plate was gently shaken at room temperature for 15 minutes and the lysate was transferred to the assay plate. 100ul per well, 3 wells per experimental design.

3. Turning on a TecanM1000 microplate reader, preheating and selecting a dual-luciferase detection system.

4. Add 10ul LARII reagent to each well, select 1-2 seconds delay, read 5-10, detect firefly luciferase activity on the microplate reader.

5. The test plate is removed and added to each well

Reagent, selecting 1-2 seconds delay, reading 5-10, detecting renilla luciferase activity on an enzyme labeling instrument.

6. And (5) counting and analyzing results.

The results of the dual luciferase assay are shown in Table 4,

TABLE 4

The results show that the siRNAs with the numbers of PP-1758, PP-17660, PP-20091, PP-20163, SP-26, SP-179, SP-2013, SP-2867, SP-3169, SP-3552, MG-83, NP-208 and NP-241 can effectively inhibit the expression of the novel coronavirus genes.

Example 5

Six siRNAs with numbers SP-26, SP-179, SP-2013, SP-2867, SP-3552 and SP-3169 and with better inhibitory effect on the S region of the novel coronavirus gene are selected to carry out concentration-dependent experiments, cell transfection methods and dual-luciferase systematic detection methods, which are shown in example 4, and the concentration ranges of the siRNAs used are 0nmol/L, 0.01nmol/L, 0.1nmol/L, 1nmol/L, 10nmol/L and 100 nmol/L. The results of the dual-luciferase assay are shown in fig. 2-7, and the results show that the concentration of siRNA has a great influence on the inhibition of the expression of the novel coronavirus gene, and the higher the concentration is, the better the inhibition effect is.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, the invention is intended to encompass any variations, uses, or adaptations of the invention following, in general, the principles of the invention, including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Sequence listing

<110> Suzhou Jima Gene GmbH

<120> small interfering RNA, composition and application

<160> 172

<170> SIPOSequenceListing 1.0

<210> 104

<211> 714

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

ctcgagaaag ccttgaattt aggtgaaaca tttgtcacgc actcaaaggg attgtacaga 60

aagtgtgtta aatccagaga agaaactggc ctactcatgc ctctaaaagc cccaaaagaa 120

attatcttct tagagggaga aacacttccc acagaagtgt taacagagga agttgtcttg 180

aaaactggtg atttacaacc attagaacaa cctactagtg aagctgttga agctccattg 240

gttggtacac cagtttgtat taacgggctt atgttgctcg aaatcaaaga cacagaaaag 300

tactgtgccc ttgcacctaa tatgatggta acaaacaata ccttcacact caaaggcggt 360

gcaccaacaa aggttacttt tggtgatgac actgtgatag aagtgcaagg ttacaagagt 420

gtgaatatca cttttgaact tgatgaaagg attgataaag tacttaatga gaagtgctct 480

gcctatacag ttgaactcgg tacagaagta aatgagttcg cctgtgttgt ggcagatgct 540

gtcataaaaa ctttgcaacc agtatctgaa ttacttacac cactgggcat tgatttagat 600

gagtggagta tggctacata ctacttattt gatgagtctg gtgagtttaa attggcttca 660

catatgtatt gttctttcta ccctccagat gaggatgaag aagaaggcgg ccgc 714

<210> 105

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ctcgagcaaa agatcgctga gattcctaaa gaggaagtta agccatttat aactgaaagt 60

aaaccttcag ttgaacagag aaaacaagat gataagaaaa tcaaagcttg tgttgaagaa 120

gttacaacaa ctctggaaga aactaagttc ctcacagaaa acttgttact ttatattgac 180

attaatggca atcttcatcc agattctgcc actcttgtta gtgacattga catcactttc 240

ttaaagaaag atgctccata tatagtgggt gatgttgttc aagagggtgt tttaactgct 300

gtggttatac ctactaaaaa ggctggtggc actactgaaa tgctagcgaa agctttgaga 360

aaagtgccaa cagacaatta tataaccact tacccgggtc agggtttaaa tggttacact 420

gtagaggagg caaagacagt gcttaaaaag tgtaaaagtg ccttttacat tctaccatct 480

attatctcta atgagaagca agaaattctt ggaactgttt cttggaattt gcgagaaatg 540

cttgcacatg cagaagaaac acgcaaatta atgcctgtct gtgtggaaac taaagccata 600

gtttcaacta tacagcgtaa atataagggt attaaaatac aagagggtgt ggttgattat 660

ggtgctagat tttactttta caccagtaaa acaactgtag cgtcacgcgg ccgc 714

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ctcgagaact accgaagttg taggagacat tatacttaaa ccagcaaata atagtttaaa 60

aattacagaa gaggttggcc acacagatct aatggctgct tatgtagaca attctagtct 120

tactattaag aaacctaatg aattatctag agtattaggt ttgaaaaccc ttgctactca 180

tggtttagct gctgttaata gtgtcccttg ggatactata gctaattatg ctaagccttt 240

tcttaacaaa gttgttagta caactactaa catagttaca cggtgtttaa accgtgtttg 300

tactaattat atgccttatt tctttacttt attgctacaa ttgtgtactt ttactagaag 360

tacaaattct agaattaaag catctatgcc gactactata gcaaagaata ctgttaagag 420

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actgataaat attataattt ggtttttact attaagtgtt tgcctaggtt ctttaatcta 540

ctcaaccgct gctttaggtg ttttaatgtc taatttaggc atgccttctt actgtactgg 600

ttacagagaa ggctatttga actctactaa tgtcactatt gcaacctact gtactggttc 660

tataccttgt agtgtttgtc ttagtggttt agattcttta gacaccgcgg ccgc 714

<210> 107

<211> 714

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ctcgagcata agaaagctac atgatgagtt aacaggacac atgttagaca tgtattctgt 60

tatgcttact aatgataaca cttcaaggta ttgggaacct gagttttatg aggctatgta 120

cacaccgcat acagtcttac aggctgttgg ggcttgtgtt ctttgcaatt cacagacttc 180

attaagatgt ggtgcttgca tacgtagacc attcttatgt tgtaaatgct gttacgacca 240

tgtcatatca acatcacata aattagtctt gtctgttaat ccgtatgttt gcaatgctcc 300

aggttgtgat gtcacagatg tgactcaact ttacttagga ggtatgagct attattgtaa 360

atcacataaa ccacccatta gttttccatt gtgtgctaat ggacaagttt ttggtttata 420

taaaaataca tgtgttggta gcgataatgt tactgacttt aatgcaattg caacatgtga 480

ctggacaaat gctggtgatt acattttagc taacacctgt actgaaagac tcaagctttt 540

tgcagcagaa acgctcaaag ctactgagga gacatttaaa ctgtcttatg gtattgctac 600

tgtacgtgaa gtgctgtctg acagagaatt acatctttca tgggaagttg gtaaacctag 660

accaccactt aaccgaaatt atgtctttac tggttatcgt gtaactgcgg ccgc 714

<210> 108

<211> 714

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

ctcgagcaga cacttatgcc tgttggcatc attctattgg atttgattac gtctataatc 60

cgtttatgat tgatgttcaa caatggggtt ttacaggtaa cctacaaagc aaccatgatc 120

tgtattgtca agtccatggt aatgcacatg tagctagttg tgatgcaatc atgactaggt 180

gtctagctgt ccacgagtgc tttgttaagc gtgttgactg gactattgaa tatcctataa 240

ttggtgatga actgaagatt aatgcggctt gtagaaaggt tcaacacatg gttgttaaag 300

ctgcattatt agcagacaaa ttcccagttc ttcacgacat tggtaaccct aaagctatta 360

agtgtgtacc tcaagctgat gtagaatgga agttctatga tgcacagcct tgtagtgaca 420

aagcttataa aatagaagaa ttattctatt cttatgccac acattctgac aaattcacag 480

atggtgtatg cctattttgg aattgcaatg tcgatagata tcctgctaat tccattgttt 540

gtagatttga cactagagtg ctatctaacc ttaacttgcc tggttgtgat ggtggcagtt 600

tgtatgtaaa taaacatgca ttccacacac cagcttttga taaaagtgct tttgttaatt 660

taaaacaatt accatttttc tattactctg acagtccatg tgagtcgcgg ccgc 714

<210> 109

<211> 674

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

ctcgagatgt ttgtttttct tgttttattg ccactagtct ctagtcagtg tgttaatctt 60

acaaccagaa ctcaattacc ccctgcatac actaattctt tcacacgtgg tgtttattac 120

cctgacaaag ttttcagatc ctcagtttta cattcaactc aggacttgtt cttacctttc 180

ttttccaatg ttacttggtt ccatgctata catgtctctg ggaccaatgg tactaagagg 240

tttgataacc ctgtcctacc atttaatgat ggtgtttatt ttgcttccac tgagaagtct 300

aacataataa gaggctggat ttttggtact actttagatt cgaagaccca gtccctactt 360

attgttaata acgctactaa tgttgttatt aaagtctgtg aatttcaatt ttgtaatgat 420

ccatttttgg gtgtttatta ccacaaaaac aacaaaagtt ggatggaaag tgagttcaga 480

gtttattcta gtgcgaataa ttgcactttt gaatatgtct ctcagccttt tcttatggac 540

cttgaaggaa aacagggtaa tttcaaaaat cttagggaat ttgtgtttaa gaatattgat 600

ggttatttta aaatatattc taagcacacg cctattaatt tagtgcgtga tctccctcag 660

ggttttgcgg ccgc 674

<210> 110

<211> 1500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

tggggaatgt ccaaattttg tatttccctt aaattccata atcaagacta ttcaaccaag 60

ggttgaaaag aaaaagcttg atggctttat gggtagaatt cgatctgtct atccagttgc 120

gtcaccaaat gaatgcaacc aaatgtgcct ttcaactctc atgaagtgtg atcattgtgg 180

tgaaacttca tggcagacgg gcgattttgt taaagccact tgcgaatttt gtggcactga 240

gaatttgact aaagaaggtg ccactacttg tggttactta ccccaaaatg ctgttgttaa 300

aatttattgt ccagcatgtc acaattcaga agtaggacct gagcatagtc ttgccgaata 360

ccataatgaa tctggcttga aaaccattct tcgtaagggt ggtcgcacta ttgcctttgg 420

aggctgtgtg ttctcttatg ttggttgcca taacaagtgt gcctattggg ttccacgtgc 480

tagcgctaac ataggttgta accatacagg tgttgttgga gaaggttccg aaggtcttaa 540

tgacaacctt cttgaaatac tccaaaaaga gaaagtcaac atcaatattg ttggtgactt 600

taaacttaat gaagagatcg ccattatttt ggcatctttt tctgcttcca caagtgcttt 660

tgtggaaact gtgaaaggtt tggattataa agcattcaaa caaattgttg aatcctgtgg 720

taattttaaa gttacaaaag gaaaagctaa aaaaggtgcc tggaatattg gtgaacagaa 780

atcaatactg agtcctcttt atgcatttgc atcagaggct gctcgtgttg tacgatcaat 840

tttctcccgc actcttgaaa ctgctcaaaa ttctgtgcgt gttttacaga aggccgctat 900

aacaatacta gatggaattt cacagtattc actgagactc attgatgcta tgatgttcac 960

atctgatttg gctactaaca atctagttgt aatggcctac attacaggtg gtgttgttca 1020

gttgacttcg cagtggctaa ctaacatctt tggcactgtt tatgaaaaac tcaaacccgt 1080

ccttgattgg cttgaagaga agtttaagga aggtgtagag tttcttagag acggttggga 1140

aattgttaaa tttatctcaa cctgtgcttg tgaaattgtc ggtggacaaa ttgtcacctg 1200

tgcaaaggaa attaaggaga gtgttcagac attctttaag cttgtaaata aatttttggc 1260

tttgtgtgct gactctatca ttattggtgg agctaaactt aaagccttga atttaggtga 1320

aacatttgtc acgcactcaa agggattgta cagaaagtgt gttaaatcca gagaagaaac 1380

tggcctactc atgcctctaa aagccccaaa agaaattatc ttcttagagg gagaaacact 1440

tcccacagaa gtgttaacag aggaagttgt cttgaaaact ggtgatttac aaccattaga 1500

<210> 111

<211> 500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

aaggttacaa gagtgtgaat atcacttttg aacttgatga aaggattgat aaagtactta 60

atgagaagtg ctctgcctat acagttgaac tcggtacaga agtaaatgag ttcgcctgtg 120

ttgtggcaga tgctgtcata aaaactttgc aaccagtatc tgaattactt acaccactgg 180

gcattgattt agatgagtgg agtatggcta catactactt atttgatgag tctggtgagt 240

ttaaattggc ttcacatatg tattgttctt tctaccctcc agatgaggat gaagaagaag 300

gtgattgtga agaagaagag tttgagccat caactcaata tgagtatggt actgaagatg 360

attaccaagg taaacctttg gaatttggtg ccacttctgc tgctcttcaa cctgaagaag 420

agcaagaaga agattggtta gatgatgata gtcaacaaac tgttggtcaa caagacggca 480

gtgaggacaa tcagacaact 500

<210> 112

<211> 500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

atatcaaatt tgctgatgat ttaaaccagt taactggtta taagaaacct gcttcaagag 60

agcttaaagt tacatttttc cctgacttaa atggtgatgt ggtggctatt gattataaac 120

actacacacc ctcttttaag aaaggagcta aattgttaca taaacctatt gtttggcatg 180

ttaacaatgc aactaataaa gccacgtata aaccaaatac ctggtgtata cgttgtcttt 240

ggagcacaaa accagttgaa acatcaaatt cgtttgatgt actgaagtca gaggacgcgc 300

agggaatgga taatcttgcc tgcgaagatc taaaaccagt ctctgaagaa gtagtggaaa 360

atcctaccat acagaaagac gttcttgagt gtaatgtgaa aactaccgaa gttgtaggag 420

acattatact taaaccagca aataatagtt taaaaattac agaagaggtt ggccacacag 480

atctaatggc tgcttatgta 500

<210> 113

<211> 500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

caatggccac aaattatgat ttgagtgttg tcaatgccag attacgtgct aagcactatg 60

tgtacattgg cgaccctgct caattacctg caccacgcac attgctaact aagggcacac 120

tagaaccaga atatttcaat tcagtgtgta gacttatgaa aactataggt ccagacatgt 180

tcctcggaac ttgtcggcgt tgtcctgctg aaattgttga cactgtgagt gctttggttt 240

atgataataa gcttaaagca cataaagaca aatcagctca atgctttaaa atgttttata 300

agggtgttat cacgcatgat gtttcatctg caattaacag gccacaaata ggcgtggtaa 360

gagaattcct tacacgtaac cctgcttgga gaaaagctgt ctttatttca ccttataatt 420

cacagaatgc tgtagcctca aagattttgg gactaccaac tcaaactgtt gattcatcac 480

agggctcaga atatgactat 500

<210> 114

<211> 440

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

tactgtgatc tgggactaca aaagagatgc tccagcacat atatctacta ttggtgtttg 60

ttctatgact gacatagcca agaaaccaac tgaaacgatt tgtgcaccac tcactgtctt 120

ttttgatggt agagttgatg gtcaagtaga cttatttaga aatgcccgta atggtgttct 180

tattacagaa ggtagtgtta aaggtttaca accatctgta ggtcccaaac aagctagtct 240

taatggagtc acattaattg gagaagccgt aaaaacacag ttcaattatt ataagaaagt 300

tgatggtgtt gtccaacaat tacctgaaac ttactttact cagagtagaa atttacaaga 360

atttaaaccc aggagtcaaa tggaaattga tttcttagaa ttagctatgg atgaattcat 420

tgaacggtat aaattagaag 440

<210> 115

<211> 960

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240

aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300

ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360

aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420

ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480

tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540

ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600

tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660

tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720

ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780

ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840

gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900

tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960

<210> 116

<211> 940

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 60

gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 120

tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 180

ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 240

gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 300

tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 360

cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 420

ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 480

aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 540

aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 600

ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 660

ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 720

cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 780

acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 840

ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 900

cctgttgcta ttcatgcaga tcaacttact cctacttggc 940

<210> 117

<211> 960

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

gtgtttattc tacaggttct aatgtttttc aaacacgtgc aggctgttta ataggggctg 60

aacatgtcaa caactcatat gagtgtgaca tacccattgg tgcaggtata tgcgctagtt 120

atcagactca gactaattct cctcggcggg cacgtagtgt agctagtcaa tccatcattg 180

cctacactat gtcacttggt gcagaaaatt cagttgctta ctctaataac tctattgcca 240

tacccacaaa ttttactatt agtgttacca cagaaattct accagtgtct atgaccaaga 300

catcagtaga ttgtacaatg tacatttgtg gtgattcaac tgaatgcagc aatcttttgt 360

tgcaatatgg cagtttttgt acacaattaa accgtgcttt aactggaata gctgttgaac 420

aagacaaaaa cacccaagaa gtttttgcac aagtcaaaca aatttacaaa acaccaccaa 480

ttaaagattt tggtggtttt aatttttcac aaatattacc agatccatca aaaccaagca 540

agaggtcatt tattgaagat ctacttttca acaaagtgac acttgcagat gctggcttca 600

tcaaacaata tggtgattgc cttggtgata ttgctgctag agacctcatt tgtgcacaaa 660

agtttaacgg ccttactgtt ttgccacctt tgctcacaga tgaaatgatt gctcaataca 720

cttctgcact gttagcgggt acaatcactt ctggttggac ctttggtgca ggtgctgcat 780

tacaaatacc atttgctatg caaatggctt ataggtttaa tggtattgga gttacacaga 840

atgttctcta tgagaaccaa aaattgattg ccaaccaatt taatagtgct attggcaaaa 900

ttcaagactc actttcttcc acagcaagtg cacttggaaa acttcaagat gtggtcaacc 960

<210> 118

<211> 962

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

aaaatgcaca agctttaaac acgcttgtta aacaacttag ctccaatttt ggtgcaattt 60

caagtgtttt aaatgatatc ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg 120

ataggttgat cacaggcaga cttcaaagtt tgcagacata tgtgactcaa caattaatta 180

gagctgcaga aatcagagct tctgctaatc ttgctgctac taaaatgtca gagtgtgtac 240

ttggacaatc aaaaagagtt gatttttgtg gaaagggcta tcatcttatg tccttccctc 300

agtcagcacc tcatggtgta gtcttcttgc atgtgactta tgtccctgca caagaaaaga 360

acttcacaac tgctcctgcc atttgtcatg atggaaaagc acactttcct cgtgaaggtg 420

tctttgtttc aaatggcaca cactggtttg taacacaaag gaatttttat gaaccacaaa 480

tcattactac agacaacaca tttgtgtctg gtaactgtga tgttgtaata ggaattgtca 540

acaacacagt ttatgatcct ttgcaacctg aattagactc attcaaggag gagttagata 600

aatattttaa gaatcataca tcaccagatg ttgatttagg tgacatctct ggcattaatg 660

cttcagttgt aaacattcaa aaagaaattg accgcctcaa tgaggttgcc aagaatttaa 720

atgaatctct catcgatctc caagaacttg gaaagtatga gcagtatata aaatggccat 780

ggtacatttg gctaggtttt atagctggct tgattgccat agtaatggtg acaattatgc 840

tttgctgtat gaccagttgc tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct 900

gcaaatttga tgaagacgac tctgagccag tgctcaaagg agtcaaatta cattacacat 960

aa 962

<210> 119

<211> 228

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

atgtactcat tcgtttcgga agagacaggt acgttaatag ttaatagcgt acttcttttt 60

cttgctttcg tggtattctt gctagttaca ctagccatcc ttactgcgct tcgattgtgt 120

gcgtactgct gcaatattgt taacgtgagt cttgtaaaac cttcttttta cgtttactct 180

cgtgttaaaa atctgaattc ttctagagtt cctgatcttc tggtctaa 228

<210> 120

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

atggcagatt ccaacggtac tattaccgtt gaagagctta aaaagctcct tgaacaatgg 60

aacctagtaa taggtttcct attccttaca tggatttgtc ttctacaatt tgcctatgcc 120

aacaggaata ggtttttgta tataattaag ttaattttcc tctggctgtt atggccagta 180

actttagctt gttttgtgct tgctgctgtt tacagaataa attggatcac cggtggaatt 240

gctatcgcaa tggcttgtct tgtaggcttg atgtggctca gctacttcat tgcttctttc 300

agactgtttg cgcgtacgcg ttccatgtgg tcattcaatc cagaaactaa cattcttctc 360

aacgtgccac tccatggcac tattctgacc agaccgcttc tagaaagtga actcgtaatc 420

ggagctgtga tccttcgtgg acatcttcgt attgctggac accatctagg acgctgtgac 480

atcaaggacc tgcctaaaga aatcactgtt gctacatcac gaacgctttc ttattacaaa 540

ttgggagctt cgcagcgtgt agcaggtgac tcaggttttg ctgcatacag tcgctacagg 600

attggcaact ataaattaaa cacagaccat tccagtagca gtgacaatat tgctttgctt 660

gtacagtaa 669

<210> 121

<211> 1069

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

taaattccct cgaggacaag gcgttccaat taacaccaat agcagtccag atgaccaaat 60

tggctactac cgaagagcta ccagacgaat tcgtggtggt gacggtaaaa tgaaagatct 120

cagtccaaga tggtatttct actacctagg aactgggcca gaagctggac ttccctatgg 180

tgctaacaaa gacggcatca tatgggttgc aactgaggga gccttgaata caccaaaaga 240

tcacattggc acccgcaatc ctgctaacaa tgctgcaatc gtgctacaac ttcctcaagg 300

aacaacattg ccaaaaggct tctacgcaga agggagcaga ggcggcagtc aagcctcttc 360

tcgttcctca tcacgtagtc gcaacagttc aagaaattca actccaggca gcagtagggg 420

aacttctcct gctagaatgg ctggcaatgg cggtgatgct gctcttgctt tgctgctgct 480

tgacagattg aaccagcttg agagcaaaat gtctggtaaa ggccaacaac aacaaggcca 540

aactgtcact aagaaatctg ctgctgaggc ttctaagaag cctcggcaaa aacgtactgc 600

cactaaagca tacaatgtaa cacaagcttt cggcagacgt ggtccagaac aaacccaagg 660

aaattttggg gaccaggaac taatcagaca aggaactgat tacaaacatt ggccgcaaat 720

tgcacaattt gcccccagcg cttcagcgtt cttcggaatg tcgcgcattg gcatggaagt 780

cacaccttcg ggaacgtggt tgacctacac agctgccatc aaattggatg acaaagatcc 840

aaatttcaaa gatcaagtca ttttgctgaa taagcatatt gacgcataca aaacattccc 900

accaacagag cctaaaaagg acaaaaagaa gaaggctgat gaaactcaag ccttaccgca 960

gagacagaag aaacagcaaa ctgtgactct tcttcctgct gcagatttgg atgatttctc 1020

caaacaattg caacaatcca tgagcagtgc tgactcaact caggcctaa 1069

Claims (3)

1. An siRNA, which consists of a sense strand and an antisense strand, wherein the sense strand is as shown in SEQ ID NO: 62, and the antisense strand is the nucleotide sequence shown as SEQ ID NO: 63;

wherein the content of the first and second substances,

sense strand 5'-CUAGUUAUCAGACUCAGACUA-3' (SEQ ID NO: 62),

antisense strand 5'-GUCUGAGUCUGAUAACUAGCG-3' (SEQ ID NO: 63);

the sense strand of the siRNA is SEQ ID NO: the 1 st, 2 nd, 3 rd, 19 th, 20 th and 21 th nucleotides of the 62 nucleotide sequence are all 2' -O-methyl ribonucleotides;

the phosphate groups between the 1 st, 2 nd and 3 rd nucleotides and the phosphate groups between the 19 th, 20 th and 21 th nucleotides of the nucleotide sequence of the sense strand of the siRNA are all phosphorothioate groups;

the 5' end of the sense strand of the siRNA is connected with cholesterol.

2. Use of the siRNA of claim 1 for the preparation of a medicament for the prevention and/or treatment of COVID-19.

3. A kit comprising the siRNA of claim 1, wherein said kit is functional for treating COVID-19.

Images