NL2032385B1 - EXOGENOUS ARTIFICIAL miRNA CAPABLE OF EFFECTIVELY INHIBITING REPLICATION OF PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV) AND USE THEREOF - Google Patents

EXOGENOUS ARTIFICIAL miRNA CAPABLE OF EFFECTIVELY INHIBITING REPLICATION OF PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV) AND USE THEREOF Download PDF

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NL2032385B1
NL2032385B1 NL2032385A NL2032385A NL2032385B1 NL 2032385 B1 NL2032385 B1 NL 2032385B1 NL 2032385 A NL2032385 A NL 2032385A NL 2032385 A NL2032385 A NL 2032385A NL 2032385 B1 NL2032385 B1 NL 2032385B1
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insdqualifier
insdseq
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artificial mirna
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Qian Kun
Zhu Tingfan
Wang Qianqian
Zhou Xiaobing
Shen Zijun
Deng Jianzhong
Zheng Jiangao
Qian Jinhan
Qin Aijian
Chai Wenxian
Liu Jinbiao
Wang Jian
Jin Wenjie
Ye Jianqiang
Wang Jiao
Shao Hongxia
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Univ Yangzhou
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

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Abstract

The present disclosure relates to an exogenous artificial miRNA capable of effectively inhibiting replication of porcine epidemic diarrhea virus (PEDV). The exogenous artificial miRNA includes a double-stranded nucleotide selected from the group 5 consisting of 1) and 2): 1) sequences of a sense strand and an antisense strand shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and 2) sequences of a sense strand and an antisense strand shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Compared with a siRNA targeting a gene encoding a PEDV protein, the artificial miRNA screened in the present disclosure for targeting a conserved sequence of an N gene of a virus can 10 inhibit the replication of the PEDV. The artificial miRNA can be developed as a novel biological preparation against the PEDV, and can be used for breeding a disease-resistant pig line.

Description

EXOGENOUS ARTIFICIAL miRNA CAPABLE OF EFFECTIVELY
INHIBITING REPLICATION OF PORCINE EPIDEMIC DIARRHEA VIRUS
(PEDV) AND USE THEREOF
TECHNICAL FIELD
[0001] The present disclosure belongs to the research field of genetic engineering and biological products. The present disclosure specifically relates to a preparation method and use of an exogenous artificial miRNA capable of effectively inhibiting replication of porcine epidemic diarrhea virus (PEDV).
BACKGROUND ART
[0002] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive RNA virus belonging to the genus Alphacoronavirus of the family Coronaviridae. Porcine epidemic diarrhea (PED) is a highly-contagious intestinal infectious disease caused by the PEDV.
PEDV infection can occur in pigs of all ages, and is most severe in piglets, with incidence and mortality generally reaching 100%. At present, PEDV mutant strains are predominant strains of the PEDV in China, and prevalence of the mutant strains in Chinese pig herds cannot be ignored. It is even widely believed that variant PED may be one of the most serious diarrheal diseases affecting piglets in the next few years. Like most viral diseases, there is no effective drug for the prevention and treatment of PED, and PED control mainly depends on vaccination. For decades, domestic and foreign swine disease researchers have developed PED inactivated vaccines, live vaccines, genetically- engineered vaccines and the like; however, there is no specific drug for the PED in the market, and the research and development of PED vaccines has not achieved very satisfactory results. Therefore, it is necessary and urgent to find a novel therapeutic strategy to treat PEDV infection.
[0003] RNA interference (RNAI) is an emerging gene silencing technology. The RNAI means that cells stimulate related enzyme complexes to cleave and degrade homologous mRNA using an exogenous or endogenous double-stranded small interfering RNA (siRNA or microRNA), thereby blocking gene expression at a post-transcriptional level to achieve attenuated expression or non-expression of homologous genes. In terms of anti-viral infection, RNAi inhibits almost all viral replication. However, siRNA-mediated
RNAi silencing of gene expression is achieved by binding small fragments of double-
stranded RNAs to an mRNA of a target gene in a sequence-specific manner. More and more studies have found that many viruses can evade RNAi by mutating target gene sequences and producing inhibitors under therapeutic selection pressure; and virus- targeted RNAi therapy may have a reduced efficacy due to virus mutation. However, compared with siRNA, miRNA only needs to partially bind to the target gene to function, thereby largely avoiding the failure of RNAi therapy due to virus mutation. The miRNAs do not require strict complementary base pairing with the targets, such that it may be more difficult for the virus to escape miRNA-specific silencing through mutation, which is more advantageous for the treatment of mutated viruses. Currently, siRNAs targeting viral genes are used to inhibit the replication of PEDV.
[0004] PEDV genome includes a single-stranded and positive-stranded linear RNA of 27 kb to 32 kb, with a cap structure (cap) at a 5'-end and a Poly(A) tail at a 3'-end; and the genome includes 3 non-structural proteins and 4 structural proteins. The structural proteins include: small membrane protein (Envelope, E), membrane glycoprotein (Membrane, M), nucleocapsid protein (Nucleocapsid, N), and spike protein (Spike, S).
The N protein can bind to viral RNA, providing a structural basis for the viral nucleocapsid protein, and can also bind to cell membranes and phospholipids to promote viral assembly. The N protein is highly-antigenic and conserved among similar coronaviruses, playing an important role in the replication of PEDV.
SUMMARY
[0005] The present disclosure provides an exogenous artificial miRNA sequence capable of significantly inhibiting replication and proliferation of PEDV, and aims to solve the technical problem of difficult epidemic control of the PEDV.
[0006] The present disclosure provides an exogenous artificial miRNA capable of effectively inhibiting replication of PEDV, including a double-stranded nucleotide selected from the group consisting of 1) and 2):
[0007] 1) a double-stranded nucleotide with sequences of a sense strand and an antisense strand shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and
[0008] SEQ ID NO: 1: 5-AGTACGAGTCCTATAACGGAG-3
[0009] SEQ ID NO: 2: 5-CTCCGTTATAGGACTCGTACT-3
[0010] 2) a double-stranded nucleotide with sequences of a sense strand and an antisense strand shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively;
[0011] SEQ ID NO: 3: 5-TAAACTGGCGATCTGAGCATA-3
[0012] SEQ ID NO: 4: S-TATGCTCAGATCGCCAGTTTA-3
[0013] the sequences of the double-stranded nucleotide of 1) and 2) are named as pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048, respectively.
[0014] The present disclosure further provides a pharmaceutical composition, including the exogenous artificial miRNA as an active constituent.
[0015] The present disclosure further provides use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV in preparation of a drug for treating
PED.
[0016] The present disclosure further provides use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV in a research on a function of a
PEDV N gene by genome modification.
[0017] The present disclosure further provides use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV in preparation of a biological preparation for treating PED.
[0018] The present disclosure further provides use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV in preparation of an anti-PEDV transgenic cell line.
[0019] The present disclosure further provides use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV in breeding an anti-PEDV transgenic pig.
[0020] Compared with a siRNA targeting a gene encoding a PEDV protein, the artificial miRNA screened in the present disclosure for targeting a conserved sequence of an N gene of a virus can inhibit the replication of the PEDV. The artificial miRNA can be developed as a novel biological preparation against the PEDV, and can be used for breeding a disease-resistant pig line.
[0021] In the present disclosure, the artificial miRNA is designed according to a conserved sequence of the PEDV N gene, and vero cells are transfected with an artificial miRNA expression vector; on the basis of optimizing a transfection efficiency, an inhibitory effect of the artificial miRNA on viral genes and replication thereof is detected by 50% tissue culture infective dose (TCIDso) and real-time PCR, thereby selecting the artificial miRNA capable of effectively inhibiting the PEDV.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a structural schematic diagram of an artificial miRNA expression vector; where
[0023] introduction in a 5'-3' order is as follows: an initial 5 nucleotide sequences (TGCTQ) in a Top chain were derived from a miRNA-155, an endogenous microRNA; in a bottom chain, a 5' end provides a 4-nucleotide suspension sequence that corresponds to a 4-nucleotide suspension tail of a linear structure of a pcDNA6.2-GW/EmGFP-miR plasmid vector, which can be combined into a double-stranded structure; reverse complementary 21 nucleotide sequences behind a Top chain (namely a mature miRNA sequence) may target a target sequence after being transcribed, such that the nucleotide sequences need to be complementary to a target RNA during design;
[0024] FIG. 2 shows real-time PCR detection of an N gene in PEDV HN-13 strain- infected miRNA-expressing cells; where
[0025] ordinate is a relative content of PEDV N gene amplification products in each group; Control represents normal untransfected cells; pcDNA6.2-miR-N-349 represents transfected cells of an artificial miRNA pcDNA6.2-miR-N-349 expression vector targeting a 349 site of the PEDV N gene; pcDNA6.2-miR-N-1048 represents transfected cells of an artificial miRNA pcDNA6.2-miR-N-1048 expression vector targeting a 1048 site of the PEDV N gene; and pcDNA6.2-miR-N-neg represents empty transfected cells as a control; and
[0026] FIG. 3 shows virus titer detection of the PEDV in the PEDV HN-13 strain- infected miRNA-expressing cells; where
[0027] ordinate represents virus titer (TCIDso), pcDNA6.2-miR-N-349 represents transfected cells of an artificial miRNA pcDNA6.2-miR-N-349 expression vector targeting a 349 site of the PEDV N gene; pcDNA6.2-miR-N-1048 represents transfected cells of an artificial miRNA pcDNA6.2-miR-N-1048 expression vector targeting a 1048 site of the PEDV N gene; and
[0028] pcDNA6.2-miR-N-neg represents empty transfected cells as a control.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0029] Source of biological materials: a pcDNA6.2-GW/EmGFP-miR vector is imported from Invitrogen lifetechnologies, USA. A PEDV-HN13 strain is isolated and preserved in the laboratory (strain information refers to: Zhenpeng Zhao. Epidemiological investigation of porcine epidemic diarrhea virus and preliminary establishment of a rapid detection method [D]. Yangzhou University, 2016.).
[0030] Example 1
[0031] I Design of miR RNAi 5 [0032] Sequence alignment was conducted according to existing N gene sequences of all
PEDV strains on an NCBI website; conserved sites were selected among the N genes of different strains of PEDV (Table 1); according to a design principle of artificial miRNA, two target sequences were selected for miRNA double-stranded oligonucleotide sequence design (Table 2), and an artificial miRNA expression plasmid was constructed.
[0033] Table 1 Target sequences of PEDV N gene 1 TTTCAGGATCGTGGCCGCAAA (SEQ IDNO.5) | 52.39 2 CTCCGTTATAGGACTCGTACT (SEQ ID NO.6) | 47.62
GTCGTGGCAATGGCAACAATA (SEQ ID NO.7)
GTCGTGGAGCTTCTCAGAACA (SEQ ID NO.8)
TCAAATGACCGTGGTGGTGTA (SEQ ID NO.9)
GGTGGTGTAACATCACGCGAT (SEQ ID NO.10)
TGCTGTCAAGGATGCACTTAA (SEQ ID NO.11)
GCTGTCAAGGATGCACTTAAA (SEQ ID NO.12)
TATGCTCAGATCGCCAGTTTA (SEQ ID NO.13)
GCCAGTTTAGCACCAAATGTT (SEQ ID NO.14)
[0034] Note: v indicates a target sequence selected for researches.
[0035] II. Synthesis and cloning of double-stranded oligonucleotides
[0036] According to a design principle of the artificial miRNA, an artificial miRNA sequence for PEDV was designed, and a double-stranded oligonucleotide (Table 2) sequence was constructed for an artificial miRNA expression plasmid; the constructed sequence was sent to Tsingke Biotechnology Co., Ltd. for single-stranded oligonucleotide (oligo) synthesis. Each pair of the single-stranded oligonucleotides was annealed, where a reaction system included: 5 pL of a positive-strand DNA oligo (200 uM), 5 uL of a negative-strand DNA oligo (200 uM), 2 pL of a 10= Oligo Annealing Buffer (100 mM
Tris-HCI pH 8.0, 500 mM NaCl, and 10 mM EDTA), and 8 pL of deionized water. The reaction system was incubated at 94°C for 5 min, and slowly cooled to room temperature for annealing. The double-stranded oligonucleotides were cloned into a pcDNA™ 6.2-
GW/EmGFP-miR vector (FIG. 1), and a ligation reaction product was transformed into
TOP10 competent cells. The screening of positive plasmids was conducted, monoclonal colonies were selected for amplification culture, and plasmids were extracted; extracted positive plasmids were identified by enzyme digestion; correct plasmids identified by enzyme digestion were sent to Tsingke Biotechnology Co., Ltd. (Nanjing) for sequencing using an EmGFP forward sequencing primer and an miRNA reverse sequencing primer; correctly-sequenced plasmids were named pcDNA6.2-miR-N-349 and pcDNA6.2-miR-
N-1048.
[0037] Table 2 Sequences of double-stranded oligonucleotides for artificial miRNA expression
[0038] Example 2
[0039] Transient transfection of the constructed plasmid was conducted on vero cells:
[0040] One day before transfection, the vero cells were digested and inoculated into a 12-well cell plate at 410° cells/well. When a cell density reached 70% to 80%, transfection was conducted, and each plasmid was transfected repeatedly in 2 to 3 wells; the cells were divided into pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048 transfection groups and an empty transfection control group; plasmid transfection was conducted using LipofectamineTM 3000 as a transfection reagent. Transfection efficiency was observed after culturing for 24 h, 48 h and 72 h. The results showed that the cells had the highest transfection efficiency at 48 h after infection.
[0041] Detection of an inhibitory effect of PEDV replication:
[0042] Cells were infected with a PEDV-HNI3 strain at the highest transfection efficiency, with a multiplicity of infection (MOI) of 0.1. The cells were harvested 48 h after infection. The collected cells were subjected to total RNA extraction with an
Axyprep total RNA preparation kit (AXYGEN), and digested with a gDNA Eraser (TaKaRa Bio) to remove a cellular genomic DNA. The relative quantitative RT-PCR detection of PEDV N gene transcription was conducted using an 18S gene as an internal reference according to instructions of a SYBR PremixEx Taq™ (TaKaRa Bio (Dalian)
Co., Ltd.). The forward and reverse primer sequences of the internal reference 18S were: 5-TCAGATACCGTCGTAGTTCC-3 (SEQ ID NO: 15) and 5-
TTCCGTCAATTCCTTTAAGTT-3 (SEQ ID NO: 16); and specific primer sequences of the N gene were: 5S-CGATGATCTGGTGGCTGCTGTC-3 (SEQ ID NO: 17) and 5-
TTCCTGCTTAGGCTTCTGCTGTTG-3 (SEQ ID NO: 18). A 20 uL PCR reaction system included: 10 uL of a SYBR Premix ExTaq II (2x), 0.8 pL for each of forward and reverse primers, 0.4 uL of a ROX Reference Dye (50x), 2 uL of a cDNA template obtained by reverse transcription, and 6 pL of dH20. An amplification program included: initial denaturation at 95°C for 30 sec; 95°C for 5 sec and 60°C for 34 sec, conducting 40 cycles; 95°C for 15 sec, 60°C for 60 sec, and 95°C for 15 sec. A PCR product was analyzed by 7500 real-time PCR System software to determine a relative expression level of the PEDV N gene among each group. Meanwhile, the 18s gene was set as a reference gene, and each group was replicated 3 times. The results showed that at 48 h after PEDV infection, the expression of PEDV N gene in vero cells transiently transfected with the pcDNAG6.2-miR-N-349 and the pcDNA6.2-miR-N-1048 was significantly inhibited than that in empty-transfected cells, with an inhibition rate of near 90% (FIG. 2); and the pcDNA6.2-miR-N-349 had the optimal inhibitory effect.
[0043] Cell supernatants were collected 48 h after PEDV infection and viral TCID: assays were conducted on vero cells. The results showed that the inhibitory effect of pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048 on PEDV was significantly different from that of the empty control group 48 h after the PEDV-HN13 strain-infected cells were infected, with a TCIDso of 10 -2.06 and 10 -2.23, respectively; while the
TCID: of the empty control group was 10 -5.09. It can be seen that pcDNA6.2-miR-N- 349 has the optimal inhibitory effect (FIG. 3).
SEQUENCE LISTING
<110> Yangzhou University <120> EXOGENOUS ARTIFICIAL miRNA CAPABLE OF EFFECTIVELY
INHIBITING
REPLICATION OF PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV) AND
USE
THEREOF
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<400> 3 taaactggcg atctgagcat a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of BOTTOM of miR-N-1048 <400> 4 tatgctcaga tcgccagttt a 21
<210> 5 <211> 21
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<400> 7 gtcgtggcaa tggcaacaat a 21
<210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Target sequences of PEDV N gene No.4 <400> 8 gstcgtggagc ttctcagaac a 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Target sequences of PEDV N gene No.5 <400> 9 tcaaatgacc gtggtggtet a 21
<210> 10 <211> 21 <212> DNA <213> Artificial Sequence
<220> <223> Target sequences of PEDV N gene No.6
<400> 10 ggtggtgtaa catcacgcga t 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Target sequences of PEDV N gene No.7 <400> 11 tgctgtcaag gatgcactta a 21
<210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Target sequences of PEDV N gene No.8 <400> 12 gctgtcaagg atgcacttaa a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence
<220> <223> Target sequences of PEDV N gene No.9 <400> 13 tatgctcaga tcgccagttt a 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Target sequences of PEDV N gene No.10
<400> 14 gccagtttag caccaaatgt t 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of internal reference 18S <400> 15 tcagataccg tegtagttec 20
<210> 16 <211> 21
<212> DNA <213> Artificial Sequence <220> <223> Reverse primer of internal reference 18S <400> 16 ttccgtcaat tcctttaagt t 21
<210> 17 <211> 22 <212> DNA <213> Artificial Sequence
<220> <223> Primer 1 of N gene <400> 17 cgatgatctg gtggctgetg tc 22 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer 2 of N gene
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SL </SeguenceDala>
Sz <SequenceData sequenceIDNumber="án> 83 <INSDSeqr
G4 <INSDSeq length»>21</INSDSeqg length» <INSDSeq moltype>DNA</INSDSeg moltype> 36 <INSDSeq divislion»PAT</INSDSeqg division» 37 <INSDSeg feature~tablex 38 <INSDPsaturer 38 <IN3DFeature key>source</IN3DFeature key> 130 <IN3DFeature location>1..21</INSDFeature locations
LOL <INSDFsature qualsg>
LOE <INSDQualifier>
Lox <INSDoualifier name>mol type“ /INSDQualifier name> 10d <INSDQualifisr value>other DNA</INSDQualifier valuer 105 </INSDOualifier> ijs <INSDOualifier id="g8">
La <IN3DQualifier name>note</INSDQualifier name> 108 <INSDQualifiler value>DNA sequence of BOTTOM of miR-N-1048</1INSDQuelifier value>
LOG «</INSDOQualifier»>
LLG <INSDQualifler id=ng¥7 > iii <INSDQualifier name>organism</INSDQualifier name> 112 <IN3DQualifier value>synthetic construct</INSDQualifier value» iis </INSDOQuali fier 144 </INSDFesature duals» 115 </INSDFeature>
Tie </INSDSeg feature-table>
LL? <INSDSeq sequance>tatgetcagategecagttta</INSDSeq sequencer 118 </INSDSe 113 </Seguencedata> 1E0 <SequenceData samience ID umbe n="8% > 121 <INSDSeq> 122 <IN3DSeqy lengibh>21</INSDSeq length
Las <INSDSeq moltype>DNA</INSDSeg moltype» 1z4 <INSDSeq division>PAT</INSDSeg division»
Las <INSDSeq feature-table> 158 <INSDFeature> 187 <INSDFeature key>sourcec/INSDFeature key» 128 <INSDFearure location>l..21</INSDFeature location» 12% <INSDFeature guals> 130 <INSDOualifien> 3 <INSDQualifier name>mol type</INSDQualiifier name>
Laz <INSDQualifier valuerother DNA</INSDQualifier value»
13 </INSDOualifiers 134 <INSDQualiifler id="g19'> ijs <INSDQualifier name>note</INSDQualifier name> 138 <INS3DQualifier value> Target sequences of PEDV N gene
NO.1</INSDQualifisr value» 1a </INSDQuali fier» 138 <INSDQuaiifler id="g3*>
LEG <INSDOQualifier namerorganism</INSDQualifier name>
LAG <INSDgQualifier value>synthetic construct“/INSDGualifier value» 141 </INSDOualifier> 142 </IN3DFeature guala> 143 </INSDFeaturs> u 144 </INSDSegy featurs-table> ids <INSDSeq seguence>tttcaggategtggccgcaaa-/INSDSeq sequence»
HEE </INSDSeg> 147 </SequenceData> 148 <Hedgquencelata segusnceliNumec=N&"> 143 <INSDSeq> 150 <IN3DSeq length»21</INSDSeq length» 151 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 152 <IN3DSeq divisior>PAT</INSDIeqg division» 153 <INSDSeq feature-table> 154 <INSDFeature>
LE <INSDFeaturs keyrsource</INSDFeaturs Key» 158 <INSDFeature location>l..21</IN3DFeature location» 157 <INSDhFeature quels» 158 <INSDQualifier> i158 <IN3DQualifier name>mol type</INSDQualifisr name>
Lan <INSDQualifiler valuerother DNA</INGDGualifier value»
Lel <{INSDQualifier»
Led <INSDQualifier id="gid>
LES CINSDQualifisr name>note</INSDQvali fier name> 164 <INSDQualifier value>Target sequences of PEDV N gene
No.2</INSDOQualifier value» 145 </INSDQualifier> u 1484 <INSDOualifier Ld=Yglin>
La <IN3DQualifier namerorganism“/INSDQuali fier name>
Los <INSDQualifier valuersynthetic construct</INSDQualifier values»
TEs </INSDOualifiers
LEG </INSDFeaturs guals> 171 </INSDFeaturer 172 </INBDSeq feature-table> 173 <IN3DSeqg sequenzs>eteegttataggactcgtact</INSDSeqy sequencer 174 </INSDSeg> iF </Zequencebatar
HE <Sequencebata seguenasibNumbar=snits 177 AINSDSeq 17a <INSDSeq length>21</IN3DSeq length» ijs <INSDSeq moltype>DNA</INSDSeq moltype> inn <IN3DSeq division»PAT</INSD3eq division»
LEL <INSDSeq fearure-tabier inn <INSDFeaiburs> 183 <INSDFeature key>source</INIDFeature key>
Tid <INSDFeature location>l..21</INSDFeature location
LEI <INSDFeature qguals> u iss <INSDQualifier»>
LET <INSDQualifier name>mol type</iNSDQualifier name> i188 <INSDuuelifier value>other DNA</INSDOualifier valued iss </INSDQualifier> u 180 <INSDQualifier id="gidnx>
Lel <INSDQualifier name»note</INSDQualifier named
Led <INSDQualifier valuerTarget sequences of PEDV N gene
No. 3</INSDgualifier value» 193 </INSDOualifier> 10d <INSDOualifier id="qL3t> ijb <IN3DQualifier namevorganism“/INSDQualifier name> 138 <INSDQualifier valuersynthetic construct</INSDQuallifier value» 1a </INSDQualifier> 183 </INSDFeature quals>
L&G </IN3DFeature> 200 “/INSDSeqg fesature-table> 201 <IN3D3eq sequence>gtegtggcaatggcaacaata</INSDSegq sequencer 202 </INSDSeq> u 203 </SeguencaData> 204 <SequenceData zaquencalDNumben=ngn> 20% <INSDSeqd»> 208 <INSDSeq length»>21</INSDSeqg length» wi’ <INSDSeq moltype>DNA</INSDSeg moltype> 208 <INSDSeq divislion»PAT</INSDSeqg division» 203 <INSDSeg Iearturertabie» 210 <INSDPsaturer
Zil <IN3DFeature key>source</IN3DFeature key> 212 <IN3DFeature location>1..21</INSDFeature locations 243 <INSDFsature qualsg> 24 <INSDuuelifier> gls <INSDoualifier name>mol type“ /INSDQualifier name>
LS <INSDQualifisr value>other DNA</INSDQualifier valuer 227 </INSDOualifier> 218 <INSDOualifier id="qgiór> 218 <IN3DQualifier name>note</INSDQualifier name> 226 <INSDQualifiler valuerTarget sequences of PEDV N gene
No. 4</INSDQualifier value» 221 </IN3DQualifier>
Ad <INSDQualifler ia="gi5> el “INSDoualifier name>organism</INSDQualifier name>
Zid <IN3DQualifier value>synthetic construct /INSDQualifier values 225 </THNSDOQualifiar> u 228 </INSDFesature duals» 227 </INSDFeature> 225 </INSDSeg feacture-taebhle> men <INSDSeq sequsncergtegtggagettctcagaaca“/INSDSeq sequencer 3E </INSDSeg> 231 </Seguencedata> 222 <SequenceData semiencelDNumber="S%> 233 <INSDSeq> 234 <IN3DSeqy lengibh>21</INSDSeq length 23% <INSDSeq moltype>DNA</INSDSeg moltyper 236 <INSDSeg division>PAT</INShIeqg division» ws’ <INSDSeq feature-table>
L238 <INZDFeature> 233 <INSDFeature key>sourcec/INSDFeature key» 240 <INSDFearure location>l..21</INSDFeature location» 241 <INSDFeature guals> 242 <INSDQualifier> 2473 <INSDQualifier name>mol type</INSDQualiifier name> 244 <INSDQualifier valuerother DNA</INSDQualifier value»
HAL </INSDOualifiers
ZAG <INSDQualiifler id="g18"> dT <INSDQualifier name>note</INSDQualifier name> 248 <INS3DQualifier value>Target sequences of PEDV N gene
No.5</INSDQualifisr value» 249 </INSDQuali fier» 250 <INSDQuaiifier id="gijx> 250 <INSDOQualifier namerorganism</INSDQualifier name>
PAY <INSDQualiflsr value>synthetic construct /INSDQualifier value> 252 </INSDOQualifier> 254 </IN3DFeature guala> 255 </THSDFeatura> u 258 </INSDSegy featurs-table> 25% <INSDSeq seguencertcaaatgaccgtggtggtgta-/INSDSeq sequence> 203 </INSDSeg>
LEG </SequenceData>
Ze <SequenceData segusnceliNumec=MNLGM> 251 <INSDSeqg> 262 <IN3DSeq length»21</INSDSeq length» 263 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 264 <IN3DSeq divisior>PAT</INSDIeqg division» 28% <INSDSeq feature-table>
Zet <INSDFeature>
“dé <INSDFeaturs keyrsource</INSDFeaturs Key»
ZEE <INSDFeature location>l..21</IN3DFeature location» 283 <INSDhFeature quels» 270 <INSDQualifier> 271 <IN3DQualifier name>mol type</INSDQualifisr name> 292 <INSDGualifier valuerother DNA /INSDGuelifier value» 273 </INSDQualifier> IJ
Zia <INSDQualifier ia=stgald¥> wis CINSDQualifisr name>note</INSDQvali fier name> 278 <INSDQualifier value>Target sequences of PEDV N gene
No. 6</INSDOQualifier value» 277 </INSDQualifier> u
SE <INSDoualifier 1d="gle"> 250 <IN3DQualifier namerorganism“/INSDQuali fier name> 2848 <INSDQualifier valuersynthetic construct</INSDQualifier valued 251 </INSDOualifiers
ZHI </INSDFeaturs guals> 282 </INSDFeaturer 284 </INBDSeq feature-table> 285 <IN3DSeqg sequencsrggtggtgtaacatcacgegat-/INSDSeq sequence» 286 </INSDSeq> 287 </Zequencebatar 233 <Sequencebata seguengsiiNuombar="iilis “6e <INSDSeg>
Zal “INSDSeq length>21</IN3DSeq Length> 231 <INSDSeq moltype>DNA</INSDSeq moltype> 232 <IN3DSeq division»PAT</INSD3eq division» 233 <INSDSeq fearure-tabier 294 <INSDFeabture> 28% <INSDFeature key>source</INIDFeature key> 298 <INSDFPeature locationrl..21</INSDFeature location wad <INSDFeature qguals> u das <INSDQualifier»> 233 <INSDQualifier name>mol type</iNSDQualifier name> 200 <INSDuuelifier value>other DNA</INSDOualifier valued
ZOL </INSDQualifier> u 302 <INSDQualifier id='"g22"x> 305 <INSDQualifier name»note</INSDQualifier named 304 <INSDQualifier valuerTarget sequences of PEDV N gene
No. 7</INSDgualifier value» 305 </INSDOualifier> 208 <INSDOualifier id="q21">
S07 <IN3DQualifier namerorganism</INSDQualifiesr name> 208 <INSDQualifier valuersynthetic construct</INSDQuallifier value» 300 <{INSDQualifier»
SLO </INSDFeature quals>
SAL </INSDFeature> u 317 </INSDSeg feature-iablex> 212 <IN3D3eq sequence>tgctgtcaaggatgcacttaa</INSDSegq sequencer
Sid </INSDSeq> u zlib </SeguenceDala> 318 <SequenceData zaquencalDNumber="3i2%>
BL? <INSDSeqr
RRS <INSDSeq length»>21</INSDSeqg length»
SLS <INSDSeq moltype>DNA</INSDSeg moltype>
Sz <INSDSeq divislion»PAT</INSDSeqg division» zal <INSDSeg Iearturertabie»
SEZ <INSDPsaturer 223 <IN3DFeature key>source</IN3DFeature key> 324 <IN3DFeature location>1..21</INSDFeature locations 32 <INSDFsature qualsg> 326 <INSDuuelifier>
Sj <INSDoualifier name>mol type“ /INSDQualifier name> 378 <INSDQualifisr value>other DNA</INSDQualifier valuer 373 </INSDOualifier> 230 <INSDOualifier 1d=VgR4an> 234 <IN3DQualifier name>note</INSDQualifier name> 332 <INSDQualifiler valuerTarget sequences of PEDV N gene
No.8«/INSDQualifier value»
IRE </INSDOualifier>
354 <INSDQualifler ia="g43> 335 <INSDQualifier name>organism</INSDQualifier name> 238 <IN3DQualifier value>synthetic construct</INSDQualifier value» 337 </INSDOQuali fier 338 </INSDFesature duals» 339 </INSDFeature> 340 </INSDSeg feature-tabhle>
RE <INSDSeq sequance>getgtecaaggatgeacttaaa</INSDSeq sequencer 347 </INSDSe 342 </Seguencedata>
REE <SequenceData seguencelDihumber="13"> 245 <INSDSeq> 348 <IN3DSeqy lengibh>21</INSDSeq length 347 <INSDSeq moltype>DNA</INSDSeg moltyper sds <INSDSeg division>PAT“/INSDSeq division»
REESE <INSDSeq feature-table>
IEG <INSDFeature> 251 <INSDFeature key>sourcec/INSDFeature key» 252 <INSDFearure location>l..21</INSDFeature location» 253 <INSDFeature guals> 354 <INSDOualifier> 355 <INSDQualifier name>mol type“/INSDQualifier name>
IRE <INSDQualifier valuerother DNA</INSDQualifier value»
SLT </INSDOualiLfier»> 358 <INSDQualiifler id="gZ8'> 353 <INSDQualifier name>note</INSDQualifier name> 260 <INS3DQualifier value>Target sequences of PEDV N gene
No.9</INSDQualifier valua> 351 </INSDQuali fier» u 38D <INSDQuaiifier id="g25x>
BOS <INSDQualifier namerorganism</INSDQualifier name> 384 <INSDQualiflsr value>synthetic construct /INSDQualifier value> 385 </INSDOualifier> 288 </INBDFeature gvals> 367 </THSDFeatura> 388 </INSDSegy featurs-table> zoo <INSDSeq seguence>tatgctcagatcgccagttta-/INSDSeq sequence» </INSDSeg>
Sl </SequenceData> 37 <SequenceData seguanoellNumbe="14%>
ST <INSDSeqg> 27d <IN3DSeq length»21</INSDSeq length» 27H ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 378 <IN3DSeq divisior>PAT</INSDIeqg division» 3 <INSDSeq feature-table>
SUG <INSDFeature>
RAS <INSDFeaturs keyrsource</INSDFeaturs Key»
IGG <INSDFeature location>l..21</IN3DFeature location»
SE: <INSDhFeature quels» 252 <INSDQualifier> 283 <IN3DQualifier name>mol type</INSDQualifisr name> 283 <INSDQualifler valuerother DNA</INSTGualifier value»
IRE </INSDQualifier>
REE <INSDQualifier ia="gli¥> a7 CINSDQualifisr name>note</INSDQvali fier name>
IEE <INSDQualifier value>Target sequences of PEDV N gene
No.10</INSD0ualifier value» 289 </INSDQualifier> u 250 <INSDOualifier ld='g2jn> 381 <INSDQualifier namerorganism“/INSDQuali fier name>
Gn <INSDQualifier valuersynthetic construct</INSDQualifier valued
KER </INSDOualifiers 344d </INSDFeaturs guals> 335 </TNSDFeaturer 236 </INBDSeq feature-table> 237 ZINSDSeq sequencsrgecagtttagcaccaaatgtt-/INSDSeq sequencer 308 </INSDSeg> 390 </SegusnceData»> 400 <SequenceData zsegvencelnNumbser="ijx>
AGL AINSDSeq 40% <INSDSeq length>20</IN3DSeq length» 202 <INSDSeq moltype>DNA</INSDSeq moltype> 404 <IN3DSeq division»PAT</INSD3eq division» 405 <INSDSeq feature-table> 40g <INSDFeabture> 407 <INSDFeature key>source</INIDFeature key>
Aus <INSDFeature location>l..20</INSDFeature location> 40% <INSDFeature qguals> u 410 <INSDQualifier»> 411 <INSDQualifier name>mol type</iNSDQualifier name> 412 <INSDuuelifier value>other DNA</INSDOualifier valued 41a </TNSDQualifisc> u 4iá <INSDQualifier id='"g30"x> 445 <INSDQualifier namernote</INSDQualifier named 414 <INSDQualifier valuerForward primer of internal reference 18S</INSDQualifier value» 417 </INSDQualifiers> 418 <INSDOualifier id="q28%> 413 <IN3DQualifier namerorganism</INSDQualifiesr name> 420 <INSDQualifier valuersynthetic construct</INSDQuallifier value» 421 <{INSDQualifier»
Anz </INSDFeature quals>
A </INSDFeaturer 474 “/INSDSeqg fesature-table> 425 <“INSDSeg sequencertcagatacegtegtagttee</IN3SDSeq sequence» 428 </INSDSegqs u da? </SeguenceDala> 328 <SequenceData zaquencalDNumber="18"> 429 <INSDSeq>
A430 <INSDSeq length»>21</INSDSeqg length» 431 <INSDSeq moltype>DNA</INSDSeg moltype> 43% <INSDSeq divislion»PAT</INSDSeqg division» 432 <INSDSeg feature~tablex di <INSDFeaturer 425 <IN3DFeature key>source</IN3DFeature key> 336 <IN3DFeature location>1..21</INSDFeature location 437 <INSDFsature qualsg> 4735 <INSDuuelifier> 430 <INSDoualifier name>mol type“ /INSDQualifier name> dan <INSDQualifisr value>other DNA</INSDQualifier valuer 441 </INSDOualifier> 442 <INSDOualifier id="qgZar> 343 <IN3DQualifier name>note</INSDQualifier name> 343 <INSDQualifiler value» Reverse primer of internal reference 18s</INSDQualifier value 445 </INSDOualifier> 446 <INSDQualifler ia="gSl> 447 <INSDQualifier name>organism</INSDQualifier name> 448 <IN3DQualifier value>synthetic construct /INSDQualifier values 449 </INSDQuali fier» u 450 </INSDFesature duals» <“/INSDFeature> 45% </INSDSeg feature-tabhle> 453 <INSDSeq sequance>tteegtecaattectttaagtt</INSDSeq sequencer 454 </INSDSe 455 </Seguencedata> 458 <SequenceData seguencelDihumber="17"> 457% <INSDSeq> 458 <INSDSeq lengibh»22</INSDSeq length 450 <INSDSeq moltype>DNA</INSDSeg moltype»
A460 <INSDSeq division>PAT</INSDSeg division» 4871 <INSDSeq feature-table> 46% <INSDFeature> 462 <INSDFeature key>sourcec/INSDFeature key» 464 <INSDFearure location>l..22</INSDFeature location» dab <INSDFeature guals> 488 <INSDOualifien> 447 <INSDQualifier name>mol type</INSDQualiifier name> 463 <INSDQualifier valuerother DNA</INSDQualifier value»
48% </INSDOualifiers 470 <INSDQualiifler id="g34"> 471 <INSDQualifier name>note</INSDQualifier name> 472 <INS5DQualifier value>Primer 1 of N gene</INSDQualifisr value> 473 </INSDQuali fier» 474 <INSDQuaiifier ia="gl39>» 475 <INSDOQualifier namerorganism</INSDQualifier name> 476 <INSDQualiflsr value>synthetic construct /INSDQualifier value> 477 </INSDOualifier> 478 </IN3DFeature guala> 479 </INSDFeaturer u 380 </INSDSegy featurs-table> 481 <INSDSeq sequence>cgatgatctggtggetgetgte</INSDSey sequences
ASZ </INSDSeg> 455 </SequenceData> 464 <SequenceData segusnceliNumec=MNLS®> 488 <INSDSeq> 488 <IN3DSeq length»24</INSDSeq length» asi ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 385 <INSDSeag divisior>PAT</INSDIeqg division» 480 <INSDSeq feature-table>
AGO <INSDFeature> del <INSDFeaturs keyrsource</INSDFeaturs key>
AO <INSDFeature location>l..24</IN3DFeature location» 452 <INSDhFeature quels» 434 <INSDQualifier> 4355 <IN3DQualifier name>mol type</INSDQualifisr name> 484 <INSDQualifiler valuerother DNA</INGDGualifier value» 487 </INSDQualifier> 405 <INSDQuelifier id="g3ó"> 44% CINSDQualifisr name>note</INSDQvali fier name>
S04 <INSDQualifier valuse>Primer 2 of N gene</INSDQualifier value» 56 </INSDQualifier> u 502 <INSDOualifier ld="g35"> 5373 <IN3DQualifier namerorganism“/INSDQuali fier name> 504 <INSDQualifier valuersynthetic construct</INSDQualifier valued 205 </INSDOualiLfier»>
S08 </INSDFeaturs guals> 507 </INSDFearurex» 568 </INBDSeq feature-table> 509 <INSDSegq seguence>tteetgettaggettetgetgttg</INSDSeqg sequence» 510 </INSDSeg>
LL </SegusnceData»>
SLE </STi6SeguenceListing>
DLS

Claims (7)

ConclusiesConclusions 1. Exogeen artificieel miRNA dat in staat is tot het effectief remmen van replicatie van epidemisch varkensdiarreevirus (PEDV), waarbij het miRNA een dubbelstrengs nucleotide omvat dat geselecteerd is uit de groep die bestaat uit 1) en 2): 1) een dubbelstrengs nucleotide met sequenties van een sense-streng en een antisense-streng die respectievelijk in SEQ ID NR: 1 en SEQ ID NR: 2 getoond zijn; en 2) een dubbelstrengs nucleotide met sequenties van een sense-streng en een antisense-streng die respectievelijk in SEQ ID NR: 3 en SEQ ID NR: 4 getoond zijn.1. Exogenous artificial miRNA capable of effectively inhibiting replication of epidemic swine diarrhea virus (PEDV), wherein the miRNA comprises a double-stranded nucleotide selected from the group consisting of 1) and 2): 1) a double-stranded nucleotide with sequences of a sense strand and an antisense strand shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and 2) a double-stranded nucleotide having a sense strand and an antisense strand sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. 2. Farmaceutische samenstelling, die het exogene artificiële miRNA volgens conclusie 1 als een actief bestanddeel omvat.A pharmaceutical composition comprising the exogenous artificial miRNA according to claim 1 as an active ingredient. 3. Gebruik van het exogene artificiële miRNA volgens conclusie 1 in bereiding van een medicijn voor het behandelen van epidemische varkensdiarree (PED).Use of the exogenous artificial miRNA according to claim 1 in the preparation of a medicament for treating epidemic porcine diarrhea (PED). 4. Gebruik van het exogene artificiële miRNA dat in staat is tot het effectief remmen van replicatie van PEDV volgens conclusie 1 in een onderzoek naar een functie van een PEDV-N-gen middels genoommodificatie.4. Use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV according to claim 1 in a study of a function of a PEDV N gene by means of genome modification. 5. Gebruik van het exogene artificiële miRNA dat in staat is tot het effectief remmen van replicatie van PEDV volgens conclusie 1 in bereiding van een biologische bereiding voor het behandelen van PED.Use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV according to claim 1 in preparation of a biological preparation for treating PED. 6. Gebruik van het exogene artificiële miRNA dat in staat is tot het effectief remmen van replicatie van PEDV volgens conclusie 1 in bereiding van een transgene anti-PEDV-cellijn.Use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV according to claim 1 in preparation of a transgenic anti-PEDV cell line. 7. Gebruik van het exogene artificiële miRNA dat in staat is tot het effectief remmen van replicatie van PEDV volgens conclusie 1 in het fokken van een transgeen anti-PEDV-varken.Use of the exogenous artificial miRNA capable of effectively inhibiting replication of PEDV according to claim 1 in breeding a transgenic anti-PEDV pig.
NL2032385A 2022-07-06 2022-07-06 EXOGENOUS ARTIFICIAL miRNA CAPABLE OF EFFECTIVELY INHIBITING REPLICATION OF PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV) AND USE THEREOF NL2032385B1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112760320A (en) * 2020-12-02 2021-05-07 扬州大学 Exogenous artificial miRNA for effectively inhibiting porcine epidemic diarrhea virus replication and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112760320A (en) * 2020-12-02 2021-05-07 扬州大学 Exogenous artificial miRNA for effectively inhibiting porcine epidemic diarrhea virus replication and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHENPENG ZHAO: "Epidemiological investigation of porcine epidemic diarrhea virus and preliminary establishment of a rapid detection method", 2016, YANGZHOU UNIVERSITY

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