CN112760320B - Exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus and application thereof - Google Patents
Exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C12N2310/141—MicroRNAs, miRNAs
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Abstract
The invention belongs to the field of genetic engineering and biological product research, and in particular relates to an exogenous artificial miRNA for effectively inhibiting replication of Porcine Epidemic Diarrhea Virus (PEDV), wherein the exogenous artificial miRNA is double-stranded nucleotide of the following 1) or 2): 1) The sequences of the sense strand and the antisense strand are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; 2) The sequences of the sense strand and the antisense strand are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively. Compared with siRNA aiming at PEDV protein coding genes, the artificial miRNA aiming at the virus N gene conserved sequence screened by the invention can inhibit PEDV replication, can be developed as a novel biological agent for resisting PEDV, and can be used for breeding disease-resistant pig strains.
Description
Technical Field
The invention belongs to the fields of gene engineering and biological product research. In particular to the preparation and the use of exogenous artificial miRNA which effectively inhibits replication of Porcine Epidemic Diarrhea Virus (PEDV).
Background
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 infectious intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV). PEDV infection can occur in pigs of all ages, with infection being most severe in piglets, and morbidity and mortality often reaching 100%. At present, the PEDV mutant strain is a dominant strain which is popular with PEDV in China, the popularity of the mutant strain in pig groups in China is not neglected, and even the mutant PED is widely considered to be one of the most serious diarrhea diseases which endanger piglets in the next few years. As with most viral diseases, there is no specific drug for the prevention of PED, mainly relying on vaccination. For decades, domestic and foreign swine researchers have researched PED inactivated vaccines, live vaccines, genetic engineering vaccines and the like, but no specific drug aiming at porcine epidemic diarrhea exists in the market at present, and the research and development of the vaccine are not ideal. Therefore, it is necessary and urgent to find a new therapeutic strategy for the treatment of PEDV infection.
RNA interference (RNAi) is an emerging gene silencing technology, which means that cells utilize exogenous or endogenous double-stranded small interfering RNA (siRNA or microRNA) to excite related enzyme complexes to cut and degrade homologous mRNA, so that the expression of genes is blocked at the posttranscriptional level, and the reduced expression or non-expression of the homologous genes is achieved. RNAi has an inhibitory effect on almost all viral replication in terms of antiviral infection. However, siRNA mediated RNAi silencing gene expression is achieved by binding small double stranded RNA to the mRNA of the target gene in a sequence-specific manner. More and more studies have found that many viruses can escape RNAi by mutation of the target gene sequence and production of inhibitors under therapeutic selection pressure, and that the therapeutic effect of RNAi against the virus may be reduced by mutation of the virus. However, compared to siRNA, miRNA can function only by binding to the target gene moiety, and thus RNAi treatment failure due to viral variation can be largely avoided. The lack of stringent base complementary pairing of a miRNA with its target may make it more difficult for viral mutations to evade specific silencing of the miRNA, which would be more advantageous for treatment of viruses susceptible to mutation. Currently, siRNA against viral genes are all used in studies to inhibit PEDV replication.
The PEDV genome consists of a linear single-stranded positive strand RNA of 27-32kb in size with a 5 'cap structure (cap) and a 3' Poly (a) tail whose genome consists essentially of 3 nonstructural proteins and 4 structural proteins. Structural proteins include four of small Membrane proteins (E), membrane glycoproteins (M), nucleocapsid proteins (N), and Spike proteins (S). Wherein, the N protein can combine with virus RNA to provide a structural basis for virus nucleocapsid protein, and can combine cell membrane and phospholipid to promote virus assembly. The N protein has strong antigenicity and is conserved in the similar coronaviruses, and has important effect on the replication of PEDV.
Disclosure of Invention
The invention provides an exogenous artificial miRNA sequence capable of remarkably inhibiting replication and proliferation of porcine epidemic diarrhea virus, and aims to solve the technical problem of difficult epidemic prevention of porcine epidemic diarrhea virus.
The exogenous artificial miRNA capable of inhibiting replication and proliferation of the porcine epidemic diarrhea virus provided by the invention is one of the following double-stranded nucleotide sequences:
1) The sequences of the sense strand and the antisense strand are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
SEQ ID NO.1：5-AGTACGAGTCCTATAACGGAG-3
SEQ ID NO.2：5-CTCCGTTATAGGACTCGTACT-3
2) The sequences of the sense strand and the antisense strand are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;
SEQ ID NO.3：5-TAAACTGGCGATCTGAGCATA-3
SEQ ID NO.4：5-TATGCTCAGATCGCCAGTTTA-3
the double-stranded nucleotide sequences of the above 1) and 2) are named pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048 respectively.
The invention also provides a pharmaceutical composition comprising the exogenous artificial miRNA as an active ingredient.
The invention discloses application of exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus in preparation of a medicament for treating porcine epidemic diarrhea.
The invention discloses application of exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus in PEDV N gene function research through genome modification.
The invention discloses application of exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus in preparation of biological preparations for treating porcine epidemic diarrhea.
The invention discloses application of exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus in preparation of an anti-PEDV transgenic cell line.
The invention discloses application of exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus in preparation of anti-PEDV transgenic pigs.
Compared with siRNA aiming at PEDV protein coding genes, the artificial miRNA aiming at the virus N gene conserved sequence screened by the invention can inhibit PEDV replication, can be developed as a novel biological agent for resisting PEDV, and can be used for breeding disease-resistant pig strains.
According to the invention, artificial miRNA is designed according to the conserved sequence of PEDV N gene, vero cells are transfected by an artificial miRNA expression vector, and on the basis of optimizing the transfection efficiency, the infection dose (TCID) of tissue cells is 50 percent 50 ) And real-time PCR detection of the inhibition effect of artificial miRNA on viral genes and replication thereof, thereby screening artificial miRN capable of effectively inhibiting PEDVA。
Drawings
FIG. 1 is a schematic diagram of the structure of an artificial miRNA expression vector.
The following is introduced in 5'-3' order: the initial 5 nucleotide sequence (TGCTG) in the Top strand is derived from miRNA-155, an endogenous microRNA. The 5' -end of the Bottom strand provides a 4-nucleotide hanging sequence corresponding to the 4-nucleotide hanging tail of the linear structure of the pcDNA6.2-GW/EmGFP-miR plasmid vector, and can be combined into a double-strand structure. The reverse complement 21 nucleotide sequence behind the Top strand (i.e., the mature miRNA sequence) will be directed against the target sequence when transcribed and thus designed to be complementary to the target RNA.
FIG. 2 shows the real-time PCR detection of N gene in HN-13 strain PEDV infected miRNA expression cells.
The ordinate is the relative content of the amplified products of PEDV N genes in each group; control represents normal untransfected cells; pcDNA6.2-miR-N-349 represents an artificial miRNA pcDNA6.2-miR-N-349 expression vector transfected cells aiming at a PEDV N gene 349 site; pcDNA6.2-miR-N-1048 represents a cell transfected by an artificial miRNA pcDNA6.2-miR-N-1048 expression vector aiming at a 1048 site of PEDV N gene; pcDNA6.2-miR-N-neg represents an empty transfected cell control.
FIG. 3 shows the detection of PEDV titer in HN-13 strain PEDV infected miRNA-expressing cells.
The ordinate represents viral Titer (TCID) 50 ) The method comprises the steps of carrying out a first treatment on the surface of the pcDNA6.2-miR-N-349 represents an artificial miRNA pcDNA6.2-miR-N-349 expression vector transfected cells aiming at a PEDV N gene 349 site; pcDNA6.2-miR-N-1048 represents a cell transfected by an artificial miRNA pcDNA6.2-miR-N-1048 expression vector aiming at a 1048 site of PEDV N gene;
pcDNA6.2-miR-N-neg represents an empty transfected cell control.
Detailed Description
Biological material source: pcDNA6.2-GW/EmGFP-miR vector: introduced from us Invitrogen life technologies company. PEDV-HN13 strain was isolated and maintained by the laboratory (strain information reference: zhao Zhenpeng. Preliminary establishment of epidemiological investigation and rapid detection methods for porcine epidemic diarrhea virus [ D ]. University of dulcimer, 2016.).
Example 1
1. MiR RNAi design
According to the sequence of all the strain N genes of PEDV existing in NCBI website, sequence comparison is carried out, the conserved part between the strain N genes of PEDV is selected (table 1), then according to the design principle of artificial miRNA, two target sequences are selected for miRNA double-stranded oligonucleotide sequence design (table 2), and the artificial miRNA expression plasmid is constructed.
TABLE 1 PEDV N Gene target sequence
Note that V represents the target sequence selected for investigation
2. Double-stranded oligonucleotide synthesis and cloning
According to the design principle of the artificial miRNA, an artificial miRNA sequence aiming at PEDV is designed, an artificial miRNA expression plasmid double-stranded oligonucleotide (table 2) sequence is constructed, and the sequence is sent to Beijing engine company for single-stranded oligonucleotide (oligo) synthesis. Annealing each pair of single-stranded oligonucleotides, wherein the reaction system is as follows: positive strand DNA oligo (200. Mu.M) 5. Mu.L, negative strand DNA oligo (200. Mu.M) 5. Mu.L, 10X Oligo Annealing Buffer 2. Mu.L (100 mM Tris-HCl pH 8.0, 500mM Nacl,10mM EDTA), deionized water 8. Mu.L. The reaction was incubated at 94℃for 5min and then allowed to anneal by slow cooling to room temperature. Cloning of double-stranded oligonucleotides into pcDNA TM 2-GW/EmGFP-miR vector (FIG. 1), and ligation reaction product was transformed into TOP10 competent cells. Screening positive plasmids, selecting monoclonal bacterial colony for amplification culture, extracting plasmids, carrying out enzyme digestion identification on the extracted positive plasmids, carrying out enzyme digestion identification on correct plasmids, and sequencing by using EmGFP forward sequencing primers and miRNA reverse sequencing primers to Nanjing qing biological science and technology Co., ltd, wherein the plasmids with correct sequencing are named pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048 respectively.
TABLE 2 double stranded oligonucleotide sequences for artificial miRNA expression
Example 2
Transiently transfecting the constructed plasmid into vero cells:
the day before transfection vero cells were digested, 4X 10 5 The cells were seeded into 12-well cell plates. Transfection when cell density reaches 70% -80%, each plasmid is transfected repeatedly for 2-3 holes; dividing cells into pcDNA6.2-miR-N-349, pcDNA6.2-miR-N-1048 transfection group and no-load transfection control group, and using Lipofectamine TM 3000 as transfection reagent. Transfection efficiency was observed after further culturing for 24h, 48h, 72 h. The results show that: the transfection efficiency was highest 48h after infection.
PEDV replication inhibition effect assay:
cells were infected with PEDV-HN13 strain at the time of highest transfection efficiency, with an infection index moi=0.1. Cells were collected 48h post infection. The collected cells were subjected to extraction of total RNA from the cells according to Axyprep total RNA preparation kit (AXYGEN Co.) and digested with gDNA Eraser (TaKaRa Co.) to remove genomic DNA from the cells. With 18S gene as reference, according to SYBR Premix Ex Taq TM (Boseki Biotechnology Co., ltd.) instructions, the relative quantitative RT-PCR detection of PEDV N gene transcription was performed. The sequence of the reference 18S forward and reverse primers is as follows: 5-TCAGATACCGTCGTAGTTCC-3 (SEQ ID NO. 15) and 5-TTCCGTCAATTCCTTTAAGTT-3 (SEQ ID NO. 16); the sequence of the N gene specific primer is as follows: 5-CGATGATCTGGTGGCTGCTGTC-3 (SEQ ID NO. 17) and 5-TTCCTGCTTAGGCTTCTGCTGTTG-3 (SEQ ID NO. 18). The 20 mu LPCR reaction system is: SYBR Premix ExTaq II (2×) 10. Mu.L, each of the forward and reverse primers was 0.8. Mu.L, ROX Reference Dye (50×) 0.4. Mu.L, and the cDNA template obtained by reverse transcription was 2. Mu.L, dH 2 O6. Mu.L. The amplification procedure was 95℃and pre-denatured for 30s;95 ℃ for 5s,60 ℃ for 34s,40 cycles; 95℃15s,60℃60s,95℃15s. The PCR products were analyzed for the relative expression levels of PEDV N genes between groups by 7500real-time PCR System software. Meanwhile, 18s genes are set as reference genes, and 3 repeats are carried out on each group. The results show that: PEDV N gene expression of pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048 transiently transfected vero cells at 48h after PEDV infection has remarkable inhibition compared with no-load transfected cell control groupThe inhibition rate was close to 90% (FIG. 2), with pcDNA6.2-miR-N-349 having the best inhibition.
Cell supernatants were collected 48h post-PEDV infection and virus TCID was performed on vero cells 50 The measurement results show that after 48 hours of infection of cells infected by the PEDV-HN13 strain, the inhibition effect of pcDNA6.2-miR-N-349 and pcDNA6.2-miR-N-1048 on PEDV is obviously different from that of an idle control group, and the TCID thereof 50 Respectively 10 -2.06 、10 -2.23 While no-load control group TCID 50 Is 10 -5.09 Thus, pcDNA6.2-miR-N-349 was found to have the best inhibitory effect (FIG. 3).
SEQUENCE LISTING
<110> university of Yangzhou
<120> exogenous artificial miRNA effective in inhibiting replication of porcine epidemic diarrhea virus and use thereof
<130>
<160> 18
<170> PatentIn version 3.3
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agtacgagtc ctataacgga g 21
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ctccgttata ggactcgtac t 21
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taaactggcg atctgagcat a 21
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tatgctcaga tcgccagttt a 21
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tttcaggatc gtggccgcaa a 21
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ctccgttata ggactcgtac t 21
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gtcgtggcaa tggcaacaat a 21
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gtcgtggagc ttctcagaac a 21
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tcaaatgacc gtggtggtgt a 21
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ggtggtgtaa catcacgcga t 21
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tgctgtcaag gatgcactta a 21
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gctgtcaagg atgcacttaa a 21
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ttccgtcaat tcctttaagt t 21
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Claims (6)
1. An exogenous artificial miRNA effective for inhibiting replication of porcine epidemic diarrhea virus, characterized by the following double-stranded nucleotides 1) or 2):
1) The sequences of the sense strand and the antisense strand are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
2) The sequences of the sense strand and the antisense strand are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.
2. A pharmaceutical composition comprising as an active ingredient the exogenous artificial miRNA of claim 1.
3. The use of the exogenous artificial miRNA of claim 1 for preparing a medicament for treating porcine epidemic diarrhea.
4. The use of exogenous artificial miRNA effective for inhibiting replication of porcine epidemic diarrhea virus according to claim 1 for the preparation of a biological agent for treating porcine epidemic diarrhea.
5. Use of an exogenous artificial miRNA effective for inhibiting replication of porcine epidemic diarrhea virus according to claim 1 for the preparation of an anti-PEDV transgenic cell line.
6. The use of exogenous artificial miRNA effective for inhibiting replication of porcine epidemic diarrhea virus according to claim 1 for the preparation of anti-PEDV transgenic pigs.
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| CN102296069A (en) * | 2011-08-31 | 2011-12-28 | 扬州大学 | Untranslated region specific artificial micro RNA (miRNA) capable of effectively inhibiting replication of porcine reproductive and respiratory syndrome (PRRS) virus strains |
| CN106957847A (en) * | 2017-05-16 | 2017-07-18 | 吉林大学 | Effectively suppress the siRNA and purposes of piglet epidemic diarrhea virus |
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| CN102296069A (en) * | 2011-08-31 | 2011-12-28 | 扬州大学 | Untranslated region specific artificial micro RNA (miRNA) capable of effectively inhibiting replication of porcine reproductive and respiratory syndrome (PRRS) virus strains |
| CN106957847A (en) * | 2017-05-16 | 2017-07-18 | 吉林大学 | Effectively suppress the siRNA and purposes of piglet epidemic diarrhea virus |
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