CN117210467A - MiRNAs for resisting porcine reproductive and respiratory syndrome and application thereof - Google Patents
MiRNAs for resisting porcine reproductive and respiratory syndrome and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to miRNAs for resisting porcine reproductive and respiratory syndrome and application thereof. The miRNAs are miR-361-3p, and the nucleotide sequence of the miR-361-3p is shown as SEQ ID No. 1. The miR-361-3p can target a PRRSV gene region, the conservation of the target region is 100%, and the miR-361-3p can obviously inhibit PRRSV replication. The method provides a new idea for developing the anti-PRRSV infection drug.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to miRNAs for resisting porcine reproductive and respiratory syndrome and application thereof.
Background
miRNAs are non-coding RNAs with the length of about 19-25nt, are multifunctional small molecules and are widely involved in regulating and controlling cell gene expression; miRNAs exert regulatory effects by targeting the 3' utr region of genes. In the nucleus, miRNAs are transcribed into pri-miRNA by their own promoters or promoters sharing their host genes, drosha RNase III endonuclease cleaves the double strand at the lower end of the stem loop at a site near the base of the primary stem loop, releasing about 60-70 nt of stem loop intermediate, called miRNA precursor (Pre-miRNA), the transfer protein exotin 5 transports the Pre-miRNA to the cytoplasm, pre-miRNA is cleaved by RNase III enzyme Dicer to form about 18-25 nt of double strand RNA, one strand is degraded in the cytoplasm, and the other strand is mature miRNA; the mature miRNA forms RISC induction silencing complex with host protein to play the role of miRNA inhibiting gene expression. mirnas control gene expression by targeting the 3' untranslated region (3'Untranslated Region,3'UTR) and coding region of a particular mRNA. In particular, mirnas tend to recruit Argonaute (AGO) protein complexes to complementary target mrnas by targeting the 3' utr of the host gene mRNA, resulting in translational inhibition or degradation of the mRNA. Animal mirnas are often partially matched gene 3'utr inhibitory translations, which induce mRNA degradation if the miRNA matches the target gene 3' utr more closely.
Recent studies have shown that host miRNAs not only regulate expression of host genes, but also can directly target viral genes to exert regulatory effects. For example, in an infection with infectious salmon anemia virus of the orthomyxoviridae family (Infectious salmon anemiavirus, ISAV), salmon miR-148a/b and miR-152 exert antiviral effects by directly targeting the HA, P3 and NP genes of ISAV; host miR-138 inhibits viral replication by targeting RNA of HSV-1 early protein ICP 0; the host miR-1207-5p is reported to have a potential target in the S gene of SARS-CoV-2. Notably, swine miR-221-5p inhibits viral replication by targeting the PEDV genome and activating NF- κB pathways; thus, host miRNAs can not only regulate host gene expression, but also can directly target viral RNAs to regulate viral replication.
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which belongs to genus Betaarterivirus suid of the family arterividae, is a enveloped single-stranded positive-strand RNA virus. PRRSV genomes are about 15kb long and comprise a 5' UTR, at least 11 Open Reading Frames (ORFs), a 3' -UTR, and a 3' poly (a) tail. PRRSV has the following two genotypes: PRRSV-1 of European origin (Betaarterivirus suid 1) and PRRSV-2 of North American origin (Betaarterivirus suid 2). Porcine Reproductive and Respiratory Syndrome (PRRS) was first discovered in the united states in 1987; in 2006, HP-PRRSV strains were developed in multiple provinces in China, resulting in death of a large number of pigs; after 2012, the NADC30-like variant strain appears in China; after 2015, NADC30 like virus began to epidemic; the NADC30-like strain may occur due to recombination of North American NADC30 strain and Chinese HP-PRRSV strain. Although not leading to high mortality, the high recombination rate of the NADC30-like strain with other viral strains leads to altered virulence, mainly leading to sow abortion, so that vaccinated pigs may be ill by infection with the NADC30-like strain, which is prevalent in swine herds. Studies have shown that recombination caused by co-infection with different strains or genotypes makes epidemiology of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) strains more complex and diverse.
Because the epidemic strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) are constantly changed, the existing vaccine can not completely protect the invasion of wild strains, and a certain range of epidemic strains exist each year, so that serious economic loss is caused. The current method for effectively preventing and treating Porcine Reproductive and Respiratory Syndrome (PRRS) mainly relies on the immunocompetence of Chinese herbal medicines such as astragalus, houttuynia cordata, honeysuckle and the like for raising pigs, so that an antiviral effect is achieved. However, since PRRSV infection can cause immunosuppression in pigs, the therapeutic efficacy of immune-enhancing related therapeutic agents is poor. Thus, there is no ideal anti-Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection drug for the prevention and treatment of Porcine Reproductive and Respiratory Syndrome (PRRS). Considering that miRNAs exert an obvious antiviral effect on targeted viral genes, prevention and treatment of porcine reproductive and respiratory syndrome by inhibiting replication of porcine reproductive and respiratory syndrome virus is a direction of urgent research. At present, there are no swine-derived miRNAs that are directly resistant to porcine reproductive and respiratory syndrome virus.
Disclosure of Invention
In order to solve the problem that the prior art does not have the swine-origin miRNAs directly resisting the porcine reproductive and respiratory syndrome virus, the invention adopts the following technical scheme:
the invention provides miRNAs for resisting porcine reproductive and respiratory syndrome, wherein the miRNAs are miR-361-3p, and the nucleotide sequence of the miR-361-3p is shown as SEQ ID No. 1.
Preferably, the miR-361-3p is used for inhibiting replication of porcine reproductive and respiratory syndrome virus.
Preferably, the miR-361-3p directly targets the porcine reproductive and respiratory syndrome virus, the targeting region is nsp1211870-11893 of the porcine reproductive and respiratory syndrome virus, and nsp1111337-11365 of the porcine reproductive and respiratory syndrome virus, so that replication of the porcine reproductive and respiratory syndrome virus is inhibited.
The invention provides application of miRNAs for resisting porcine reproductive and respiratory syndrome in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome.
Preferably, the medicament is an oral formulation.
Preferably, the pharmaceutical dosage forms include solutions, capsules and granules.
Preferably, the medicament takes miR-361-3p as an active ingredient and is assisted by pharmaceutically acceptable auxiliary materials or carriers.
Preferably, the effective content of miR-361-3p in the medicine is 8-12 nM/g. Wherein the effective content of miR-361-3p in the most preferred medicine is 10nM/g.
The miR-361-3p can be prepared into oral administration preparations such as tablets, powder, pills, solutions, capsules and granules. Among them, solutions, capsules and granules are preferable.
The miR-361-3p is matched with different auxiliary materials or carriers according to different dosage forms, and is processed into a dosage form for animals through a preparation process of the corresponding dosage form. The corresponding auxiliary materials of the solution comprise any one or more of micro powder silica gel and distilled water; the corresponding auxiliary materials of the capsule comprise any one or more of starch, sodium hydroxymethyl cellulose and methyl cellulose; the granule corresponding auxiliary materials comprise sodium bicarbonate, citric acid, starch, sodium hydroxymethyl cellulose, and methylcellulose. The carrier comprises any one of microsphere, nanoparticle and liposome.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides miRNAs for resisting porcine reproductive and respiratory syndrome and application thereof, wherein the miRNAs are miR-361-3p, have the capacity of inhibiting porcine reproductive and respiratory syndrome virus replication, can provide a material basis for preparing medicines for treating and preventing porcine reproductive and respiratory syndrome virus infection, and further can promote construction of related medicine systems.
2. The miRNAs (miR-361-3 p) provided by the invention target PRRSV nsp11 and PRRSV nsp12, and the targeting areas are respectively: the nsp1211870-11893 of the porcine reproductive and respiratory syndrome virus and the nsp1111337-11365 of the porcine reproductive and respiratory syndrome virus further inhibit replication of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), provide a new thought for developing medicaments for resisting infection of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and can promote and infect construction of related medicament systems of the porcine reproductive and respiratory syndrome virus.
Drawings
FIG. 1 is a diagram showing analysis of miR-361-3p targeted PRRSV gene regions of the invention;
FIG. 2 shows that miR-361-3p inhibits p of the inventionGLLuciferase activity of 3-promter-PRRSV, wherein the representative differences are very pronounced, P<0.01；
Fig. 3 is a graph of miR-361-3P inhibiting PRRSV replication, wherein x represents the difference is very significant, P <0.05;
fig. 4 is a graph of miR-361-3P inhibiting PRRSV replication, wherein x represents a very significant difference, P <0.01.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Commercial reagents and cells required to be used in the examples of the present invention
1. Cells and plasmids
DH 5. Alpha. Competent cells and firefly luciferase reporter vectors were purchased from Bio Inc.
HEK293t cells give away for the university of stone university Cheng Jinliang professor.
MARC-145 cells give away to the Shanghai veterinary institute Yafeng of China academy of agricultural sciences.
miR-361-3p homolog is chemically synthesized by Shanghai Biochemical company.
The luciferase reporter vector plasmid pGL-promter-PRRSV was synthesized by Shanghai Biotechnology Co.
pRL-TK plasmid was purchased from Shanghai Biotechnology Co.
2. Main reagent
DMEM medium, fetal bovine serum, diabody were purchased from beijing sonebao technologies limited; reverse transcription kit and fluorescent quantitative detection kit were purchased from Takara corporation.
The dual luciferase reporter assay kit was purchased from novzan biotechnology limited.
3. Main instrument
Micropipettes: shanghai Baibo kang instruments Co., ltd.
Desk-top high-speed centrifuge: the products of the science and technology company.
PCR amplification instrument: yaRui technology (China) limited company product.
Gel imager: beijing Liuyi company product.
Nucleic acid electrophoresis apparatus: jinan Ailaibao instruments and devices limited.
Pure water meter: shanghai instruments, inc. of China.
-80 ℃ low temperature refrigerator: sanyo science instruments Co.
Constant temperature shaking incubator: shanghai Dibosi Co.
Ultra-clean bench: jiangsu Di instruments and technology Co., ltd.
Constant temperature water bath: the biological technology limited company of European Lebo in south China.
Electric heating constant temperature incubator: zhongyan Lihua Instrument technology Co., ltd.
An electronic balance: (Chinese) Switzerland.
Example 1
1. Screening of miR-361-3p
According to reported related documents of miRNAs, selecting 27 miRNAs with different expression of PRRSV infection, selecting the currently popular nadc-30like strain sequence as a target by comparing and analyzing the PRRSV epidemic strain sequence, selecting the miRNAs with different expression of PRRSV infection as target molecules, respectively analyzing the miRNAs targeting PRRSV gene sequence and the region thereof by using miRanda software, and sequencing according to the scores; and miR-361-3p capable of targeted inhibition of PRRSV replication is screened, and the nucleotide sequence of the miR-361-3p is shown in SEQ ID NO. 1: CCCCCAGGUGUGAUUCUGAUUUGC.
miR-361-3p targets 2 regions of PPRSV gene altogether, and the targeting regions are PRRSV nsp1211870-11893,PRRSV nsp1111337-11365 respectively, and the specific situation is shown in figure 1.
2. Dual luciferase activity assay
HEK293t cells with higher transfection efficiency were inoculated into 6-well plates, cultured overnight (. Gtoreq.12 h), 300ng of luciferase reporter vector plasmid pGL-master-PRRSV, 6ng of pRL-TK plasmid, and miR-361-3p 10. Mu.L were co-transfected into HEK293t cells, 300ng of luciferase reporter vector plasmid pGL-master-PRRSV, 6ng of pRL-TK plasmid, and 10. Mu.L of miRNANC as a miRNANC group (control group) were co-transfected for 24h, and promoter activity was detected using the Novain biological double luciferase reporter gene system. And data variance analysis was performed using GraphPadVersion 5.01 software, the results of which are shown in fig. 2.
As can be seen from fig. 2, transfection of miR-361-3p significantly inhibited luciferase activity compared to the mirrnanc group.
3. Fluorescent quantitative detection and TCID50
MARC-145 cells were inoculated into 6-well plates, cultured overnight (. Gtoreq.12 h), miR-361-3p 10. Mu.L of transfected cells were used as the miRNANC group (control group) simultaneously, 10. Mu.L of transfected miRNANC was used as the miRNANC group, after 12h of transfection, after 24h of infection of PRRSV, cell supernatants and cell samples were collected, RNA was extracted for fluorescent quantitative PCR detection of PRRSV orf7, and virus titer was determined using the cell supernatants. The results are shown in FIGS. 3 and 4.
Wherein, the fluorescent quantitative PCR detection comprises the following steps: MARC-145 cell total RNA was extracted by Trizol method, cDNA fractions were prepared according to Table 1, and reverse transcription reaction was performed after light mixing of the reaction system. cDNA reaction procedure (reaction on fluorescent quantitative PCR apparatus): the reaction product was stored at 37℃for 20min (reverse transcription reaction), 85℃for 30sec (reverse transcriptase inactivation) and 4 ℃.
TABLE 1cDNA reverse transcription reaction System
| The components | System of |
| 5 XPimeScript RT premix | 2μL |
| Total RNA (500 ng) | XμL |
| RNasedH2O free | 8-XμL |
A fluorescence quantitative reaction system was prepared in accordance with Table 2, and a total of 20. Mu.L was obtained. After gentle mixing, a PCR reaction was performed. Reaction procedure (Bio-Rad CFX96 Touch): after 30s at 95 ℃, 40 cycles are carried out, 5s at 95 ℃ and 30s at 62 ℃; a melting curve program was added. Briefly, the expression level of β -actin was used as an internal control. The relative expression of target gene is calculated by taking beta-actin as reference. The primer sequences are shown in Table 3.
TABLE 2 real-time fluorescent quantitative PCR reaction system
| The components | System of |
| TBGreenPremixExTaqII(TliRNaseHPlus)(2X) | 10μL |
| PCR forward primer (10. Mu.M) | 0.4μL |
| PCR reverse primer (10. Mu.M) | 0.4μL |
| cDNA template | 0.8μL |
| ddH2O | 8.4μL |
TABLE 3 primer sequences
| Name of the name | Sequence (5 '. Fwdarw.3') |
| β-actinqpcr-f | CGGGAAATCGTGCGTGAC |
| β-actinqpcr-r | ATGCCCAGGAAGGAAGGTTG |
| Prrsvorf7F | ATCCAGACTGCCTTCAAT |
| Prrsvorf7R | AACTCCACAGTGTAACTTATC |
TCID50: calculated according to the Reed-Muench method. The specific operation is as follows: MARC-145 cells were inoculated in advance into 96-well plates, after overnight (. Gtoreq.12 h) culture, cell culture supernatants were diluted 8 times at 10-fold ratio, 8 replicates were made per dilution, 100. Mu.L of each dilution was inoculated with one void, 8 replicates were made per dilution, and after 48h culture at 37 ℃, lesions were observed according to the formula: TCID50 = log of the highest dilution of virus above 50% lesions + distance ratio, TCID50 is calculated.
The fluorescence quantitative detection result is identical with the TCID50 result, and the over-expression of miR-361-3p can obviously inhibit PRRSV replication, so that the miR-361-3p can play an antiviral role by targeting a PRRSV gene region.
The invention also provides a medicine for preventing and treating porcine reproductive and respiratory syndrome, which is oral, wherein the dosage form of the medicine comprises solution, capsule and granule. The miR-361-3p is matched with different auxiliary materials or carriers according to different dosage forms, and is processed into a dosage form for animals through a preparation process of the corresponding dosage form. The corresponding auxiliary materials of the solution comprise any one or more of micro powder silica gel and distilled water; the corresponding auxiliary materials of the capsule comprise one or more of starch, sodium hydroxymethyl cellulose and methyl cellulose; the corresponding auxiliary materials of the granule comprise any one or more of sodium bicarbonate, citric acid, starch and sodium hydroxymethyl cellulose, and any one or more of methylcellulose. Common carriers include any one of microspheres, nanoparticles, and liposomes.
The preparation method of the solution will be described below by taking the solution as an example:
the miR-361-3p is prepared and packaged into an oral administration preparation, wherein a safe and effective amount of miRNAs shown in a formula (I) can be extracted and then administered to pigs in a solution form. Alternatively, the disclosed miRNAs can be prepared and packaged in unit dosage form, wherein each physically discrete unit contains a safe and effective amount of the miRNAs of formula (I). When prepared in unit dosage form, the effective content of miR-361-3p in the solution disclosed by the invention is 10nM/g.
Mixing chemically synthesized 100nM miRNAs (miR-361-3 p) with 80g starch, 20g sucrose and 100mL sterilized water, and packaging to obtain a solution for preventing and treating pig breeding and respiratory syndrome.
To verify the effect of the prepared solution, 30 pigs suffering from the pig breeding and respiration syndrome in the local pig farm in Xinjiang Shihe city are selected, and are fed with the prepared solution for 7 days except normal food every day, wherein the single medicine amount per day is not more than 5mg. After 7 days, the porcine reproductive and respiratory syndrome virus is detected, and the number of pigs infected with the porcine reproductive and respiratory syndrome is counted again.
Only 13 pigs out of 30 had the porcine reproductive and respiratory syndrome. The result shows that the solution prepared by the method can treat the porcine reproductive and respiratory syndrome, and the treatment effective rate is 56.7%.
The experimental result proves that the miRNAs provided by the invention are miR-361-3p, have the capability of inhibiting the reproduction of porcine reproductive and respiratory syndrome virus, can provide a material basis for preparing medicines for treating and preventing porcine reproductive and respiratory syndrome virus, and can further promote the construction of related medicine systems.
The miRNAs (miR-361-3 p) provided by the invention target PRRSVnsp11 and PRRSVnsp 12, and the targeting areas are respectively: the nsp1211870-11893 of the porcine reproductive and respiratory syndrome virus and the nsp1111337-11365 of the porcine reproductive and respiratory syndrome virus further inhibit replication of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), provide a new thought for developing medicaments for resisting infection of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and can promote and infect construction of related medicament systems of the porcine reproductive and respiratory syndrome virus.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. The miRNAs for resisting the porcine reproductive and respiratory syndrome are miR-361-3p, and the nucleotide sequence of the miR-361-3p is shown as SEQ ID No. 1.
2. The miRNAs against porcine reproductive and respiratory syndrome of claim 1, wherein miR-361-3p is for inhibiting replication of porcine reproductive and respiratory syndrome virus.
3. The miRNAs against porcine reproductive and respiratory syndrome of claim 2, wherein miR-361-3p is directly targeted against porcine reproductive and respiratory syndrome virus, wherein the targeting region is nsp1211870-11893 of porcine reproductive and respiratory syndrome virus, and wherein nsp1111337-11365 of porcine reproductive and respiratory syndrome virus further inhibits replication of porcine reproductive and respiratory syndrome virus.
4. The use of miRNAs against porcine reproductive and respiratory syndrome as claimed in claim 1 in the manufacture of a medicament for the prevention and treatment of porcine reproductive and respiratory syndrome.
5. The use according to claim 4, wherein the medicament is an oral formulation.
6. The use according to claim 5, wherein the pharmaceutical dosage forms comprise solutions, capsules and granules.
7. The use according to claim 6, characterized in that the medicament comprises miR-361-3p as an active ingredient according to claim 1, supplemented with pharmaceutically acceptable excipients or carriers.
8. A medicament for preventing and treating porcine reproductive and respiratory syndrome, which is characterized by comprising miR-361-3p in claim 1 and pharmaceutically-acceptable auxiliary materials or carriers.
9. The medicine according to claim 8, wherein the auxiliary materials comprise any one or more of silica gel micropowder, starch, sodium hydroxymethyl cellulose, methylcellulose, sodium bicarbonate and citric acid; the carrier comprises any one of microsphere, nanoparticle and liposome.
10. The medicament according to claim 8, wherein the effective content of miR-361-3p in the medicament is 8-12 nM/g.
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