AU2020101084A4 - A long non-coding RNA porcine Lnc-000649 and its application - Google Patents
A long non-coding RNA porcine Lnc-000649 and its application Download PDFInfo
- Publication number
- AU2020101084A4 AU2020101084A4 AU2020101084A AU2020101084A AU2020101084A4 AU 2020101084 A4 AU2020101084 A4 AU 2020101084A4 AU 2020101084 A AU2020101084 A AU 2020101084A AU 2020101084 A AU2020101084 A AU 2020101084A AU 2020101084 A4 AU2020101084 A4 AU 2020101084A4
- Authority
- AU
- Australia
- Prior art keywords
- lnc
- porcine
- quantitative real
- time pcr
- long non
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a long non-encoding RNA porcine
Lnc-000649 and its application, the cDNA nucleotide sequence
corresponding to the long non-encoding RNA porcine Lnc-000649 is
shown in SEQ ID NO: 1. The invention also provides a pair of primers
which is shown in SEQ ID NO : 2 and SEQ ID NO : 3, and a kit for
quantitative real-time PCR of porcine Lnc-000649 gene. Meanwhile, a
porcine Lnc-000649 gene overexpression vector is also provided in the
invention. A new porcine LncRNA, named Lnc-000649, is first identified
in the invention, which is significantly down regulated after PRRSV
infection. Further research showed that overexpression of the LncRNA
could significantly inhibit the proliferation of PRRSV in cells. This
provides a new design target for the research and development of new
prevention and control drugs for PRRS, and lays a technical foundation
for PRRS disease-resistance breeding.
Description
A LONG NON-CODING RNA PORCINE LNC-000649 AND ITS APPLICATION
FIELD OF THE INVENTION The invention relates to the field of genetic engineering, in particular to a long non-encoding RNA porcine Lnc-000649 and its application.
BACKGROUND OF THE INVENTION Porcine Reproductive and Respiratory Syndrome (PRRS), also known as porcine blue-eared disease, is a highly harmful infectious immunosuppressive disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is characterized by reproductive disorders (e.g abortion, stillbirth and mummification) of pregnant sows and respiratory diseases of pigs of all ages, especially piglets. PRRSV have strict host and cell tropism, it invades host cells relied on cell-specific receptors, such as CD163. PRRSV only infect pigs, not other species, and mainly infect fully differentiated cells in monocyte macrophage system, especially the porcine alveolar macrophages (PAMs). PRRSV can proliferate in cells like PAMs, PK-CD163, Marc-145 in vitro. The current use of vaccines and other means failed to achieve satisfactory results because PRRSV have the characteristics of rapid and complex variation, immunosuppression and antibody dependent enhancement, so it is imperative to cultivate new antiviral pig breeds by genetic breeding. Although domestic and foreign scholars have successfully obtained the gene-edited pigs that can resist PRRS by knocking out the key sequence of CD163 gene in recent years, but the way of industrialization is still unclear. Therefore, it is urgent to find the target molecule of new drug design, develop effective new control technology and cultivate new antiviral breeds. Long non-coding RNAs (LncRNA) have a length of more than 200 nucleotides, lack of specific and complete open reading frame, and have little or no protein coding function. These RNAs are widely involved in gene transcription regulation and epigenetic regulation, including the regulation of methylation, chromatin remodeling, genomic imprinting and dosage compensation. LncRNA can be used as a biomarker for disease diagnosis to develop precision drugs because their expression profile has strong cell type specificity, tissue specificity and developmental stage specificity. Studies have shown that LncRNA play a role in immune response and host response to viral infection. The pathogen may hijack the host cell by the characteristics that LncRNA can control the process of gene transcription. However, compared with mRNA and miRNA, there are few reports about the role and mechanism of LncRNA in PRRSV infection. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
SUMMARY OF THE INVENTION It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. The invention relates to a long non-encoding RNA porcine Lnc-000649 and its application. The invention used high-throughput sequencing technology to analyze the whole transcriptome changes in PRRSV-infected and normal PAMs, and identified a novel porcine LncRNA, named porcine Lnc-000649, which was significantly down regulated after PRRSV infection. Further research showed that overexpression of the porcine Lnc-000649 could significantly inhibit the proliferation of PRRSV in cells. This provides a new design target for the research and development of new prevention and control drugs for PRRS and lays a technical foundation for PRRS disease-resistance breeding. According to a first aspect, the invention relates to a long non-coding RNA porcine Lnc-000649, wherein a cDNA nucleotide sequence corresponding to the porcine Lnc-000649 is shown in SEQ ID No: 1. In one embodiment of the invention, based on the complete transcriptome sequencing results of PAMs, a novel long non-coding RNA molecule named porcine
Lnc-000649 was identified by a large number of bioinformatics analysis of the sequencing results of total RNA samples of PRRSV-infection and normal PAMs, and the length of its cDNA nucleotide sequence is 1483bp. According to a second aspect, the invention relates to a long non-coding RNA porcine Lnc-000649 overexpression vector, wherein the overexpression vector comprises the cDNA nucleotide sequence defined in the invention. In an embodiment of the inventiont, it is a vector called Lnc-000649-pcDNA3.1, and the construction method of Lnc-000649-pcDNA3.1 comprises: PCR amplification which uses PAMs cDNA as template and the primer shown in SEQ ID No: 6-7; Double enzyme digestion of PCR products and pcDNA3.1 expression vector by Hind III and XbaI; After recovery, purification and connection, then transforming the Lnc-000649-pcDNA3.1 into E. coli. According to a third aspect, the invention relates to use of the long non-coding RNA porcine Lnc-000649 according to the invention or its overexpression vector according to the invention in the preparation of a PRRSV inhibitor. In one embodiment, the invention provides the use of the porcine Lnc-000649 gene and its overexpression vector in the preparation of PRRSV inhibitor. After porcine Lnc-000649 is overexpressed in PAMs by Lnc-000649-pcDNA3.1, the proliferation of PRRSV is significantly inhibited. The results showed that the porcine Lnc-000649 could inhibit the proliferation of PRRSV, and can be used as an inhibitor of PRRSV, for example, it can be used to prepare kit or drug, etc. According to a fourth aspect, the invention relates to a pair of primers for quantitative real-time PCR of porcine Lnc-000649, wherein a nucleotide sequence of a forward primer is shown in SEQ ID No: 2, and a nucleotide sequence of a reverse primer is shown in SEQ ID No: 3. According to a fifth aspect, the invention relates to a quantitative real-time PCR kit for long non-coding RNA porcine Lnc-000649, wherein the kit comprises the pair of primers according to the invention. As an embodiment, the quantitative real-time PCR kit comprises RNA extraction reagent, reverse transcription system and quantitative real-time PCR system, wherein the quantitative real-time PCR system comprises: qPCR mix, ddH20, cDNA synthesized by the reverse transcription system and the primer-pair for quantitative real-time PCR shown above. As a further embodiment, the quantitative real-time PCR system is as follows: qPCR mix lOpL, forward primer (10pM) 0.5pL, reverse primer (10pM) 0.5pL, cDNA template l pL, sterilized ddH 20 8pL, total volume is 20pL. According to a sixth aspect, the invention relates to a method of using the quantitative real-time PCR kit according to the invention, wherein the method comprises the following steps: Step 1. extract RNA from a sample and then synthesize cDNA by reverse transcription; Step 2. amplify the cDNA by quantitative real-time PCR using the pair of primers according to the invention, and calculate relative expression of long non-coding RNA porcine Lnc-000649 gene by 2-ACT method; and Step 3. detect the long non-coding RNA porcine Lnc-000649 and its expression in the sample according to PCR amplification products and the relative gene expression. As an embodiment, the reaction condition of quantitative real-time PCR in step
2 is: pre-denaturation at 95°C for 5min, denaturation at 95°C for 15s, annealing at
°C for 15s, extension at 72 °C for 15s, and cycle 40 times.
The invention relates to: 1. A new porcine LncRNA, named Lnc-000649, is first identified in the invention, which is significantly down regulated after PRRSV infection. Further research showed that overexpression of the LncRNA could significantly inhibit the proliferation of PRRSV in cells. This provides a new design target for the research and development of new prevention and control drugs for PRRS, and lays a technical foundation for PRRS disease-resistance breeding. 2. The invention provides a pair of primers for quantitative real-time PCR and a quantitative real-time PCR kit for porcine Lnc-000649 gene, and provides the nucleotide sequence of primer-pair and method for quantitatively detecting the relative expression of porcine Lnc-000649, which can be used to detect the existence and expression level of porcine Lnc-000649. 3. The long non-coding RNA porcine Lnc-000649 and its overexpression vector in the invention can be used as a PRRSV inhibitor, In one embodiment, the overexpression of porcine Lnc-000649 gene in the natural target cells infected by PRRSV can significantly inhibit the proliferation of PRRSV; wherein, the overexpression vector Lnc-000649-pcDNA3.1 that comprises the full-length sequence of porcine Lnc-000649, can be used to stably express the porcine Lnc-000649 gene, and the quantitative real-time PCR can reflect the expression level of Lnc-000649. These technologies can be applied to related fields.
BRIEF DESCRIPTION OF THE FIGURES Figure 1: PCR amplification result of the full-length sequence of porcine Lnc-000649 in PAMs in embodiment 1 of the invention; wherein lane M referred to DL2000 DNA marker, lane 1, lane 2 and lane 3 were full-length amplification fragments of porcine Lnc-000649; Figure 2: The quantitative real-time PCR result of porcine Lnc-000649 in PAMs in embodiment 2 of the invention; Figure 3: The structure diagram of pcDNA3.1 empty vector used in embodiment 3 of the invention; Figure 4: The double enzyme digestion identification agarose gel electrophoresis map of Lnc-000649-pcDNA3.1, the porcine Lnc-000649 overexpression vector constructed in embodiment 3 of the invention; In the map, lane M referred to 1KB DNA ladder, lane 1 was a circular Lnc-000649-pcDNA3.1 vector before double enzyme digestion; lane 2 was the linearized pcDNA3.1(+) empty vector fragment and the porcine Lnc-000649 fragment after the Lnc-000649-pcDNA3.1 vector was double enzyme digested by Hind III and XbaI; Figure 5: The structure diagram of Lnc-000649-pcDNA3.1 constructed in embodiment 3 of the invention;
Figure 6: The quantitative real-time PCR result of PRRSV RNA in embodiment 3 of the invention. As shown in the reference mark, Lnc-00649 represented the cell transfected by Lnc-000649-pcDNA3.1 overexpression vector; pcDNA3.1 represented the cell transfected by pcDNA3.1(+) empty vector; Null represented the blank control without any transfection vector, **P<0.0 1.
Embodiment 1: Discovery and preparation of long non-coding RNA porcine Lnc-000649 full-length sequence I. Discovery of porcine Lnc-000649 gene The nucleotide sequence of cDNA corresponding to the porcine Lnc-000649 was shown in SEQ ID No: 1. The porcine Lnc-000649 was a novel LncRNA found in the whole transcriptome sequencing of porcine PAMs and screened by a large number of bioinformatics analysis such as genome splicing, coding potential analysis, gene expression levels in PRRSV-infected and uninfected cells, etc. The novel LncRNA was named porcine Lnc-000649, and the length of the cDNA nucleotide sequence corresponding to porcine Lnc-000649 is 1483bp. II. Preparation of full-length sequence of porcine Lnc-000649 1. Isolation and culture of PAMs (1) Select 4-5 weeks old PRRSV-free, PCV2-free, and PRV-free Large White piglets were selected for euthanasia and the lungs were separated, then slowly infused 400-500mL phosphate-buffered saline (PBS) buffer with penicillin and streptomycin to the lung in batches, massaged the filled lung every time, and gently blew with a straw to disperse the cell clusters and mucus lump, then collected washing solution filtered with sterile gauze, and centrifugated at 1600r/min for min to colletc cells. (2) The collected cells were washed with RPMI 1640 medium containing double antibody, then centrifuged at 1600r/min for 10min, and repeated the washing step for 1-2 times. (3) The washed cells were resuspended in RPMI 1640 medium with 10% fetal bovine serum and then cultured in a 10cm dish at 37°C and 5%CO 2 for 2 h. (4) After removing the non-adherent cells, the remaining cells could be digested and transferred into the 6-well plate for subsequent test. The surplus cells could be resuspended with cell cryopreservation solution and then frozen separately. 2. Total RNA extraction from PAMs The total RNA of PAMs was extracted by Trizol and chloroform, the specific steps were as follows: (1) After cleaning the cells in the 6-well plate with PBS for 1-2 times, added 1mL Trizol into each well to crack cells on ice for 5min, blew them evenly with the pipette gun, and then moved them into the sterile 1.5mL RNAase-free centrifuge tube; (2) Added 0.2mL chloroform per 1mL of homogenate, and shook violently for s, then placed the centrifuge tube at room temperature for 15min;
(3) Centrifuged at 12000g for 10min at 4°C;
(4) Extracted the the upper layer solution carefully and put it into another 1.5mL RNAase-free centrifuge tube, then added 0.5 times volume of isopropanol and mixed well, placed at test temperature for 5-10min.
(5) Centrifuged at 12000g for 10min at 4°C, RNA was deposited at the bottom
of the tube, and discarded the supernatant. (6) Added 1mL 75% ethanol, shook the centrifuge tube gently to suspend the precipitate;
(7) Centrifuged at 8000g for 5min at 4°C and discarded the supernatant;
(8) Added mL absolute ethanol, shook the centrifuge tube gently to suspend the precipitate;
(9) Centrifuged at 8000g for 5min at 4°C, discarded the supernatant and dry at room temperature; (10) Used 30-50pL RNAase-free deionized water to dissolve RNA, and stored at -80°C. (11) Used the NanoDrop 1000 micro spectrophotometer to detect the concentration and OD value of the RNA, and detected the integrity of RNA by 1% agarosegel electrophoresis.
3. Amplification and identification of full-length sequence of porcine
Lnc-000649 (1) Reverse transcription was carried out with Thermo ScientificTM
RevertAidTMFirst Strand cDNA Synthesis Kit to synthesis cDNA template using
1pg RNA; (2) The reverse transcribed cDNA was diluted 5 times with RNAase-free
deionized water and used as PCR template.
(3) The full-length sequence of Lnc-000649 was obtained by PCR. The PCR primer-pair are shown in SEQ ID No:4 and SEQ ID No:5. The annealing
temperature of PCR was 55°C, and the extension time was 90s.
(4) The obtained PCR product of the target fragment (shown in Figure 1) was
sent to Wuhan Qingke innovation Biotechnology Co., Ltd. for sequencing, and the
sequencing result was completely consistent with the expected sequence. Therefore,
it could be confirmed that the Lnc-000649 really exists in PAMs.
Embodiment 2: The quantitative real-time PCR kit and method of using the
porcine Lnc-000649 In this embodiment, a pair of primers for quantitative real-time PCR was
designed to quantitatively detect the expression level of Lnc-000649 in cells or
tissues.
1. Isolation and culture of PAMs
The primary PAMs were isolated and cultured from the lungs of 4-5 weeks old
healthy Large White pigs with PRRSV-free, PCV2-free and PRV-free. The specific steps were the same as embodiment 1. 2. PRRSV infection and control PAMs were divided into two groups, one group was inoculated with PRRSV virus, the other group was the control group, and each group had three replicates. The PRRSV infection group: put PRRSV stock solution on ice to melt slowly, used MOI=0.5 PRRSV to infect cells, the cell culture medium was RPMI 1640 culture medium with 2% fetal bovine serum, and collected cells 24 hours after infection. The control group: Treated cells with RPMI 1640 medium with 2% fetal bovine serum, and collected cells after 24 hours. 3. Extraction of total RNA of cells The total RNA of cells was extracted by Trizol and chloroform. The specific steps were the same as embodiment 1. 4. The quantitative real-time PCR (1) Reverse transcription was carried out with Thermo ScientificTM RevertAidTMFirst Strand cDNA Synthesis Kit to synthesis cDNA template using 1pg RNA; (2) The quantitative real-time PCR system (20pL) was as follows: qPCR mix 1Oul, forward primer (10pM) 0.5pL, reverse primer (10IM) 0.5pL, cDNA template 1pL, sterilized ddH20 8pL. The nucleotide sequence of forward primer was shown in SEQ ID NO: 2, and the nucleotide sequence of reverse primer was shown in SEQ ID NO: 3. The qPCR mix was obtained from THUNDERBIRD SYBR qPCR Mix Kit of Takara. (3) Analysed the expression of cDNA by quantitative real-time PCR using LightCycler480 Real-Time PCR System of Roche and taking p-actin as internal reference, then calculated the relative expression by2-^C' method. Each experiment, three independent experiments were performed in triplicate. The reaction condition of quantitative real-time PCR comprised: pre-denaturation at 95°C for 5min, denaturation at 95°C for 15s, annealing at 55°C for 15s, extension at 72 °C for 15s, and cycle 40 times. The expression levels of Lnc-000649 in PRRSV infected PAMs and control PAMs were shown in Figure 2. The expression level of Lnc-000649 in PRRSV-infected PAMs was significantly lower than that in uninfected PAMs (P < 0.01).
Embodiment 3: Overexpression of Lnc-000649 inhibits PRRSV proliferation in cells 1. Construction of an overexpression vector comprising the full-length sequence of porcine Lnc-000649 (1) Two endonucleases Hind III and XbaI were used to digest the pcDNA3.1(+) vector. The pcDNA3.1(+) vector was purchased from Invitrogen company, and its structure diagram was shown in Figure 3. The reaction system was 50pL, which was
digested at 37°C for 2 hours. Then the enzyme products were purified and
recovered. (2) The two ends of the primer-pair shown in SEQ ID No:4 and SEQ ID No:5
sequences were added with Hind III ( CAAGCTT ) and XbaI (CTCTAGA)
recognition site and protective base respectively to obtain the primer-pair shown in SEQ ID No: 6 and SEQ ID No: 7. (3) PCR amplification of the full-length sequence of porcine Lnc-000649 containing Hind III and XbaI restriction endonuclease recognition site with the primers shown in SEQ ID No: 6 and SEQ ID No: 7 and template of PAMs'cDNA, then, the endonucleases Hind III and XbaI were used for double enzyme digestion.
The enzyme digestion system was 50pL, which was digested at 37°C for 2 hours.
Then the enzyme products were purified and recovered. (4) T4 DNA ligase was used to connect pcDNA3.1(+) vector after double
enzyme digestion with fragment obtained by PCR amplification overnight at 16°C.
(5) The connection products were transformed into the competent cells, the positive clones were identified by PCR and sent to Wuhan Qingke innovation Biotechnology Co., Ltd. for sequencing. The result was shown in SEQ ID No: 1. The DNA of recombinant vector of the positive clones was extracted with the Omega E.Z.N.A.TM Endo-Free Plasmid Mini KitSpin and the specific steps were shown in the instructions of the kit, then the concentration of the plasmid was detected. At the same time, Hind III and XbaI restriction endonuclease were used to digest the vector to verify the correctness of the inserted fragment (shown in Figure. 4). The vector was named Lnc-000649-pcDNA3.1, and its structure diagram was shown in Figure 5. 2. Isolation and culture of PAMs The protocol was the same as embodiment 1. 3. Cell transfection (1) Diluted Lnc-000649-pcDNA3.1 vector and empty pcDNA3.1(+) vector with 125pL Opti-MEM medium respectively, and then added 5pL P3000; (2) Diluted P3000 with 250pL Opti-MEM medium and mixed it well; (3) Added 125pL mixture of step (2) to the two tubes of mixture of step (1) respectively, and incubated at room temperature for 5min; (4) 250 pL mixture of step (3) was transferred into the PAMs. There were 3 repetitions for each experimental group and the cells were divided into three groups: (A) pcDNA3.1(+) empty vector group; (B) Lnc-000649-pcDNA3.1 vector group; (C) Blank control group without any vector. 4. PRRSV infection After 24 hours of transfection, cells were incubated with MOI=0.5 PRRSV, and the culture medium was replaced by RPMI 1640 with 2% fetal bovine serum. Cells were collected at 24 hours after infection, and digested by Trizol, then the total RNA was extracted for quantitative real-time PCR. 5. Quantitative real-time PCR of PRRSV RNA The reverse transcription of PRRSV RNA was carried out by Thermo ScientificTM RevertAidTMFirst Strand cDNA Synthesis Kit. The relative expression level of PRRSV RNA in cells was detected by primer-pair: PRRSV-F and PRRSV-R; and THUNDERBIRD SYBR qPCR Mix Kit of Takara, wherein the nucleotide sequence of PRRSV-F was 5'-CGCCAGTGTACATCACCATC-3', the nucleotide sequence of PRRSV-R was 5'-CATTGCCAAACACCACTTTG-3'. The reaction condition of quantitative real-time PCR was: pre-denaturation at 95°C for 5min, denaturation at 95°C for 15s, annealing at 55°C for 15s, extension at 72°C for 15s, and cycle 40 times. The results showed that compared with the PAMs transfected with pcDNA3.1(+) empty vector, the expression level of Lnc-000649 in the PAMs transfected with Lnc-000649-pcDNA3.1 vector was up-regulated by 5214 times, which indicated that the porcine Lnc-000649 overexpression cell model was successfully constructed. On this basis, the content of PRRSV RNA in Lnc-000649 overexpression cell model and control cells that were infected by PRRSV was detected by quantitative real-time PCR 24 hours later. The results showed that the expression level of PRRSV RNA in Lnc-000649 overexpression cell model was significantly lower than that in PAMs transfected with pcDNA3.1(+) empty vector and untreated cells (P< 0.01) (shown in Figure. 6). This indicated that Lnc-000649 can inhibit the proliferation of PRRSV. In conclusion, the long non-coding RNA porcine Lnc-000649 overexpression vector can stably express the porcine Lnc-000649 gene. And Lnc-000649 can inhibit the proliferation of PRRSV, which can be used as PRRSV inhibitor. In the invention, "long non-coding RNA porcine Lnc-000649" and "porcine Lnc-000649" are different names of the same RNA. Throughout the description and claims of the specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. The above embodiments are only preferred embodiments of the invention and do not limit the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the invention shall be included in the protection scope of the invention.
Claims (6)
1. A long non-coding RNA porcine Lnc-000649, wherein a cDNA
nucleotide sequence corresponding to the long non-coding RNA porcine
Lnc-000649 is shown in SEQ ID No: 1.
2. A long non-coding RNA porcine Lnc-000649 overexpression
vector, wherein the overexpression vector comprises the cDNA
nucleotide sequence defined in claim 1.
3. The overexpression vector according to claim 2, wherein the
overexpression vector is Lnc-000649-pcDNA3.1, and a construction
method of Lnc-000649-pcDNA3.1 comprises: PCR amplification which
uses a PAMs cDNA as template and a primer shown in SEQ ID No: 6-7;
double enzyme digestion of PCR products and pcDNA3.1 vector by Hind
III and XbaI; after recovery, purification and connection, then
transforming the Lnc-000649-pcDNA3.1 into E. coli.
4. Use of the long non-coding RNA porcine Lnc-000649 according
to claim 1 or its overexpression vector according to claim 2 or claim 3 in
the preparation of a PRRSV inhibitor.
5. A pair of primers for quantitative real-time PCR of porcine
Lnc-000649, wherein a nucleotide sequence of a forward primer is shown
in SEQ ID No: 2, and a nucleotide sequence of reverse primer is shown in
SEQ ID No: 3.
6. A quantitative real-time PCR kit for long non-coding RNA porcine Lnc-000649, wherein the kit comprises the pair of primers according to claim 5.
7. The quantitative real-time PCR kit according to claim 6, wherein
the quantitative real-time PCR kit also comprises RNA extraction reagent,
reverse transcription system and quantitative real-time PCR system,
wherein the quantitative real-time PCR system comprises qPCR mix,
ddH20, cDNA synthesized by the reverse transcription system and the
pair of primers according to claim 5.
8. A method of using the quantitative real-time PCR kit according to
claim 6 or claim 7, wherein the method comprises the following steps:
Step 1. extract RNA from a sample and then synthesize cDNA by
reverse transcription;
Step 2. amplify the cDNA by quantitative real-time PCR using the
pair of primers according to claim 5, and calculate relative expression of
long non-coding RNA porcine Lnc-000649 gene by 2-ACT method; and
Step 3. detect the long non-coding RNA porcine Lnc-000649 and its
expression in the samples according to PCR amplification products and
the relative gene expression.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911233862.8A CN110885823B (en) | 2019-12-05 | 2019-12-05 | Long-chain non-coding RNA pig Lnc-000649 and application thereof |
| CN2019112338628 | 2019-12-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020101084A4 true AU2020101084A4 (en) | 2020-07-23 |
Family
ID=69750540
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020101084A Ceased AU2020101084A4 (en) | 2019-12-05 | 2020-06-23 | A long non-coding RNA porcine Lnc-000649 and its application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110885823B (en) |
| AU (1) | AU2020101084A4 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114934051A (en) * | 2022-05-26 | 2022-08-23 | 华南农业大学 | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells |
| CN115068611A (en) * | 2022-06-24 | 2022-09-20 | 天津市农业科学院 | Application of ssc-miR-374b-3p in preparation of PDCoV proliferation resistant medicine |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930527A (en) * | 2021-12-09 | 2022-01-14 | 常州市动物疫病预防控制中心(常州市畜牧兽医站) | Non-coding RNA lnc-ALVE-env1 expression detection primer, kit and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399313B (en) * | 2016-09-29 | 2019-10-18 | 西北农林科技大学 | A kind of microRNA sample virus tiny RNA sequence of anti-PRRSV and application thereof and detection method |
| CN107586780B (en) * | 2017-10-25 | 2019-10-11 | 中国农业科学院兰州兽医研究所 | A kind of long-chain non-coding RNA inhibiting porcine reproductive and respiratory syndrome virus |
| CN107828788B (en) * | 2017-11-02 | 2020-03-27 | 中国农业科学院兰州兽医研究所 | Application of lncRNA related to PRRSV infection and siRNA thereof in inhibiting virus replication |
-
2019
- 2019-12-05 CN CN201911233862.8A patent/CN110885823B/en active Active
-
2020
- 2020-06-23 AU AU2020101084A patent/AU2020101084A4/en not_active Ceased
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114934051A (en) * | 2022-05-26 | 2022-08-23 | 华南农业大学 | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells |
| CN114934051B (en) * | 2022-05-26 | 2023-11-28 | 华南农业大学 | LncRNA TAB2-AS and application thereof in porcine ovarian granulosa cells |
| CN115068611A (en) * | 2022-06-24 | 2022-09-20 | 天津市农业科学院 | Application of ssc-miR-374b-3p in preparation of PDCoV proliferation resistant medicine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110885823B (en) | 2021-06-22 |
| CN110885823A (en) | 2020-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020101084A4 (en) | A long non-coding RNA porcine Lnc-000649 and its application | |
| CN107502608B (en) | Construction method and application of sgRNA and ALDH2 gene-deleted cell strain for knocking out human ALDH2 gene | |
| JP5735927B2 (en) | Re-engineering the primary structure of mRNA to enhance protein production | |
| CA2769289C (en) | Development of universal cancer drugs and vaccines | |
| Zhu et al. | Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus-and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors | |
| Gao et al. | MicroRNA-2187 modulates the NF-κB and IRF3 pathway in teleost fish by targeting TRAF6 | |
| CN106399313B (en) | A kind of microRNA sample virus tiny RNA sequence of anti-PRRSV and application thereof and detection method | |
| Su et al. | Circular RNA circSamd4a regulates antiviral immunity in teleost fish by upregulating STING through sponging miR-29a-3p | |
| Zhang et al. | Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses | |
| Wang et al. | Expression pattern analysis of antiviral genes and inflammatory cytokines in PEDV-infected porcine intestinal epithelial cells | |
| Pan et al. | Grass carp reovirus encoding circular RNAs with antiviral activity | |
| CN111500576B (en) | Application of siRNA in preparation of blocker for inhibiting highly pathogenic porcine reproductive and respiratory syndrome virus infection | |
| Pham et al. | MicroRNA gga-miR-10a-mediated transcriptional regulation of the immune genes in necrotic enteritis afflicted chickens | |
| Li et al. | Interferon-stimulated genes inhibit caprine parainfluenza virus type 3 replication in Madin-Darby bovine kidney cells | |
| Cai et al. | Swine Interferon-Inducible transmembrane proteins potently inhibit african swine fever virus replication | |
| CN111840310B (en) | Application of ssc-miR-151-3p in preparation of medicine for regulating replication of porcine reproductive and respiratory syndrome virus | |
| Li et al. | LSM14A inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication by activating IFN-β signaling pathway in Marc-145 | |
| Lin et al. | Gga-miR-200b-3p promotes macrophage activation and differentiation via targeting monocyte to macrophage differentiation-associated in HD11 cells | |
| CN111676222A (en) | shRNA for inhibiting Mettl3 gene expression, recombinant adeno-associated virus thereof and application thereof | |
| CN111808858B (en) | siRNA sequence and application of target thereof in improving PEDV (porcine reproductive and respiratory syndrome Virus) toxicity | |
| CN110229816B (en) | Construction method and application of sgRNA (ribonucleic acid) for knocking out RBP4 gene and RBP4 gene deletion cell strain | |
| AU2021105546A4 (en) | MiRNA against lung injury caused by porcine reproductive and respiratory syndrome virus and its application | |
| Zhang et al. | Downregulation of miR-122 by porcine reproductive and respiratory syndrome virus promotes viral replication by targeting SOCS3 | |
| Guo et al. | Deep sequencing analysis reveals a TMV mutant with a poly (A) tract reduces host defense responses in Nicotiana benthamiana | |
| CN110684797A (en) | TCV-based VIGS vector capable of simultaneously silencing 2 endogenous genes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGI | Letters patent sealed or granted (innovation patent) | ||
| MK22 | Patent ceased section 143a(d), or expired - non payment of renewal fee or expiry |