CN115125250A - Pig NONO protein knockout gene, related plasmid, cell line, preparation method and application - Google Patents
Pig NONO protein knockout gene, related plasmid, cell line, preparation method and application Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology, in particular to gene knockout plasmids and a cell line, wherein the level of IFN-beta mRNA generated after PRV infection of the cell line is obviously reduced compared with PAM-KNU cells; in addition, the cell line promotes PRV proliferation and can be used for the production of PRV proliferation and PRV vaccines.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a pig NONO protein knockout gene, a related plasmid, a cell line, a preparation method and an application.
Background
Not comprising a POU domainOctapolynucleotide binding protein (NONO, also known as P54) nrb ) Is a member of the Drosophila behavior/human splicing protein (DBHS) family. The human NONO gene encodes 471 amino acids, and the protein structure mainly comprises four parts, two RNA recognition motifs (RRM 1, RRM 2) connected in series at the N terminal can recognize RNA combination and splicing sites; a C-terminal Coiled coil domain (Coiled coil) comprising approximately 100 amino acids; there is a conserved domain NOPS between RRM2 and the Coiled coil domain, comprising 52 amino acids; near the C-terminus, there is a helix-turn-helix (HTH) structure and an acidic/basic amino acid rich region that together form a DNA Binding Domain (DBD) capable of binding double-stranded DNA.
The NONO protein is a multifunctional protein and can participate in various biological processes in cells, and independently or in cooperation with other transcription factors, the NONO protein participates in processes such as transcription regulation and mRNA transport and the like through the action of combining DNA or RNA; the NONO protein can regulate the splicing of mRNA by combining with molecules such as RNA polymerase II or exonuclease and the like; the DNA binding domain of the NONO protein can be directly combined with the DNA damaged and broken in the cell nucleus, and is favorable for accurate and rapid repair of double-stranded DNA; in the aspect of regulating innate immunity, the NONO protein enhances the combination of cGAS on DNA, promotes the expression of IFN-beta and inhibits the infection of HIV.
At present, the reports of the pig NONO (sNONO) protein are less, and in order to research the functions of the pig NONO (sNONO) protein more deeply, particularly the influence of the sNONO on antiviral immunity, a method for knocking out the sNONO gene from a pig cell genome is needed to be developed, so that a foundation is laid for the subsequent research on the pig NONO protein.
Disclosure of Invention
In order to research the related functions of sNONO more deeply, a gene knockout plasmid Px459M-sNONO-KO and a cell line sNONO KO-PAM-KNU which stably knock out an sNONO gene from a genome are provided, and a foundation is laid for researching the functions of sNONO.
The invention also provides a preparation method of the gene knockout plasmid Px459M-sNONO-KO and the cell line sNONO KO-PAM-KNU.
The invention also provides application of the cell line sNONO KO-PAM-KNU.
The invention is obtained by the following steps:
the nucleotide sequence of the pig NONO protein knockout gene is shown as the nucleotide sequence of a sequence 2 in a sequence table.
Compared with the nucleotide sequence of the NONO protein, the nucleotide deletion between 450 and 966 bp of the pig NONO protein knockout gene.
A pig NONO protein knockout gene related plasmid contains a nucleotide sequence of a sequence 1 in a sequence table.
The pig NONO protein knockout gene related plasmid is prepared from Px459M plasmidHind III cleavage site and distanceHind III furthest from the cleavage siteBbs IThe nucleotide sequence inserted between the enzyme cutting sites is shown as the nucleotide sequence of the sequence 1 in the sequence table.
A preparation method of pig NONO protein knockout gene related plasmid is obtained by the following steps:
(1) two pairs of primers were designed with the following nucleotide sequences:
sNONO-KO-3Fwd: 5’-CACCAGGAAGGTTTCGGACCGTAA-3’，
sNONO-KO-3Rev: 5’-AAACTTACGGTCCGAAACCTTCCT-3’，
sNONO-KO-4Fwd: 5’-CACCGGCGTCAAGAAGAACTTCGG-3’，
sNONO-KO-4Rev: 5’-AAACCCGAAGTTCTTCTTGACGCC-3’，
phosphorylating and annealing primers sNONO-KO-3Fwd and sNONO-KO-3Rev to obtain sNONO-KO-3P, phosphorylating and annealing primers sNONO-KO-4Fwd and sNONO-KO-4Rev to obtain sNONO-KO-4P;
(2) construction of Gene knockout plasmids
Use ofBbs I enzyme digestion plasmids Px459M and EZ-Guide-XH are respectively recovered by glue to obtain a vector fragment 1 and a vector fragment 2, sNONO-KO-3P is connected to the vector fragment 1 to obtain Px459M-sNONO-KO-3P, sNONO-KO-4P is connected to the vector fragment 2 to obtain EZ-Guide-XH-sNONO-KO-4P, and the use of the enzyme digestion plasmids Px459M and EZ-Guide-XH is used forHind III/Xho I, respectively carrying out double enzyme digestion on Px459M-sNONO-KO-3P and EZ-Guide-XH-sNONO-KO-4P to obtain a vector fragment 3 and a gene fragment 4, and connecting the vector fragment 3 with the gene fragment 4And obtaining the porcine NONO protein knockout gene related plasmid Px 459M-sNONO-KO.
The phosphorylation and annealing operations were optimized as follows: the reaction system contains 10 mu moL/L of sNONO-KO-3Fwd and sNONO-KO-3Rev or sNONO-KO-4Fwd and sNONO-KO-4Rev, 10 XT 4 ligase buffer 1 mu L, T4 PNK 1U, H 2 O6 muL, 10 muL in total; the reaction conditions are as follows: 30 min at 37 ℃ and 5 min at 95 ℃, and the temperature of the PCR is reduced to 25 ℃ in a gradient way at the speed of 0.1 ℃ per second, 5 min at 25 ℃ and 5 min at 4 ℃.
The pig NONO protein knockout gene related plasmid is transfected with PAM-KNU cells, puromycin-containing culture medium is used for screening, and the surviving resistant cells are cloned to obtain the pig NONO protein knockout gene related cell line.
The pig NONO protein knockout gene related plasmid and the pig NONO protein knockout gene related cell line are applied to preparation of vaccines.
After PRV is infected by the pig NONO protein knockout gene-related cell line, IFN-beta mRNA expression is inhibited.
The pig NONO protein knockout gene-related cell line promotes the proliferation of PRV wild type and vaccine strains, and can be applied to PRV vaccine production.
The invention has the beneficial effects that:
(1) the sNONO KO-PAM-KNU cell line constructed by the invention obviously reduces the expression level of IFN-beta under the condition of PRV infection, and can be applied to the research of regulating the innate immunity mechanism by the NONO of pigs;
(2) PRV infection experiments prove that under the same conditions, the sNONO KO-PAM-KNU cell line constructed by the invention is more favorable for proliferation of PRV wild type and vaccine strains than the PAM-KNU cell line, and the cell line can be applied to sNONO antiviral action research and PRV vaccine production.
Description of the drawings:
FIG. 1 is a plasmid map for constructing an sNONO gene knockout cell line, and the left figure is a plasmid map of Px 459M; the middle diagram is a plasmid map of EZ-Guide-XH; the right figure is a plasmid map of Px 459M-sNONO-KO;
FIG. 2 isBbs I electrophoresis picture of restriction enzyme Px459M plasmid, band obtained after restriction enzyme is carrierA body segment 1;
FIG. 3 is a drawing showingBbs I, carrying out enzyme digestion on an electrophoresis picture of an EZ-Guide-XH plasmid, wherein a band obtained after the enzyme digestion is a vector fragment 2;
FIG. 4 is a PCR identification map of the recombinant vectors Px459M-sNONO-KO-3P and EZ-Guide-XH-sNONO-KO-4P, wherein a fragment of about 400 bp is the PCR result of Px459M-sNONO-KO-3P and a fragment of about 200 bp is the PCR result of EZ-Guide-XH-sNONO-KO-4P;
FIG. 5 is a drawing showingHind III/Xho I electrophoresis images of a double-enzyme digestion recombinant vector Px459M-sNONO-KO-3P and EZ-Guide-XH-sNONO-KO-4P, wherein a vector fragment 3 is obtained by double-enzyme digestion of Px459M-sNONO-KO-3P, and a gene fragment 4 is obtained by double-enzyme digestion of EZ-Guide-XH-sNONO-KO-4P;
FIG. 6 shows Px459M-sNONO-KO plasmidHind III/Xho I, double enzyme digestion identification map;
FIG. 7 is an identification of sNONO KO-PAM-KNU cell line, wherein the PCR result of the PAM-KNU cell line amplifying sNONO full length is 1413 bp; the PCR result of the sNONO KO-PAM-KNU cell line for amplifying the full length of sNONO is 853 bp;
FIG. 8 shows the sequencing result of sNONO gene of sNONO KO-PAM-KNU cell line, and swine NONO is the sequence of the sNONO gene of PAM-KNU cell line; the brine NONO KO is an sNONO KO-PAM-KNU cell line NONO gene sequence;
FIG. 9 shows PRV SD1404 infected sNONO KO-PAM-KNU and PAM-KNU cell lines, respectively, and IFN- β expression levels were measured in both cells;
FIG. 10 shows PRV SD1404 infected sNONO KO-PAM-KNU and PAM-KNU cell lines, respectively, sampled at defined time points, and PRV titer determined;
FIG. 11 shows PRV Bartha-K61 vaccine strains infected with sNONO KO-PAM-KNU and PAM-KNU cell lines, respectively, sampled at defined time points, and tested for PRV titers.
Detailed Description
The technical solution of the present application will be specifically described below with reference to specific examples.
Example 1: construction of pig NONO gene knockout plasmid Px459M-sNONO-KO
1.1 primer design
Two pairs of primers were designed, respectively, sNONO-KO-3Fwd/sNONO-KO-3Rev and sNONO-KO-4Fwd/sNONO-KO-4Rev with primers at the 5' endBbs I, the nucleotide sequence after enzyme digestion is as follows:
sNONO-KO-3Fwd: 5’-CACCAGGAAGGTTTCGGACCGTAA-3’，
sNONO-KO-3Rev: 5’-AAACTTACGGTCCGAAACCTTCCT-3’，
sNONO-KO-4Fwd: 5’-CACCGGCGTCAAGAAGAACTTCGG-3’，
sNONO-KO-4Rev: 5’-AAACCCGAAGTTCTTCTTGACGCC-3’，
the two pairs of guide RNAs are edited simultaneously on two exon regions of the sNONO gene, and the mRNA transcribed from the edited gene is reduced by about 500 bp compared with the normal NONO mRNA.
1.2 phosphorylation and annealing of primers
The primers sNONO-KO-3Fwd/sNONO-KO-3Rev and sNONO-KO-4Fwd/sNONO-KO-4Rev are diluted to 100 mu M, and the two pairs of primers are respectively phosphorylated and annealed to obtain sNONO-KO-3P and sNONO-KO-4P. The reaction system is 10 mu L, each 10 mu M of sNONO-KO-3Fwd/sNONO-KO-3Rev and sNONO-KO-4Fwd/sNONO-KO-4Rev is contained, 10 xT 4 ligase buffer 1 mu L, T4 PNK 1U, H 2 O6 muL. The reaction conditions are as follows: 30 min at 37 ℃ and 5 min at 95 ℃, and the temperature of PCR is reduced to 25 ℃ in a gradient way, wherein the temperature is reduced by 0.1 ℃ per second, 5 min at 25 ℃ and 5 min at 4 ℃.
1.3 construction and identification of Gene knockout plasmids
Plasmid maps of knock-out plasmids Px459M and EZ-Guide-XH are shown in FIG. 1, usingBbs I enzyme digestion of plasmid Px459M and EZ-Guide-XH, respectively glue recovery to obtain vector fragment 1 and vector fragment 2, the results are shown in figure 2 and figure 3, sNONO-KO-3P is connected to vector fragment 1 to obtain Px459M-sNONO-KO-3P, sNONO-KO-4P is connected to vector fragment 2 to obtain EZ-Guide-XH-sNONO-KO-4P, DH5 alpha competent bacteria are transformed, overnight culture is carried out at 37 ℃, single colony is picked up in LB culture medium with ampicillin resistance, overnight shaking culture is carried out at 37 ℃, plasmid is extracted, Px459M-sNONO-KO P is subjected to PCR identification by using sNONO-KO-3Fwd and CAG-R, CAG-R primer sequence is as follows:
CAG-R: 5’-GTACTGGGCACAATGCCAG-3’
EZ-Guide-XH-sNONO-KO-4P Using sNONO-KO-4Fwd and M13F Universal primers (c: (R))5'-TGTAAAACGACGGCCAGT-3') were subjected to PCR, and the results are shown in FIG. 4. The plasmid with positive identification result is sequenced by Qingdao engine company, and the sequence is analyzed by DNAstar software. By usingHind III/Xho I double-enzyme digestion Px459M-sNONO-KO-3P and EZ-Guide-XH-sNONO-KO-4P respectively to obtain a vector fragment 3 and a gene fragment 4, the result is shown in figure 5, the gene fragment 4 is connected to the vector fragment 3, the obtained connection product is transformed into DH5 alpha competent bacteria, the overnight culture is carried out at 37 ℃, a single colony is selected to be arranged in an LB culture medium with ampicillin resistance, the overnight shaking culture is carried out at 37 ℃, plasmids are extracted, and the plasmid is usedHind III/Xho The result of double enzyme digestion identification is shown in figure 6, then the DNA fragment is subjected to sequencing by Qingdao engine company, DNAstar software is used for sequence analysis, the Px459M-sNONO-KO plasmid is obtained, the plasmid map is shown in figure 1, and the sequence inserted in the plasmid is shown in sequence 1.
Example 2: construction of pig NONO gene knockout cell line sNONO KO-PAM-KNU
2.1 testing of the puromycin resistance of PAM-KNU cells
Digesting PAM-KNU cells, diluting with cell culture medium to make the density of the cells 1 × 10 4 cells/mL, 1 mL per well, were plated onto 12-well cell culture plates in 5% CO 2 And cultured overnight in an incubator at 37 ℃. Cell tolerance tests were performed the following day using puromycin concentrations: 1.2, 4, 6, 8 and 10 mug/mL. The puromycin concentration causing total cell death by day 4 of the screening was used as the optimal drug action concentration for monoclonal screening of PAM-KNU cells. The optimal puromycin action concentration screened by the method is 4 mug/mL.
2.2 knock-out plasmid Px459M-sNONO-KO transfected PAM-KNU cells
Digesting PAM-KNU cells, diluting to 1X 10 using cell culture medium 4 cells/mL were seeded onto 12-well cell culture plates and cultured overnight in a cell incubator according to Lipofectamine ® 3000 (Invitrogen) instructions for transfection procedure, 50. mu.L of 37 ℃ preheated OPTI-MEM serum-free medium was added to a sterilized 1.5 mL centrifuge tube, then 2. mu. L P3000 and 1. mu.g of recombinant plasmid Px459M-sNONO-KO were added and mixed gently; in anotherAdding 50 muL of OPTI-MEM serum-free culture medium preheated at 37 ℃ into a sterilized 1.5 mL centrifuge tube, and then adding 3 muL of Lipofectamine ® 3000 transfection reagent, mix gently, mix reagents in two 1.5 mL centrifuge tubes, incubate for 10 min at room temperature, then add to the medium of the cell to be transfected, put into the cell incubator to continue culturing. Untransfected cells were also set as controls.
2.3 screening of sNONO KO-PAM-KNU cell line
24 hours after transfection, cells were screened using cell culture medium containing 4 μ g/mL puromycin. By day 4 of the selection, untransfected control cells all died, and viable resistant cell clones appeared in PAM-KNU cells transfected with Px 459M-sNONO-KO. Discarding a screening culture medium in resistant cells, replacing the screening culture medium with a normal cell culture medium for continuous culture, selecting cell clones, diluting the cells according to a limiting dilution method, then inoculating the cells into a 96-well cell culture plate to ensure that the number of cells in each well is about 1, placing the cells into a cell culture box for culture, digesting the cells in the 96-well plate after the cells grow to a monolayer, respectively performing amplification culture to 24-well plates, after the cells grow to the monolayer, digesting the cells, taking 50% of the cells out of each well for identification, and continuously culturing the rest cells. The cell line identified as positive was expanded and named sNONO KO-PAM-KNU cell line.
Example 3: identification of sNONO KO-PAM-KNU cell line
3.1 PCR identification
Extracting RNA from cells taken out of a 24-well plate and PAM-KNU cells used as a control, carrying out reverse transcription on the RNA to form cDNA, and carrying out PCR identification by using a primer for amplifying an sNONO gene sequence by using the reverse transcribed cDNA as a template, wherein the primer sequence is as follows:
sNONO-Fwd: 5’-ATGCAGAGCAATAAAACT-3’
sNONO-Rev: 5’-TTAGTATCGGCGACGTTTGTTTGGA-3’
the PCR reaction system is 25 mu L, contains 1 mu L of cDNA, 200 mu moL/L of dNTP, 2.5 mu L of 10 XPCR Buffer, 400 nmoL/L of sNONO-Fwd and sNONO-Rev respectively, and TaKaRa Taq TM 0.25U. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; then 35 cycles were performed, cycleThe conditions are 95 ℃ for 45 s, 60 ℃ for 45 s and 72 ℃ for 2 min; then, the PCR product was extended at 72 ℃ for 10 min and subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 7, in which the electrophoresis band of the selected positive sNONO KO-PAM-KNU cells was 896 bp, and the electrophoresis band of the selected positive sNONO KO-PAM-KNU cells was 1413 bp.
3.2 Gene sequencing identification
And (3) carrying out gel recovery on the band with the electrophoresis size of 896 bp in the steps, handing the recovered product to Qingdao engine company for sequencing, wherein the result is shown in figure 8, and the nucleotide sequence is shown in a sequence 2 in a sequence table. Compared with the NONO gene sequence (see the sequence 3 in the sequence table), the base deletion is found to occur between 450 and 966 bp.
Example 4: application of sNONO KO-PAM-KNU cell line
4.1 Effect of sNONO on PRV-induced IFN- β expression levels
Digesting sNONO KO-PAM-KNU and PAM-KNU cells, inoculating the cells to a 12-hole cell culture plate, culturing the cells until the cells grow full of a monolayer, infecting PRV with 1 MOI, collecting the cells at 3 h, 6 h, 9 h and 12 h respectively, extracting total RNA by using a TRIzol reagent, carrying out reverse transcription by using a PrimeScript RT reagent kit with gDNA Eraser kit of Takara, synthesizing cDNA, carrying out real-time fluorescence quantitative PCR analysis by using sIFN-beta and beta-actin primers, wherein the primer sequences are as follows:
sIFN-β-F：5’-TGCATCCTCCAAATCGCTCT-3’
sIFN-β-R：5’-ATTGAGGAGTCCCAGGCAAC-3’
sβ-actin-F：5’-TCTGGCACCACACCTTCT-3’
sβ-actin-R：5’-GATCTGGGTCATCTTCTCAC-3’
as shown in FIG. 9, the level of IFN- β mRNA in sNONO KO-PAM-KNU after PRV infection is significantly lower than that in PAM-KNU cells, which proves that sNONO KO-PAM-KNU can be applied to the research on sNONO regulation of innate immunity.
4.2 application of sNONO KO-PAM-KNU cells in PRV proliferation
Inoculating sNONO KO-PAM-KNU and PAM-KNU cells into a 12-hole plate, infecting 1 MOI PRV wild strain PRV SD1404 after the cells grow into a monolayer, collecting 100 mu L of supernatant at 3 h, 6 h, 9 h and 12 h respectively, and collecting the supernatant on PK-15 cellsDetermination of TCID for PRV at Each time Point 50 Referring to FIG. 10, PRV SD1404 was found to have a greater proliferative capacity on sNONO KO-PAM-KNU cells than on PAM-KNU cells, so that the sNONO KO-PAM-KNU cell line could be applied to relevant studies on the proliferation of PRV.
4.3 application of sNONO KO-PAM-KNU cells in vaccine production
Inoculating sNONO KO-PAM-KNU and PAM-KNU cells into a 12-hole plate, infecting 1 MOI PRV Barthar-K61 vaccine strain when the cells grow into a single layer, respectively collecting 100 mu L of supernatant at 3 h, 6 h, 9 h and 12 h, and determining TCID at each time point on PK-15 cells 50 Referring to FIG. 11, PRV Barthar-K61 strain was found to have a stronger proliferation potency on sNONO KO-PAM-KNU cells than on PAM-KNU cells, so that the sNONO KO-PAM-KNU cell line could be applied to vaccine production of PRV.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Sequence listing
<110> institute of veterinary and animal husbandry of academy of agricultural sciences of Shandong province
<120> pig NONO protein knockout gene, related plasmid, cell line, preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 565
<212> DNA
<213> artificial sequence
<400> 1
caccaggaag gtttcggacc gtaagtttta gagctagaaa tagcaagtta aaataaggct 60
agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tagagctaga 120
aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa 180
tggctctaga gtcgacacta gtctcgagga gggcctattt cccatgattc cttcatattt 240
gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa 300
gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 360
aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 420
cttggcttta tatatcttgt ggaaaggacg aaacaccggc gtcaagaaga acttcgggtt 480
ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 540
accgagtcgg tgctttttta agctt 565
<210> 1
<211> 896
<212> DNA
<213> artificial sequence
<400> 1
atgcagagca ataaaacttt taacttggag aagcaaaacc acactccaag gaaacatcat 60
cagcatcacc atcagcagca ccaccagcag caacagcagc agccaccgcc tccaccaata 120
cctgcaaacg ggcagcaagc cagcagccag aatgaaggct taactattga cctgaagaat 180
tttaggaaac caggagagaa gaccttcacc caacgtagcc ggctctttgt gggcaatctt 240
cctcctgaca tcactgagga ggaaatgagg aaactatttg agaaatacgg gaaggcaggc 300
gaagtcttca ttcacaagga taaaggcttt ggctttatcc gcttggaaac acgaacccta 360
gcggagattg ccaaagtgga actggacaac atgccactcc gtggaaagca gctgcgtgtg 420
cgctttgcct gccatagtgc atcccttatc ggaggatgga agagctgcat aaccaagaag 480
tgcaaaaacg aaagcagttg gagctcagac aggaggagga acgcaggcgc cgtgaggaag 540
agatgcggcg gcagcaagaa gaaatgatgc ggcgacagca ggaaggattc aagggaacct 600
tccctgatgc gagagagcag gagatacgga tgggccagat ggctatggga ggtgctatgg 660
gcataaacaa cagaggcgcc atgccccctg ctcctgtgcc agctggtaca ccagctcctc 720
caggacctgc cactatgatg ccagatggaa ccttgggatt gaccccacca acaactgaac 780
gctttggcca agctgctaca atggaaggaa ttggggcaat tggtggaacc cctccagcat 840
tcaaccgtgc agctcctgga gctgaatttg ctccaaacaa acgtcgccga tactaa 896
<210> 1
<211> 1413
<212> DNA
<213> artificial sequence
<400> 1
atgcagagca ataaaacttt taacttggag aagcaaaacc acactccaag gaaacatcat 60
cagcatcacc atcagcagca ccaccagcag caacagcagc agccaccgcc tccaccaata 120
cctgcaaacg ggcagcaagc cagcagccag aatgaaggct taactattga cctgaagaat 180
tttaggaaac caggagagaa gaccttcacc caacgtagcc ggctctttgt gggcaatctt 240
cctcctgaca tcactgagga ggaaatgagg aaactatttg agaaatacgg gaaggcaggc 300
gaagtcttca ttcacaagga taaaggcttt ggctttatcc gcttggaaac acgaacccta 360
gcggagattg ccaaagtgga actggacaac atgccactcc gtggaaagca gctgcgtgtg 420
cgctttgcct gccatagtgc atcccttacg gtccgaaacc ttcctcagta tgtgtccaac 480
gaactgctgg aggaagcctt ttctgtgttc ggccaggtag agagggctgt agtcatcgtg 540
gatgatcgag gaaggccctc agggaaaggc attgtcgagt tttcagggaa gccagctgct 600
cgcaaagctc tggacagatg cagtgaaggc tccttcctgc taaccacatt tcctaggcct 660
gtgactgtgg agcccatgga ccagttagat gatgaagagg gacttccaga gaagttggtt 720
ataaagaacc agcaatttca caaggagcga gagcagccac ccaggtttgc acagcctggc 780
tcttttgagt atgagtatgc tatgcgctgg aaggcactta ttgagatgga gaagcagcag 840
caggaccaag tggaccgaaa catcaaggaa gcccgtgaga agctggagat ggagatggag 900
gctgctcgcc atgaacatca ggtcatgcta atgaggcagg atttgatgag gcgtcaagaa 960
gaacttcgga ggatggaaga gctgcataac caagaagtgc aaaaacgaaa gcagttggag 1020
ctcagacagg aggaggaacg caggcgccgt gaggaagaga tgcggcggca gcaagaagaa 1080
atgatgcggc gacagcagga aggattcaag ggaaccttcc ctgatgcgag agagcaggag 1140
atacggatgg gccagatggc tatgggaggt gctatgggca taaacaacag aggcgccatg 1200
ccccctgctc ctgtgccagc tggtacacca gctcctccag gacctgccac tatgatgcca 1260
gatggaacct tgggattgac cccaccaaca actgaacgct ttggccaagc tgctacaatg 1320
gaaggaattg gggcaattgg tggaacccct ccagcattca accgtgcagc tcctggagct 1380
gaatttgctc caaacaaacg tcgccgatac taa 1413
Claims (10)
1. The pig NONO protein knockout gene is characterized in that a nucleotide sequence is shown as a nucleotide sequence of a sequence 2 in a sequence table.
2. The NONO protein knockout gene in swine as claimed in claim 1, wherein the nucleotide sequence of the NONO protein has a deletion of between 450 and 966 bp bases.
3. A pig NONO protein knockout gene related plasmid contains a nucleotide sequence of a sequence 1 in a sequence table.
4. The porcine NONO protein knockout gene-related plasmid of claim 3, wherein the plasmid is selected from the group consisting of Px459M plasmidHind III cleavage site and distanceHind III furthest from the cleavage siteBbs IThe nucleotide sequence inserted between the enzyme cutting sites is shown as the nucleotide sequence of the sequence 1 in the sequence table.
5. A preparation method of pig NONO protein knockout gene related plasmid is characterized by comprising the following steps:
(1) two pairs of primers were designed with the following nucleotide sequences:
sNONO-KO-3Fwd: 5’-CACCAGGAAGGTTTCGGACCGTAA-3’，
sNONO-KO-3Rev: 5’-AAACTTACGGTCCGAAACCTTCCT-3’，
sNONO-KO-4Fwd: 5’-CACCGGCGTCAAGAAGAACTTCGG-3’，
sNONO-KO-4Rev: 5’-AAACCCGAAGTTCTTCTTGACGCC-3’，
phosphorylating and annealing primers sNONO-KO-3Fwd and sNONO-KO-3Rev to obtain sNONO-KO-3P, phosphorylating and annealing primers sNONO-KO-4Fwd and sNONO-KO-4Rev to obtain sNONO-KO-4P;
(2) construction of Gene knockout plasmids
Use ofBbsI enzyme digestion plasmids Px459M and EZ-Guide-XH are respectively recovered by glue to obtain a vector fragment 1 and a vector fragment 2, sNONO-KO-3P is connected to the vector fragment 1 to obtain Px459M-sNONO-KO-3P, sNONO-KO-4P is connected to the vector fragment 2 to obtain EZ-Guide-XH-sNONO-KO-4P, and the use of the enzyme digestion plasmids Px459M and EZ-Guide-XH is used forHind III/XhoI, performing double enzyme digestion on Px459M-sNONO-KO-3P and EZ-Guide-XH-sNONO-KO-4P respectively to obtain a vector fragment 3 and a gene fragment 4, and connecting the vector fragment 3 and the gene fragment 4 to obtain a pig NONO protein knockout gene-related plasmid Px 459M-sNONO-KO.
6. The method according to claim 5, wherein the phosphorylation and annealing operations are as follows: the reaction system contains 10 mu moL/L of sNONO-KO-3Fwd and sNONO-KO-3Rev or sNONO-KO-4Fwd and sNONO-KO-4Rev, 10 XT 4 ligase buffer 1 mu L, T4 PNK 1U, H 2 O6 muL, 10 muL in total; the reaction conditions are as follows: 30 min at 37 ℃ and 5 min at 95 ℃, and the temperature of PCR is reduced to 25 ℃ in a gradient way, wherein the temperature is reduced by 0.1 ℃ per second, 5 min at 25 ℃ and 5 min at 4 ℃.
7. A cell line related to pig NONO protein knockout gene, wherein the plasmid related to pig NONO protein knockout gene of claim 5 or 6 is used for transfecting PAM-KNU cells, puromycin-containing culture medium is used for screening, and the surviving resistant cell clone is the cell line related to pig NONO protein knockout gene.
8. Use of the pig NONO protein knockout gene of claim 1 or 2, the pig NONO protein knockout gene associated plasmid of claim 3 or 4, or the pig NONO protein knockout gene associated cell line of claim 7 in the preparation of a vaccine.
9. The use according to claim 8, wherein the porcine NONO protein knockout gene related cell line inhibits IFN- β mRNA expression following PRV infection.
10. Use according to claim 8, characterized in that the cell line promotes PRV proliferation.
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| JP2014039509A (en) * | 2012-08-23 | 2014-03-06 | Osaka Univ | Anticancer substance screening method |
| CN103667430A (en) * | 2012-09-18 | 2014-03-26 | 上海吉凯基因化学技术有限公司 | Application and relevant medicament of nucleotide octamer binding protein expression gene |
| CN109456995A (en) * | 2018-11-08 | 2019-03-12 | 杜以军 | Gene knockout plasmid, cell line and preparation method and application |
| KR20200022187A (en) * | 2018-08-22 | 2020-03-03 | 울산대학교 산학협력단 | Composition for enhancing radiation sensitivity comprising expression or activity inhibitor of NONO |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2014039509A (en) * | 2012-08-23 | 2014-03-06 | Osaka Univ | Anticancer substance screening method |
| CN103667430A (en) * | 2012-09-18 | 2014-03-26 | 上海吉凯基因化学技术有限公司 | Application and relevant medicament of nucleotide octamer binding protein expression gene |
| KR20200022187A (en) * | 2018-08-22 | 2020-03-03 | 울산대학교 산학협력단 | Composition for enhancing radiation sensitivity comprising expression or activity inhibitor of NONO |
| CN109456995A (en) * | 2018-11-08 | 2019-03-12 | 杜以军 | Gene knockout plasmid, cell line and preparation method and application |
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