CN109055385A - A kind of effective gene order and its application for inhibiting II type PRRSV infection - Google Patents
A kind of effective gene order and its application for inhibiting II type PRRSV infection Download PDFInfo
- Publication number
- CN109055385A CN109055385A CN201810952139.4A CN201810952139A CN109055385A CN 109055385 A CN109055385 A CN 109055385A CN 201810952139 A CN201810952139 A CN 201810952139A CN 109055385 A CN109055385 A CN 109055385A
- Authority
- CN
- China
- Prior art keywords
- seq
- pig
- gene
- gene order
- sequence
- 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.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 49
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 title claims abstract description 35
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 28
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 13
- 101100382101 Sus scrofa CD163 gene Proteins 0.000 claims abstract description 18
- 101150087379 CD163 gene Proteins 0.000 claims abstract description 6
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 18
- 238000011144 upstream manufacturing Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 238000000338 in vitro Methods 0.000 claims description 8
- 210000002950 fibroblast Anatomy 0.000 claims description 7
- 230000008685 targeting Effects 0.000 claims description 7
- 210000003754 fetus Anatomy 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 101150072531 10 gene Proteins 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims description 2
- 238000012239 gene modification Methods 0.000 claims description 2
- 229940097275 indigo Drugs 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 claims 1
- 210000001132 alveolar macrophage Anatomy 0.000 abstract description 7
- 108091033409 CRISPR Proteins 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 230000002685 pulmonary effect Effects 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 2
- 230000009456 molecular mechanism Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 241000282898 Sus scrofa Species 0.000 description 53
- 210000004027 cell Anatomy 0.000 description 23
- 108020004414 DNA Proteins 0.000 description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 description 13
- 230000000295 complement effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 101150038500 cas9 gene Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 101100129922 Caenorhabditis elegans pig-1 gene Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101100520057 Drosophila melanogaster Pig1 gene Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001147 anti-toxic effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000032625 disorder of ear Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000000630 fibrocyte Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of gene orders for effectively inhibiting II type PRRSV infection, for the gene order that II type PRRSV infection can be effectively suppressed after a kind of replacement the 7th exon of pig CD163, application of the sequence in disease-resistant research is also disclosed simultaneously, utilizes CRISPR/Cas9 technology；Gene order the 5th exon genes sequence of pig CD163 as shown in SEQ ID 1 is successfully replaced into the gene order of the 7th exon of pig CD163, and obtains the pulmonary alveolar macrophage of the positive colony pig of CD163 gene order such as SEQ ID 2；It can be used for illustrating the molecular mechanism of PRRSV infection and effectively inhibit the pig population of PRRSV infection to provide theoretical basis for subsequent acquisition, reduce because PRRSV gives pig breeding industry bring massive losses.
Description
Technical field
It is a kind of replacement pig CD163 the invention discloses a kind of gene order for effectively inhibiting II type PRRSV infection
The gene order of II type PRRSV infection can be effectively suppressed after seven exons, while also disclosing the sequence in disease-resistant research
Using belonging to technical field of bioengineering.
Background technique
Porcine reproductive and respiratory syndrome (PRRS) is also known as pig blue-ear disease, is by porcine reproductive and respiratory syndrome virus
(PRRSV) global infectious disease caused by, low, the pregnant sow miscarriage that is characterized primarily by fertility of sow of PRRS, piglet and
Pneumonia occurs for growing and fattening pigs, and along with respiratory disorder, piglet slow growth etc.；In addition, can also bring serious immunosupress and
Persistent infection.The disease has been found within 1996 for the first time in China, and since 2006, the appearance of the pathogenic variant of height is so that the disease exists
Chinese large-scale outburst, can all cause serious economic loss to pig breeding industry every year.According to the difference of genotype, PRRSV master
It wants that two hypotypes: I type (Europe class) and II type (U.S. state type) can be divided into.Since PRRSV strain speed of mutation is fast；PRRSV with
There are antibody dependents to enhance phenomenon between host；These factors result in worldwide that vaccine immunity protection mechanism cannot
Effectively immunoprotection is provided to swinery.
Summary of the invention
The invention discloses a kind of gene orders for effectively inhibiting II type PRRSV infection, and are prepared for containing this section of gene
The clone pig of sequence.The experimental results showed that the PAMs replaced after the 7th exon of pig CD163 with this section of gene order can be effective
Inhibit the infection of II type PRRSV in vitro.
A kind of gene order effectively inhibiting II type PRRSV infection disclosed by the invention, it is characterised in that:
Gene order is as shown in SEQ ID 1；CD163 gene order after gene modification as: shown in SEQ ID 2；Can effectively it expand
Increase the upstream primer sequence F1 of the target gene as: shown in SEQ ID 3, downstream primer sequence R1 as: shown in SEQ ID 4；Energy
Effectively amplification the 7th exon upstream gene 5-HA of CD163 upstream primer sequence F2 as: shown in SEQ ID 5, downstream primer
Sequence R2 as: shown in SEQ ID 6, the gene order 5-HA that amplifies as: shown in SEQ ID 7；CD163 can effectively be expanded
The upstream primer sequence F3 of seven exon downstream gene 3-HA is such as: SEQ ID 8, downstream primer sequence R3 such as 9 institute of SEQ ID
Show, the gene order amplified as: shown in SEQ ID 10.By SEQ ID 1, SEQ ID 7, SEQ ID 10 recombination connection are obtained
Sequence as shown in SEQ ID 19.
The present invention can effectively edit the DNA sequence dna of the sgRNA of pig CD163 gene cluster, it is characterised in that: the DNA of sgRNA1
Sequence as shown in SEQ ID 11, the DNA sequence dna of complementary strand as: shown in SEQ ID 12, the RNA sequence of sgRNA1 such as SEQ ID
Shown in 13, the RNA sequence of complementary strand as: shown in SEQ ID 14；The DNA sequence dna of sgRNA2 is as shown in SEQ ID 15, complementary strand
DNA sequence dna as: shown in SEQ ID 16, the RNA sequence of sgRNA1 as shown in SEQ ID 17, the RNA sequence of complementary strand such as:
Shown in SEQ ID 18.
A kind of preparation method of gene order effectively inhibiting II type PRRSV infection of the present invention, including following step
It is rapid:
1) pig CD163 gene order is recalled in NCBI gene pool；
2) base sequence such as SEQ ID 3 respectively, upstream primer F1 shown in SEQ ID 4 are obtained using Primer5 software design
With downstream primer R1；
3) base sequence objective gene sequence as shown in SEQ ID 1 is obtained using PCR amplification in vitro method；
4) pig CD163 gene order is recalled in NCBI gene pool；
5) base sequence is obtained respectively such as SEQ ID 5, SEQ ID 6, SEQ ID 8, SEQ ID using Primer5 software design
Upstream primer F2 shown in 9, R2 and downstream primer F3, R3；
6) base sequence such as SEQ ID 7 is obtained using PCR amplification in vitro method；Target gene upstream and downstream shown in SEQ ID 10
Gene；
7) by above-mentioned base sequence obtained such as SEQ ID 1, SEQ ID 7, gene of SEQ ID 10 etc. is than being added containing SEQ
In the PCR system of ID 5, SEQ ID 9；
8) by round pcr, vitro recombination connects these three segments, obtains sequence homologous replacement as shown in SEQ ID 19 and carries
Body；
9) by the sgRNA plasmid of building: SEQ ID 11, SEQ ID 12, SEQ ID 14, SEQ ID 15 and homologous targeting vector
Into porcine fetus fibroblasts PFF, and by limiting dilution assay, screening and identification obtains positive ho-mology and replaces 19 cotransfection of SEQ ID
The porcine fetus fibroblasts changed, positive fibroblasts CD163 gene order is as shown in SEQ ID 2.
A kind of gene order effectively inhibiting II type PRRSV infection of the present invention.It is characterized by: the gene
Gene order is obtained as shown in SEQ ID 2 after fixed point replacement the 7th exon of pig CD163 of sequence SEQ ID 1.Isolated
CD163 gene order primary pulmonary alveolar macrophage of positive colony pig as shown in SEQ ID 2 can effectively inhibit II type PRRSV
Duplication, and illustrate PRRSV infection Mechanism Study to be subsequent and the swinery of PRRSV infection can be effectively suppressed provide reason to obtain
By basis.
The positive effect of the present invention is: CRISPR/Cas9 technology is utilized, by gene order pig as shown in SEQ ID 1
The 5th exon genes sequence of CD163 successfully replaces the gene order of the 7th exon of pig CD163, and obtains CD163
The pulmonary alveolar macrophage of the positive colony pig of gene order such as SEQ ID 2；Can be used for illustrating PRRSV infection molecular mechanism and
Effectively inhibit the pig population of PRRSV infection to provide theoretical basis for subsequent acquisition, reduces because PRRSV is huge to pig breeding industry bring
Big loss.
Detailed description of the invention:
Fig. 1: for assessing the sequencing peak figure of the sgRNA cutting efficiency in the site CD163 exon7；
Fig. 2: the sequencer map and RFLP electrophoretogram of fixed point target practice pig fetal cell positive colony；
Fig. 3: absolute fluorescence quantitative PCR result.
Specific embodiment
By following embodiment further illustrate description the present invention, do not limit the invention in any way, without departing substantially from
Under the premise of technical solution of the invention, easy to accomplish any of those of ordinary skill in the art made for the present invention changes
Dynamic or change is fallen within scope of the presently claimed invention.
Embodiment 1
The preparation of the gene order of II type PRRSV infection can be effectively suppressed as shown in SEQ ID 1
1) pig CD163 gene order is recalled in NCBI gene pool；
2) base sequence such as SEQ ID 3 respectively, upstream primer F1 shown in SEQ ID 4 are obtained using Primer5 software design
With downstream primer R1；
3) base sequence objective gene sequence as shown in SEQ ID 1 is obtained using PCR amplification in vitro method；
Embodiment 2
The design of 1-1, sgRNA sequence and the building of PX330 expression vector
2 sgRNA sequences for being directed to the site pig CD163 exon7 are designed and synthesized.The sgRNA sequence that above-mentioned design is completed is closed
At；The DNA sequence dna of 4 single-stranded sgRNA forms 2 targeting pig CD163 exon7 different locis after annealing respectively
The oligonucleotide chain of sgRNA；Then the oligonucleotide is entered into PX330 plasmid vector repeatedly.
The sequence of this 2 sgRNA and its sequence of action site are respectively as follows:
SgRNA-1 sequence: 5-GGAAACCCAGGCTGGTTGGA-3
The sequence of SgRNA-1 action site: 5-TCCAACCAGCCTGGGTTTCC-3
SgRNA-2 sequence: 5-GAGTAGCACCCCGCCCTGAC-3
The sequence of SgRNA-2 action site: 5-GTCAGGGCGGGGTGCTACTC-3
Wherein, according to the present invention
The RNA sequence of SgRNA-1 are as follows: 5-GGAAACCCAGGCUGGUUGGA-3
The complementary series of the RNA sequence of SgRNA-1 are as follows: 5-UCCAACCAGCCUGGGUUUCC-3
Wherein, according to the present invention
The RNA sequence of SgRNA-2 are as follows: 5- GAGUAGCACCCCGCCCUGAC -3
The complementary series of the RNA sequence of SgRNA-2 are as follows: 5- GUCAGGGCGGGGUGCUACUC -3.
The assessment and screening of 1-2, efficient sgRNA
After 2 kinds of PX330-sgRNA expression vector sequence verifications to building, extracts purpose plasmid and carries out ethanol precipitation,
Will certain density three kinds of PX330-sgRNA expression vectors after purification, be introduced by way of electroporation transfection pig fetus at
In fibrocyte, after transfection 72 hours, the genome of group of cells is extracted, then with the primer of the detection mutation efficiency of specificity
PCR reaction is carried out, PCR product obtained is sent into sequencing cutting by the analysis entry evaluation sgRNA to sequencing peak figure
Efficiency is cut, while remaining PCR product is used to connect carrier T or each sgRNA is accurately assessed to target base by T7E1 analysis
The cutting efficiency of cause (referring to Fig. 1, for assessing the sequencing peak figure of 2 difference sgRNA cutting efficiencies)；Fig. 1 is the result shows that above-mentioned
Corresponding to sgRNA1, SEQ ID 13 and SEQ ID 14 corresponding to SEQ ID 11 and SEQ ID 12 obtained
SgRNA2 sequence can effectively guide cas9 albumen to cut target site in pig cell.
Embodiment 3
The building of homologous targeting vector
According to the efficient sgRNA filtered out and construct homologous targeting vector (sequence such as 19 institute of SEQ ID matched with the sgRNA
Show), the major components of the targeting vector are successively are as follows: sequence upstream homology arm as shown in SEQ ID 7, pig sequence such as SEQ ID
The exon of CD163 the 5th shown in 1, sequence skeleton carrier of downstream homology arm and prokaryotic expression as shown in SEQ ID 10.It should
Site-directed integration target practice plasmid and the sgRNA collective effect filtered out can carry out special gene to the site pig CD163 exon7
Then transformation can very easily analyze foreign gene in conjunction with the method for PCR and RFLP and integrate simultaneously in the sgRNA recognition site
The feasibility of expression.The site-directed integration targeting vector constructed in the present invention, base sequence and SEQ ID 19 are consistent.
Embodiment 4
The preparation of CD1163 genotype positive colony porcine alveolar macrophage as shown in SEQ ID 2
Electrotransfection buffering is added when after washing 2 ~ 3 times with DPBS, abandon supernatant close to when covering in recovery porcine fetus fibroblasts
Then PX330 plasmid and sequence such as sequence plasmid as shown in SEQ ID 19 are proportionally added into cell and buffer by liquid
In, after being mixed gently with pipettor, electric revolving cup is put on electroporation apparatus and carries out by moving into mixed liquor in pole cup gently
Electric shock operation.After the completion of electric shock, electric revolving cup is stood after ten minutes, and the mixed liquor in electric revolving cup is transferred in Tissue Culture Dish.Most
The Tissue Culture Dish is placed in 37 DEG C of carbon dioxide incubators afterwards and is cultivated.After culture 12 hours, liquid is changed.After electrotransfection 72h, make
Pig PFF cell is taped against in 100mm Tissue Culture Dish by the method for Method of Limited Dilution with trypsin digestion cell, replacement one in 2 ~ 3 days
Secondary cell culture fluid.After 8 ~ 10 days after cell clone grows up to, cell clone is uniformly marked under fluorescence microscope, so
Afterwards these labeled clone's pickings are entered in 24 porocyte culture plates to be then incubated for.It is long to the cell in 24 orifice plates after 2 ~ 3 days
To certain convergence degree, pass through the side of PCR sequencing and RFLP after a quarter of these cells is cracked with NP40 lysate again
Method further verifies pig measurement the 7th exon of pig CD163 fixed point replacement event.
The positive PFFs that above-mentioned screening is obtained obtains external reconstructed embryo by SCNT, and is implanted into replace-conceive sow
In utero, positive F is obtained after about 114 days0Clone pig.Part tail tissue is taken to mention genome identification (such as resulting offspring's clone pig
Shown in Fig. 2).Fig. 2's the result shows that the present invention has been successfully prepared 3 positive colony pigs.After clone pig long to 3 weeks, not
Entire lung is removed in the case where scratching tracheae and lung, and gas tube orifice is tightened immediately, it is closed, it puts into clean sack, low temperature
Transport laboratory back.Entire lung is cleaned with sterile PBS in super-clean bench, removes dirt.By upper 2/3rds part of tracheae
Excision；It is injected with the PBS of 50ML from gas tube orifice, in triplicate.Recovered liquid is blown and beaten repeatedly with suction pipe, keeps its cell mass fastly and glutinous
The dispersion of liquid block.Filtering, 500g, 15min.Supernatant is removed, precipitating is resuspended with PBS.Resulting cell be exactly CD1163 genotype such as
Positive colony porcine alveolar macrophage shown in SEQ ID 2.
In summary: the present invention is successfully obtained using Crispr/cas9 gene editing technology and somatic cell nuclear transfer technique
It is consistent containing sequence such as SEQ ID 2, the cell and individual of the gene order of II type PRRSV infection can be effectively suppressed.
Experimental example 1
The positive external II type PRRSV infection test of CD163 SRCR3 fixed point the 7th exon porcine alveolar macrophage of replacement:
Dual anti-, the 1mM sodium by above-mentioned the obtained primary pulmonary alveolar macrophage cell RPMI-1640,10%FBS, 1% of pig
1% glutamine of pyruvate, 1% nonessential amino acid culture.It is thin that cell obtained by certain density is inoculated in 12 holes
In born of the same parents' culture plate, after 4 hour cells are adherent, inhale and abandon not adherent cell.After cell adherent 12 hours, it is inoculated with II type
PRRSV virus (MOI=1) and cell are incubated for 3 hours altogether；Virus liquid is then discarded, PBS is cleaned three times, and the new culture of 1ml is added
Liquid is put into cell incubator culture.Cell and 250 microlitres of culture solution supernatant are collected after 72 hours, and 750 microlitres of TRIZOL are added,
Extract cell and supernatant total serum IgE.Quantitative RNA amount is 1 microgram, using Bo company cDNA the first chain synthetic agent box to total serum IgE
Carry out reverse transcription.The said firm BioEasy SYBR Green I fluorescent PCR kit and bio-rad iQ5 fluorescence are used later
Quantitative PCR apparatus carries out fluorogenic quantitative detection.Pass through the quantitative result pair of cellular control unit and water control group with not virus inoculation
Than determining the antitoxic effect of cell.(referring to Fig. 3, absolute fluorescence quantitative PCR result)
The experiment of Fig. 3 cell in vitro virus infection shows can with the PAMs after this section of gene order replacement the 7th exon of pig CD163
The infection or even antiviral effect for effectively inhibiting II type PRRSV in vitro become apparent from than deleting the PAMs of the 7th exon.
Sequence table
<110>Jilin University
<120>a kind of gene order and its application for effectively inhibiting II type PRRSV infection
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 315
<212> DNA
<213>pig (Sus scrofa)
<400> 1
cccacaggca gctgagagtg gtagatggag tcactgaatg ttcaggaaga ttggaagtga 60
aattccaagg agaatgggga acaatctgtg atgatggctg ggatagtgat gatgccgctg 120
tggcatgtaa gcaactggga tgtccaactg ctgtcactgc cattggtcga gttaacgcca 180
gtgagggaac tggacacatt tggcttgaca gtgtttcttg ccatggacac gagtctgctc 240
tctggcagtg tagacaccat gaatggggaa agcattattg caatcataat gaagatgctg 300
gtgtgacatg ttcta 315
<210> 2
<211> 3348
<212> DNA
<213>pig (Sus scrofa)
<400> 2
atggacaaac tcagaatggt gctacatgaa aactctggat ctgcagactt tagaagatgt 60
tctgcccatt taagttcctt cacttttgct gtagtcgctg ttctcagtgc ctgcttggtc 120
actagttctc ttggaggaaa agacaaggag ctgaggctaa cgggtggtga aaacaagtgc 180
tctggaagag tggaggtgaa agtgcaggag gagtggggaa ctgtgtgtaa taatggctgg 240
gacatggatg tggtctctgt tgtttgtagg cagctgggat gtccaactgc tatcaaagcc 300
actggatggg ctaattttag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360
cgagggaatg agtcagctct ctgggactgc aaacatgatg gatggggaaa gcataactgt 420
actcaccaac aggatgctgg agtaacctgc tcagatggat ctgatttaga gatgaggctg 480
gtgaatggag gaaaccggtg cttaggaaga atagaagtca aatttcaagg acggtgggga 540
acagtgtgtg atgataactt caacataaat catgcttctg tggtttgtaa acaacttgaa 600
tgtggaagtg ctgtcagttt ctctggttca gctaattttg gagaaggttc tggaccaatc 660
tggtttgatg atcttgtatg caatggaaat gagtcagctc tctggaactg caaacatgaa 720
ggatggggaa agcacaattg cgatcatgct gaggatgctg gagtgatttg cttaaatgga 780
gcagacctga aactgagagt ggtagatgga gtcactgaat gttcaggaag attggaagtg 840
aaattccaag gagaatgggg aacaatctgt gatgatggct gggatagtga tgatgccgct 900
gtggcatgta agcaactggg atgtccaact gctgtcactg ccattggtcg agttaacgcc 960
agtgagggaa ctggacacat ttggcttgac agtgtttctt gccatggaca cgagtctgct 1020
ctctggcagt gtagacacca tgaatgggga aagcattatt gcaatcataa tgaagatgct 1080
ggtgtgacat gttctgatgg atcagatctg gaactgagac ttaaaggtgg aggcagccac 1140
tgtgctggga cagtggaggt ggaaattcag aaactggtag gaaaagtgtg tgatagaagc 1200
tggggactga aagaagctga tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa 1260
acatcatatc aagtttattc caaaaccaag gcaacaaaca catggctgtt tgtaagcagc 1320
tgtaatggaa atgaaacttc tctttgggac tgcaagaatt ggcagtgggg tggacttagt 1380
tgtgatcact atgacgaagc caaaattacc tgctcagccc acaggcagct gagagtggta 1440
gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga atggggaaca 1500
atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca actgggatgt 1560
ccaactgctg tcactgccat tggtcgagtt aacgccagtg agggaactgg acacatttgg 1620
cttgacagtg tttcttgcca tggacacgag tctgctctct ggcagtgtag acaccatgaa 1680
tggggaaagc attattgcaa tcataatgaa gatgctggtg tgacatgttc tagatacaca 1740
caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 1800
gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 1860
cagcagctta aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga 1920
agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catgggagat 1980
tgttccgtca ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc 2040
tgctcaggga accagagtca gacactatcc ccgtgcaatt catcatcctc ggacccatca 2100
agctctatta tttcagaaga aaatggtgtt gcctgcatag ggagtggtca acttcgcctg 2160
gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 2220
accatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 2280
tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 2340
tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 2400
ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcggagttc 2460
atgtctctca gactgatcag tgaaagcagc agagagacct gtgcagggcg cctggaagtt 2520
ttttacaacg gagcttgggg cagcgttggc aagaatagca tgtctccagc cacagtgggg 2580
gtggtatgca ggcagctggg ctgtgcagac agaggggaca tcagccctgc atcttcagac 2640
aagacagtgt ccaggcacat gtgggtggac aatgttcagt gtcctaaagg acctgacacc 2700
ctatggcagt gcccatcatc tccatggaag aagagactgg ccagcccctc agaggagaca 2760
tggatcacat gtgccaacaa aataagactt caagaaggaa acactaattg ttctggacgt 2820
gtggagatct ggtacggagg ttcctggggc actgtgtgtg acgactcctg ggaccttgaa 2880
gatgctcagg tggtgtgccg acagctgggc tgtggctcag ctttggaggc aggaaaagag 2940
gccgcatttg gccaggggac tgggcccata tggctcaatg aagtgaagtg caaggggaat 3000
gaaacctcct tgtgggattg tcctgccaga tcctggggcc acagtgactg tggacacaag 3060
gaggatgctg ctgtgacgtg ttcagaaatt gcaaagagcc gagaatccct acatgccaca 3120
ggtcgctcat cttttgttgc acttgcaatc tttggggtca ttctgttggc ctgtctcatc 3180
gcattcctca tttggactca gaagcgaaga cagaggcagc ggctctcagt tttctcagga 3240
ggagagaatt ctgtccatca aattcaatac cgggagatga attcttgcct gaaagcagat 3300
gaaacggata tgctaaatcc ctcaggagac cactctgaag tacaatga 3348
<210> 3
<211> 32
<212> DNA
<213>pig (Sus scrofa)
<400> 3
cccacaggaa actgagagtg gtagatggag tc 32
<210> 4
<211> 34
<212> DNA
<213>pig (Sus scrofa)
<400> 4
tccctgggtc tcactagaac atgtcaccag catc 34
<210> 5
<211> 22
<212> DNA
<213>pig (Sus scrofa)
<400> 5
gatgattgcg ctcttaaacc tg 22
<210> 6
<211> 19
<212> DNA
<213>pig (Sus scrofa)
<400> 6
ctcagtttcc tgtgggctg 19
<210> 7
<211> 418
<212> DNA
<213>pig (Sus scrofa)
<400> 7
gatgattgcg ctcttaacct ggcaaagatt gtctttaaaa tctgagctcc atgtcttctg 60
ctttatttct ggtgtgcctt tgactccaga ttacagtaaa tggaggactg agtatagggc 120
taaaaagtag agagaatgga tgcatattat ctgtggtctc caatgtgatg aatgaagtag 180
gcaaatactc aaaggaaaga gaaagcatgc tccaagaatt atgggttcca gaaggcaaag 240
tcccagaatt gtctccaggg aaggacaggg aggtctagaa tcggctaagc ccactgtagg 300
cagaaaaacc aagaggcatg aatggcttcc ctttctcact tttcaccctc tggcttactc 360
ctatcatgaa ggaaaatatt ggaatcatat tctccctcac cgaaatgcta tttttcag 418
<210> 8
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 8
agtgagaccc agggaatgtg 20
<210> 9
<211> 21
<212> DNA
<213>pig (Sus scrofa)
<400> 9
tcagcctaca ccacaatcac a 21
<210> 10
<211> 1452
<212> DNA
<213>pig (Sus scrofa)
<400> 10
accatgaatg gggaaagcat tattgcaatc ataatgaaga tgctggtgtg acatgttcta 60
gtgagaccca gggaatgtgt tcactttgtt cccatgccat gaagagggta gggttaggta 120
gtcacagaca tctttttaaa gccctgtctc cttccaggat acacacaaat ccgcttggtg 180
aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 240
ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 300
ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 360
aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 420
ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc aggtaagaga 480
ataagggcag ccagtgatga gccactcatg acggtgcctt aagagtgggt gtacctagga 540
gttcccattg tggctcagtg gtaacaaact cgactggtat ccatgagggt atgggtttga 600
tccctggcct tgctcaatgg gttaaggatc cagcattgct gtgagctgtg gtataggttg 660
cagactctgc tcaggtccca tgttgctgtg attgtggtgt aggctgactg ctgcagcttc 720
aatttgaccc ctagcccggg aatttccata ggccacacgt gcagcactaa ggaaggaaaa 780
aaaaaaaaaa aaaaaaaaga gtgggtgtgc ctatagtgaa gaacagatgt aaaagggaag 840
tgaaagggat tcccccattc tgagggattg tgagaagtgt gccagaatat taacttcatt 900
tgacttgtta cagggaaagt aaacttgact ttcacggacc tcctagttac ctggtgctta 960
ctatatgtct tctcagagta cctgattcat tcccagcctg gttgacccat ccccctatct 1020
ctatggctat gtttatccag agcacatcta tctaacactc cagctgatct tcctgacaca 1080
gctgtggcaa ccctggatcc tttaaccaac tgtgccaggc tggagatcaa acctaagcct 1140
ctgcagcaac ccaagctgct gcagtcagat ttttaacccc ctgtgccact gtgggtatct 1200
ccgatatttt gtatcttctg tgactgagtg gtttgctgtt tgcagggaac cagagtcaga 1260
cactatcccc gtgcaattca tcatcctcgg acccatcaag ctctattatt tcagaagaaa 1320
atggtgttgc ctgcataggt gagaatcagt gaccaaccta tgaaaatgat ctcaatcctc 1380
tgaaatgcat tttattcatg ttttatttcc tctttgcagg gagtggtcaa cttcgcctgg 1440
tcgatggagg tg 1452
<210> 11
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 11
ggaaacccag gctggttgga 20
<210> 12
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 12
tccaaccagc ctgggtttcc 20
<210> 13
<211> 20
<212> RNA
<213>pig (Sus scrofa)
<400> 13
ggaaacccag gcugguugga 20
<210> 14
<211> 20
<212> RNA
<213>pig (Sus scrofa)
<400> 14
uccaaccagc cuggguuucc 20
<210> 15
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 15
gagtagcacc ccgccctgac 20
<210> 16
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 16
gtcagggcgg ggtgctactc 20
<210> 17
<211> 20
<212> RNA
<213>pig (Sus scrofa)
<400> 17
gaguagcacc ccgcccugac 20
<210> 18
<211> 2125
<212> DNA
<213>pig (Sus scrofa)
<400> 18
gatgattgcg ctcttaacct ggcaaagatt gtctttaaaa tctgagctcc atgtcttctg 60
ctttatttct ggtgtgcctt tgactccaga ttacagtaaa tggaggactg agtatagggc 120
taaaaagtag agagaatgga tgcatattat ctgtggtctc caatgtgatg aatgaagtag 180
gcaaatactc aaaggaaaga gaaagcatgc tccaagaatt atgggttcca gaaggcaaag 240
tcccagaatt gtctccaggg aaggacaggg aggtctagaa tcggctaagc ccactgtagg 300
cagaaaaacc aagaggcatg aatggcttcc ctttctcact tttcaccctc tggcttactc 360
ctatcatgaa ggaaaatatt ggaatcatat tctccctcac cgaaatgcta tttttcagcc 420
cacaggcagc tgagagtggt agatggagtc actgaatgtt caggaagatt ggaagtgaaa 480
ttccaaggag aatggggaac aatctgtgat gatggctggg atagtgatga tgccgctgtg 540
gcatgtaagc aactgggatg tccaactgct gtcactgcca ttggtcgagt taacgccagt 600
gagggaactg gacacatttg gcttgacagt gtttcttgcc atggacacga gtctgctctc 660
tggcagtgta gacaccatga atggggaaag cattattgca atcataatga agatgctggt 720
gtgacatgtt ctagtgagac ccagggaatg tgttcacttt gttcccatgc catgaagagg 780
gtagggttag gtagtcacag acatcttttt aaagccctgt ctccttccag gatacacaca 840
aatccgcttg gtgaatggca agaccccatg tgaaggaaga gtggagctca acattcttgg 900
gtcctggggg tccctctgca actctcactg ggacatggaa gatgcccatg ttttatgcca 960
gcagcttaaa tgtggagttg ccctttctat cccgggagga gcaccttttg ggaaaggaag 1020
tgagcaggtc tggaggcaca tgtttcactg cactgggact gagaagcaca tgggagattg 1080
ttccgtcact gctctgggcg catcactctg ttcttcaggg caagtggcct ctgtaatctg 1140
ctcaggtaag agaataaggg cagccagtga tgagccactc atgacggtgc cttaagagtg 1200
ggtgtaccta ggagttccca ttgtggctca gtggtaacaa actcgactgg tatccatgag 1260
ggtatgggtt tgatccctgg ccttgctcaa tgggttaagg atccagcatt gctgtgagct 1320
gtggtatagg ttgcagactc tgctcaggtc ccatgttgct gtgattgtgg tgtaggctga 1380
ctgctgcagc ttcaatttga cccctagccc gggaatttcc ataggccaca cgtgcagcac 1440
taaggaagga aaaaaaaaaa aaaaaaaaaa agagtgggtg tgcctatagt gaagaacaga 1500
tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag tgtgccagaa 1560
tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg acctcctagt 1620
tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc ctggttgacc 1680
catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca ctccagctga 1740
tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca ggctggagat 1800
caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac cccctgtgcc 1860
actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct gtttgcaggg 1920
aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc aagctctatt 1980
atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac ctatgaaaat 2040
gatctcaatc ctctgaaatg cattttattc atgttttatt tcctctttgc agggagtggt 2100
caacttcgcc tggtcgatgg aggtg 2125
Claims (4)
1. a kind of gene order for effectively inhibiting II type PRRSV infection, it is characterised in that:
Gene order is as shown in SEQ ID 1；CD163 gene order after gene modification as: shown in SEQ ID 2.
2. a kind of preparation method of gene order for effectively inhibiting II type PRRSV infection as described in claim 1, including it is following
Step:
1) pig CD163 gene order is recalled in NCBI gene pool；
2) base sequence such as SEQ ID 3 respectively, upstream primer F1 shown in SEQ ID 4 are obtained using Primer5 software design
With downstream primer R1；
3) base sequence objective gene sequence as shown in SEQ ID 1 is obtained using PCR amplification in vitro method；
4) pig CD163 gene order is recalled in NCBI gene pool；
5) base sequence is obtained respectively such as SEQ ID 5, SEQ ID 6, SEQ ID 8, SEQ ID using Primer5 software design
Upstream primer F2 shown in 9, R2 and downstream primer F3, R3；
6) base sequence such as SEQ ID 7 is obtained using PCR amplification in vitro method；Target gene upstream and downstream shown in SEQ ID 10
Gene；
7) by above-mentioned base sequence obtained such as SEQ ID 1, SEQ ID 7, gene of SEQ ID 10 etc. is than being added containing SEQ
In the PCR system of ID 5, SEQ ID 9；
8) by round pcr, vitro recombination connects these three segments, obtains sequence homologous replacement as shown in SEQ ID 19 and carries
Body；
9) by the sgRNA plasmid of building: SEQ ID 11, SEQ ID 12, SEQ ID 14, SEQ ID 15 and homologous targeting vector
Into porcine fetus fibroblasts PFF, and by limiting dilution assay, screening and identification obtains positive ho-mology and replaces 19 cotransfection of SEQ ID
The porcine fetus fibroblasts changed, positive fibroblasts CD163 gene order is as shown in SEQ ID 2.
3. a kind of gene order of II type PRRSV infection of effectively inhibition as described in claim 1 is inhibiting II type PRRSV infection
Purposes.
4. as described in claim 1 a kind of effectively inhibit the gene order of II type PRRSV infection to turn base prepare anti-indigo plant virus
Because of the application in pig.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810952139.4A CN109055385B (en) | 2018-08-21 | 2018-08-21 | Gene sequence for effectively inhibiting II type PRRSV infection and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810952139.4A CN109055385B (en) | 2018-08-21 | 2018-08-21 | Gene sequence for effectively inhibiting II type PRRSV infection and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109055385A true CN109055385A (en) | 2018-12-21 |
| CN109055385B CN109055385B (en) | 2021-08-27 |
Family
ID=64686666
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810952139.4A Active CN109055385B (en) | 2018-08-21 | 2018-08-21 | Gene sequence for effectively inhibiting II type PRRSV infection and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109055385B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105443A (en) * | 2019-05-30 | 2019-08-09 | 河南省农业科学院 | A kind of CD163 mutant and its application |
| CN113151291A (en) * | 2020-05-05 | 2021-07-23 | 吉纳斯公司 | Method for improving swine health by targeted inactivation of CD163 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0676467A2 (en) * | 1994-04-11 | 1995-10-11 | Akzo Nobel N.V. | European vaccine strains of the porcine reproductive respiratory syndrome virus (PRRSV) |
| CN102899317A (en) * | 2012-09-18 | 2013-01-30 | 江南大学 | Method for quickly and efficiently increasing gene fragments on plasmid |
| CN106916820A (en) * | 2017-05-16 | 2017-07-04 | 吉林大学 | SgRNA and its application of porcine ROSA 26 gene can effectively be edited |
-
2018
- 2018-08-21 CN CN201810952139.4A patent/CN109055385B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0676467A2 (en) * | 1994-04-11 | 1995-10-11 | Akzo Nobel N.V. | European vaccine strains of the porcine reproductive respiratory syndrome virus (PRRSV) |
| CN102899317A (en) * | 2012-09-18 | 2013-01-30 | 江南大学 | Method for quickly and efficiently increasing gene fragments on plasmid |
| CN106916820A (en) * | 2017-05-16 | 2017-07-04 | 吉林大学 | SgRNA and its application of porcine ROSA 26 gene can effectively be edited |
Non-Patent Citations (1)
| Title |
|---|
| 金光明等: "猪繁殖与呼吸综合症免疫学研究", 《安徽农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110105443A (en) * | 2019-05-30 | 2019-08-09 | 河南省农业科学院 | A kind of CD163 mutant and its application |
| CN110105443B (en) * | 2019-05-30 | 2022-02-08 | 河南省农业科学院 | CD163 mutant and application thereof |
| CN113151291A (en) * | 2020-05-05 | 2021-07-23 | 吉纳斯公司 | Method for improving swine health by targeted inactivation of CD163 |
| CN117487855A (en) * | 2020-05-05 | 2024-02-02 | 吉纳斯公司 | Methods for improving swine health by targeted inactivation of CD163 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109055385B (en) | 2021-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105907758B (en) | CRISPR-Cas9 guide sequence and primer thereof, transgenic expression vector and construction method thereof | |
| Foroughi-Wehr et al. | On the genetic improvement of androgenetic haploid formation in Hordeum vulgare L. | |
| US9598671B2 (en) | Cyprinid herpesvirus II-sensitive brain tissue cell line of Carassius auratus gibelio and establishing method and use thereof | |
| CN107937345B (en) | A kind of fibroblastic method of pig for preparing while knocking out CD163 gene and CD13 gene | |
| CN106755091A (en) | Gene knockout carrier, MH7A cell NLRP1 gene knockout methods | |
| Koornneef et al. | Cytogenetic tools for Arabidopsis thaliana | |
| CN108103099B (en) | Anti-blue ear disease Marc-145 cell line and preparation method and application thereof | |
| CN109055385A (en) | A kind of effective gene order and its application for inhibiting II type PRRSV infection | |
| CN103461132B (en) | Megaspore culture technique is utilized to cultivate the method for breed cucumber material | |
| Yamamoto et al. | Chromosomal control of malignancy in tumors from cells transformed by polyoma virus | |
| CN103798132B (en) | A kind of method of scale initiative wheat alien translocation line | |
| CN114874991A (en) | I-type interferon receptor gene knockout bovine kidney cell line and construction method and application thereof | |
| CN104928260A (en) | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof | |
| CN101979598A (en) | Method for constructing HSV-1 BAC system carrying luciferase report genes | |
| CN108866240A (en) | For identifying primer and enzyme and its application of DAdV-3 and DAdV-A | |
| CN105713866A (en) | Human cytomegalovirus (HCMV) infectious clone as well as construction method and applications of HCMV infectious clone | |
| CN116948975A (en) | IFNAR1 gene knockout MDCK cell strain, construction method and application thereof | |
| CN102140440A (en) | Method for separating and culturing poultry bone mesenchymal stem cells by using bone marrow tissue block | |
| RU2488631C1 (en) | CELL LINE OF CALF KIDNEY Bos taurus RBT (Rene Bos Taurus) FOR REPRODUCTION OF ANIMAL VIRUSES | |
| CN107541484A (en) | Diploid red crucian caudal fin cell system 2nFC and its construction method and application | |
| KR20120048626A (en) | Method for producing double haploid plants | |
| CN108103027A (en) | The method that the reprogramming of high efficiency haemocyte realizes gene editing simultaneously | |
| Adamus et al. | Experiments of backcrossing of interspecific F1 A. cepa× A. roylei hybrids with Allium cepa | |
| CN113564165B (en) | Cell strain for intracellular editing of pseudorabies virus key genes, construction method and application thereof | |
| CN110004181B (en) | Additional CRISPR/Cas9 expression vector and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220114 Address after: 518000 Golden New Building, No. 18 Guangming Street Photoelectric North Road, Guangming New District, Shenzhen City, Guangdong Province Patentee after: SHENZHEN JINXINNONG TECHNOLOGY CO.,LTD. Address before: No.2699, Qianjin Street, Changchun City, Jilin Province, 130011 Patentee before: Jilin University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221111 Address after: Floor 5, National Science and Technology Enterprise Incubator, Building 19, No. 64, Changzhou Street, Rongchang District, Chongqing 402460 Patentee after: Chongqing Jitang Biotechnology Research Institute Co.,Ltd. Address before: 518000 Golden New Building, No. 18 Guangming Street Photoelectric North Road, Guangming New District, Shenzhen City, Guangdong Province Patentee before: SHENZHEN JINXINNONG TECHNOLOGY CO.,LTD. |
|
| TR01 | Transfer of patent right |