CN109628494A - Coronavirus resistance clone pig and preparation method thereof - Google Patents
Coronavirus resistance clone pig and preparation method thereof Download PDFInfo
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- CN109628494A CN109628494A CN201910062079.3A CN201910062079A CN109628494A CN 109628494 A CN109628494 A CN 109628494A CN 201910062079 A CN201910062079 A CN 201910062079A CN 109628494 A CN109628494 A CN 109628494A
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Abstract
The present invention relates to a kind of coronavirus resistance clone pig and preparation method thereof based on CRISPR/Cas9 gene editing technology, the preparation method of the clone pig, it is characterized in that, the following steps are included: (1) obtains the gRNA sequence of selectively targeted pAPN Exon 2 and the 3rd exon, nucleotide sequence is successively as shown in SEQ ID No.1-2；Prepare CRISPR-Cas9 the targeting vector PX330-pAPN2 and PX330-pAPN3 of corresponding gRNA sequence；(2) the targeting vector PX330-pAPN2 of acquisition or targeting vector PX330-pAPN3 are transferred in porcine fetus fibroblasts, obtain pAPN gene editing cell line；(3) body-cell neucleus transplanting operation is carried out to pAPN gene editing cell line, and reconstruct embryo is implanted into surrogate recipient, be born the clone pig by natural pregnancy.The present invention can get the clone pig of the complete loss of expression of pig coronavirus receptor pAPN, have the ability for resisting pig coronavirus infection.
Description
Technical field
The present invention relates to gene technology, in particular to a kind of coronavirus based on CRISPR/Cas9 gene editing technology
Resistance clone pig and preparation method thereof.
Background technique
CRISPR(Clustered regularly interspaced short palindromic repeats)/
9 system of CRISPR-associated protein (Cas) is a kind of adaptive immune system of prokaryotes, is bitten for resisting
Thallus and exogenous genetic material intrusion.In recent years, researcher makes it be applicable to the cell of mammal by the way that the system is transformed
With it is internal.The system is guided by one with the consistent guide RNA (gRNA) of target DNA site sequence, to mediate
Cas9 albumen specifically cuts purpose site, to form DNA double chain fracture.And the double-strand break on genome can
To be obviously improved the efficiency of subsequent gene knockout (Knockout) and gene knock-in (Knockin), so as to efficiently right
Purpose site carries out modification operation, to reach missing destination gene expression expression, or assigns target gene with new function.
CRISPR/Cas9 has currently obtained extensive and deep application in every field such as biology, medicine, agriculturals, almost can be with
Arbitrary editor is carried out to any genetic fragment of any species.
Coronavirus (Coronavirus) is a kind of important virus for influencing pig breeding industry, mainly causes diarrhea of pigs and breathing
Systemic disease especially has compared with high lethality rate piglet.The pig coronavirus of current Major Epidemic includes epidemic gastroenteritis disease
Poison (Transmissible gastroenteritis virus, TGEV), Porcine epidemic diarrhea virus (Porcine
Epidemic diarrhea virus, PEDV), porcine respiratory coronavirus (porcine respiratory
Coronavirus, PRCV) and fourth type coronavirus (Porcine deltacoronavirus, PDCoV) etc..Among these especially with
PEDV is the most serious, is widely current in recent years, normality outburst, to the lethality of nursery-age pig up to 95% or more, provisions
Pig industry causes heavy economic losses.The vaccine prevention and control of this viroid are more difficult, and immune to the passive milk of piglet is main way
Diameter, control effect are still not clear enough；And quickenings of strain variation also result in the lag of vaccine prevention and control in vain.
Research discovery pig Aminopeptidase N (Pig Aminopeptidase N, pAPN) of early period is the thin of a variety of coronavirus
Born of the same parents' receptor, including TGEV, PRCV, PDCoV etc., the receptor that PEDV infects host may also be pAPN, but still have dispute.Cell
It can become the sensitive cells of coronavirus after the horizontal non-sensitive cell importing expression pAPN of research discovery coronavirus；And it hinders
The combination of disconnected coronavirus and pAPN can inhibit its infection to host cell.Therefore as can lacking pAPN gene in pig body
Expression, it will block infection of a variety of coronavirus to pig.This kind of receptor missing pig is cultivated to have great importance to pig breeding industry.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of based on the coronal of CRISPR/Cas9 gene editing technology
Virus resistance clone pig, by lacking the expression of pAPN albumen in pig body completely for pig coronavirus receptor pAPN gene knockout,
To have the ability for resisting pig coronavirus infection；
Based on above-mentioned, the present invention also provides a kind of coronavirus resistances gram based on CRISPR/Cas9 gene editing technology
The preparation method of grand pig carries out frameshift mutation to pAPN gene using the fixed point Knockout technology of CRISPR/Cas9 System-mediated, from
And obtain the clone pig of pAPN gene knockout.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
A kind of preparation method of the coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology, including with
Lower step:
(1) the gRNA sequence of selectively targeted pAPN Exon 2 and the 3rd exon, nucleotide sequence are obtained
Successively as shown in SEQ ID No.1-2；
Prepare the CRISPR/Cas9 targeting vector of corresponding gRNA sequence, the system of the CRISPR/Cas9 targeting vector
Preparation Method are as follows: PX330 carrier is subjected to digestion with restriction enzyme BbsI；It is respectively synthesized exon 2 and the 3rd exon
GRNA normal chain and complementary chain dna, and the sequence that the cohesive terminus,cohesive termini formed with the PX330 carrier after digestion matches is taken at both ends
Column；The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA to mix respectively, then by mixed mixing sequence
Column, which are placed in temperature and are at least in 98-100 DEG C of water, boils 2.5-3.5min, is then allowed to stand and is cooled to 20-30 DEG C so that two mutually
It mends chain DNA to anneal to form double-stranded DNA, and carries the cohesive terminus,cohesive termini that can be connected with by the PX330 carrier after BbsI digestion；Annealing
The double-stranded DNA of formation is connect with by the PX330 carrier after BbsI digestion；Obtain targeting vector PX330-pAPN2 and targeting vector
PX330-pAPN3；
(2) the targeting vector PX330-pAPN2 of acquisition or targeting vector PX330-pAPN3 are transferred to pig fetus into fiber finer
In born of the same parents, the positive cell clone that pAPN is knocked out is obtained through monoclonal and screening；
(3) using positive cell as nuclear donor, in implantation stoning porcine oocytes, positive reconstruct Pig embryos are obtained；It will weigh again
Structure Pig embryos are implanted into gestation in replace-conceive Gilt Uterus, obtain the clone pig.
A kind of coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology, using it is above-mentioned based on
The preparation method of the coronavirus resistance clone pig of CRISPR/Cas9 gene editing technology obtains.
The beneficial effects of the present invention are:
The preparation method of coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology of the invention, is adopted
It is transferred in porcine fetus fibroblasts with the CRISPR/Cas9 gene editing carrier of targeting pig pAPN gene, obtains pAPN and practice shooting
Positive cell clone, be introduced into frameshift mutation in the 2nd or the 3rd exon of pAPN in positive colony, cause pAPN albumen without
Method expression；The positive colony practiced shooting using pAPN obtains reconstruct clone as nuclear transfer donor cell, by somatic cell nuclear transfer technique
Pig embryos；The clone pig of pAPN missing will be obtained in embryo transfer such as replace-conceive Gilt Uterus through gestation.The present invention can get a variety of
The clone pig of the complete loss of expression of pig coronavirus receptor pAPN, the clone pig of preparation have without pAPN protein expression and resist pig
The ability of coronavirus infection.
Detailed description of the invention
Fig. 1 is the target practice site information figure of pAPN gene structure and selection in the embodiment of the present invention 1；
Fig. 2 is the photo of clone pig in the embodiment of the present invention 3；
Fig. 3 is the pAPN protein expression of birth clone pig and wild type control pig detection comparison in the embodiment of the present invention 3
Figure.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, below in conjunction with embodiment and cooperate attached
Figure is explained.
The most critical design of the present invention is: using CRISPR/Cas9 gene editing system porcine somatic cell pAPN base
Frameshift mutation is introduced in 2nd or the 3rd exon of cause, pAPN albumen is caused to be beyond expression；It is obtained again by body-cell neucleus transplanting
Lack the clone pig of pAPN expression.
A kind of preparation method of the coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology, including with
Lower step:
(1) the gRNA sequence of selectively targeted pAPN Exon 2 and the 3rd exon, nucleotide sequence are obtained
Successively as shown in SEQ ID No.1-2；
Prepare the CRISPR/Cas9 targeting vector of corresponding gRNA sequence, the system of the CRISPR/Cas9 targeting vector
Preparation Method are as follows: PX330 carrier is subjected to digestion with restriction enzyme BbsI；It is respectively synthesized exon 2 and the 3rd exon
GRNA normal chain and complementary chain dna, and the sequence that the cohesive terminus,cohesive termini formed with the PX330 carrier after digestion matches is taken at both ends
Column；The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA to mix respectively, then by mixed mixing sequence
Column, which are placed in temperature and are at least in 98-100 DEG C of water, boils 2.5-3.5min, is then allowed to stand and is cooled to 20-30 DEG C so that two mutually
It mends chain DNA to anneal to form double-stranded DNA, and carries the cohesive terminus,cohesive termini that can be connected with by the PX330 carrier after BbsI digestion；Annealing
The double-stranded DNA of formation is connect with by the PX330 carrier after BbsI digestion；Obtain targeting vector PX330-pAPN2 and targeting vector
PX330-pAPN3；
(2) the targeting vector PX330-pAPN2 of acquisition or targeting vector PX330-pAPN3 are transferred to pig fetus into fiber finer
In born of the same parents, the positive colony that pAPN is knocked out is obtained through monoclonal and screening；
(3) using positive cell as nuclear donor, in implantation stoning porcine oocytes, positive reconstruct Pig embryos are obtained；It will weigh again
Structure Pig embryos are implanted into gestation in replace-conceive Gilt Uterus, obtain the clone pig.
As can be seen from the above description, the beneficial effects of the present invention are:
The preparation method of coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology of the invention, is adopted
It is transferred in porcine fetus fibroblasts with the CRISPR/Cas9 gene editing carrier of targeting pig pAPN gene, obtains pAPN and practice shooting
Positive cell clone, be introduced into frameshift mutation in the 2nd or the 3rd exon of pAPN in positive colony, cause pAPN albumen without
Method expression；The positive colony practiced shooting using pAPN obtains reconstruct clone as nuclear transfer donor cell, by somatic cell nuclear transfer technique
Pig embryos；The clone pig of pAPN missing will be obtained in embryo transfer such as replace-conceive Gilt Uterus through gestation.The present invention can get a variety of
The clone pig of the complete loss of expression of pig coronavirus receptor pAPN, the clone pig of preparation have without pAPN protein expression and resist pig
The ability of coronavirus infection.
Above-mentioned PX330 carrier is purchased from Addgene company；Oligonucleotide, primer and sequencing are completed by Shanghai Sangon Biotech Company；
Restriction enzyme, T4DNA ligase are purchased from NEB company；Extaq archaeal dna polymerase is purchased from Takara company；KOD-plus-
Neo archaeal dna polymerase is purchased from Toyobo company；DNA purifying, plasmid extraction, genome extraction agent box are purchased from Tiangeng company.Enzyme
It cuts, purify, connecting, converting, the conventional molecular biologicals laboratory operating procedures such as PCR amplification and plasmid extraction are detailed in " molecular cloning
(third edition) ".
Further, target practice recognition site SEQ ID No.1 and SEQ ID No.2 is located at the downstream ATG of gene coding region
The 1-2 exon most started on.
Target practice recognition site SEQ ID No.1 and SEQ ID No.2 is located at most starting for the downstream ATG of gene coding region
On 1-2 exon, it can guarantee that introducing for frameshift mutation it can express in the premature termination that destination protein pAPN is translated in this way,
To achieve the purpose that thoroughly to eliminate pAPN protein expression.
Further, CRISPR skeleton carrier PX330 is selected, and gene editing site sequence structure is entered into skeleton carrier and is obtained
PAPN special CRISPR targeting vector PX330-pAPN2 and PX330-pAPN3.
Further, CRISPR targeting vector PX330-pAPN2 and PX330-pAPN3 are transfected into pig fetus into fiber finer
In born of the same parents, by low density cell culture, pure single cell clone is obtained in the case where not needing antibiotic-screening.
Further, to the single cell clone of screening, using PCR amplification and sequencing, and peak figure analysis method mirror is sequenced
Make the cell clone with different genotype, the amplification of use and sequencing primer include gRNA2 target practice Locus Analysis in Shoots primer and
GRNA3 target practice Locus Analysis in Shoots primer, the nucleotide sequence of the gRNA2 target practice Locus Analysis in Shoots primer is successively such as SEQ ID No.5-
Shown in 6；The nucleotide sequence of the gRNA3 target practice Locus Analysis in Shoots primer is successively as shown in SEQ ID No.7-8.
Further, the edit mode that the pAPN of body cell is used is introduced on its exon 2 or 3 pair using CRISPR
Equipotential frameshift mutation, so that the expression of two equipotential pAPN genes lacks completely.
Further, gene editing pig is prepared in conjunction with somatic cell gene editor and body-cell neucleus transplanting.
Further, porcine oocytes are enucleated, the body cell of pAPN gene editing is merged into production with enucleation oocyte
The reconstruct Pig embryos of raw pAPN gene editing will reconstruct embryo implantation replace-conceive Gilt Uterus, pass through gestation birth and donorcells
The consistent clone pig of genotype.
Embodiment 1:
The building of pAPN gene knockout carrier
1, the selection and synthesis in target practice site.
CRISPR target practice site is selected according to the sequence rules of GN (18-20) GG, site need to guarantee in the outer aobvious of the downstream ATG
On son, and it is located at preceding the 1/3 of code area, to guarantee that the introducing of mutation can lead to more complete protein inactivation.Two chosen
A target practice site is located on exon 2 and exon 3, is respectively designated as gRNA2 and gRNA3.
Its length is respectively 19bp and 20bp, sequence be respectively GGGCTCCTGGCAGATGAAG and
GATGTTGAACGTGGCCTTCA.PAPN gene structure and the target practice site information of selection are as shown in Figure 1.According to selected target practice
Site synthesizes the oligonucleotide for expressing gRNA, and sequence is as shown in table 1.
Table 1
2, the building of pAPN targeting vector.
PX330 carrier is subjected to digestion with restriction enzyme BbsI；It is respectively synthesized exon 2 and the 3rd exon
GRNA normal chain and complementary chain dna, and the sequence that the cohesive terminus,cohesive termini formed with the PX330 carrier after digestion matches is taken at both ends
Column；The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA to mix respectively, then by mixed mixing sequence
Column, which are placed in boiling water, boils 3min, keeps mixed sequence motionless in boiling water, is then allowed to stand and is cooled to room temperature, so that two complementary strands
DNA anneals to form double-stranded DNA, and carries the cohesive terminus,cohesive termini that can be connected with by the PX330 carrier after BbsI digestion；Annealing is formed
Double-stranded DNA connect with by the PX330 carrier after BbsI digestion；Among the above, by PX330 carrier with restriction enzyme BbsI into
After row digestion, gel extraction purifying and vector linearization processing are carried out, the double-stranded DNA and PX330 carrier for formation of annealing are in 16
It is connected overnight under the conditions of DEG C, is transformed into competent cell Top10, form single colonie, the sequencing identification of picking single colonie through coated plate.Point
It Xing Cheng not pAPN targeting vector PX330-pAPN2 and PX330-pAPN3.
3, target practice plasmid is largely prepared.Positive plasmid converts again and forms monoclonal colonies, 1 single bacterium of each plasmid picking
It drops down onto shaken cultivation in 3ml LB culture solution with ampicillin to stay overnight, then all be inoculated with bacterium solution green into 200ml benzyl containing ammonia
Shaken cultivation 12 hours or overnight in the culture solution of mycin.Plasmid is largely prepared with endotoxin-free plasmid extraction agent box to be used for carefully
Dysuria with lower abdominal colic dye.
The screening of embodiment 2:pAPN Knockout cells
1, it more than porcine fetus fibroblasts cultures to 80% convergence degree in 30 days of primary separation, digested, be centrifuged removal
Cell culture fluid, is then resuspended cell with PBS, and adjustment cell concentration is 105A/100 μ l PBS.Take 100 μ l cell suspensions with
The mixing of 10 μ g target practice plasmids is placed in Lonza Nucleofector and carries out electroporation transfection.Electric shock procedure selection is A330.Electricity turns
Cell point is into 10 10cm culture dishes afterwards, about 1000 cells of every ware, with the culture solution of DMEM+15%FBS in 37 DEG C, 5%
CO2Incubator in cultivate.
2, it transfects cell 10 days and forms sizeable monoclonal, need picking single cell clone to 48 orifice plates at this time, to 48
Hole covers with, and about 1/10 cell is taken to carry out genotype detection, and remaining 9/10 cell, which reaches 24 holes, to be continued to cultivate.
3,1/10 cell taken out, which is centrifuged, removes culture solution, 10 μ l cell pyrolysis liquids of addition (4mg/ml Proteinase K+
0.5%NP40 is dissolved in sterile water) cell, 56 DEG C of cracking 30min, then 98 DEG C of 10min inactivated proteases K is resuspended.Take 1 μ
L lysate carries out PCR detection directly as template.PAPN2 target practice site is by pAPN2-exon2-F/pAPN2-exon2-R primer
It is expanded to ExTaq enzyme；PAPN3 target practice site is by pAPN3-exon3-F/pAPN3-exon3-R primer pair KOD-
Plus-neo enzyme is expanded.PAPN genotype identification primer sequence is as shown in table 2.
Table 2
| Title | Sequence (5 ' -3 ') |
| pAPN2-exon2-F(SEQ ID No.5) | CATCGCTCTGTCTGTGGTGTA |
| pAPN2-exon2-R(SEQ ID No.6) | GTAGAAGCCTGCCAGGTCGT |
| pAPN3-exon3-F(SEQ ID No.7) | AGGAGCTCTGCTCTCCCAAG |
| pAPN3-exon3-R(SEQ ID No.8) | GACCAGTTGGGGTCTTCTGC |
4, identification peak figure and aligned sequences is sequenced through Sanger in PCR product, determines genotype and target practice situation.Genotype point
For following 5 kinds of situations: (1) sequencing sequence is consistent with wild type pAPN, and all shows as unimodal, is accredited as wild type；(2) it surveys
Sequence sequence and wild type are compared occurs missing or insertion (Indel) at target practice site, and all shows as unimodal, is accredited as double
Equipotential knockout type (double knockout types)；(3) show as contour bimodal behind target practice site, and one has an indel, and one is wild type,
It is accredited as single equipotential knockout type (single knockout type)；(4) show as contour bimodal behind target practice site, and two have indel, also reflect
It is set to double equipotential knockout types (double knockout types)；(5) not contour bimodal, multimodal etc. is generally multiple clone mix, therefore not
Consider.
5, above-mentioned (2), (4) two kinds of genotype are further screened, and are removed missing or are inserted into the thin of the integral multiple that base number is 3
Born of the same parents clone, remaining clone are the cell clone that frameshift mutation was all practiced shooting and generated to double equipotentials.It identifies and sieves through the present embodiment
It is required positive cell that choosing, which obtains cell clone,.
Embodiment 3
The preparation of pAPN gene knock-out pig
1, positive cell is obtained using in embodiment 2 as nuclear donor, is implanted into the porcine oocytes of the maturation in vitro of stoning,
Through electro' asion and activation, reconstruct embryo is formed.By reconstruct embryo to be carried out in the receptor Gilt Uterus for be implanted into estrus synchronization of performing the operation
Gestation, every receptor move into 200 pieces or so of reconstructed embryo.30 days progress first time B ultrasound detect whether gestation after operation, hereafter fixed
Phase carries out B ultrasound and monitors Pregnancy.Embryo transfer situation and clone pig Birth Situation are shown in Table 3.Clone pig picture as shown in Figure 2 (1
Monthly age).
Table 3
According to table 3, in situation of giving a birth, amount to and obtain strong son 16, weak young 2,5, stillborn foetus.
2, the genotype detection of birth clone pig
The a small amount of clone pig ear tissue of clip, carries out extracting genome DNA with genome extraction agent box.Expand target practice position
It is sequenced after the sequence of point.PCR reaction is consistent with 2 step 3 of embodiment with condition.Clone pig pAPN genotype sequencing identification feelings
Condition is as shown in table 4.The clone pig of all birth survivals is all double equipotential homozygous knockouts, can cause frameshit prominent to pAPN gene
Become, to lack it in the expression of pig whole body.
Table 4
3, the pAPN protein expression detection of birth clone pig
The small intestine for taking both ends death clone pig (#1 and #2), row Western Blotting detects pAPN egg after cracking
White expression.Clone pig is without pAPN protein expression as the result is shown, and wild type control pig has significant expression (as schemed
3)。
In conclusion the coronavirus resistance clone pig provided by the invention based on CRISPR/Cas9 gene editing technology
Preparation method, can get the complete loss of expression of pig coronavirus receptor pAPN clone pig, the clone pig of preparation is without pAPN egg
White expression has the ability for resisting pig coronavirus infection.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright specification and accompanying drawing content are applied directly or indirectly in relevant technical field, similarly include
In scope of patent protection of the invention.
SEQUENCE LISTING
<110>Agricultural University Of South China Wen Shi food Group Plc
<120>coronavirus resistance clone pig and preparation method thereof
<130> 2019
<160> 8
<170> PatentIn version 3.5
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<211> 19
<212> DNA
<213>artificial sequence
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gggctcctgg cagatgaag 19
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<400> 2
gatgttgaac gtggccttca 20
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cttcatctgc caggagccc 19
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tgaaggccac gttcaacatc 20
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<400> 5
catcgctctg tctgtggtgt a 21
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> 6
gtagaagcct gccaggtcgt 20
<210> 7
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aggagctctg ctctcccaag 20
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<400> 8
gaccagttgg ggtcttctgc 20
Claims (9)
1. a kind of preparation method of the coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology, feature exist
In, comprising the following steps:
(1) the gRNA sequence of selectively targeted pAPN Exon 2 and the 3rd exon is obtained, nucleotide sequence is successively
As shown in SEQ ID No.1-2；
Prepare the CRISPR/Cas9 targeting vector of corresponding gRNA sequence, the preparation side of the CRISPR/Cas9 targeting vector
Method are as follows: PX330 carrier is subjected to digestion with restriction enzyme BbsI；It is respectively synthesized the gRNA of exon 2 and the 3rd exon
Normal chain and complementary chain dna, and the sequence that the cohesive terminus,cohesive termini formed with the PX330 carrier after digestion matches is taken at both ends；It will
The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA and mixes respectively, then sets mixed mixed sequence
It is at least in 98-100 DEG C of water in temperature and boils 2.5-3.5min, be then allowed to stand and be cooled to 20-30 DEG C, so that two complementary strand
DNA anneals to form double-stranded DNA, and carries the cohesive terminus,cohesive termini that can be connected with by the PX330 carrier after BbsI digestion；Annealing is formed
Double-stranded DNA connect with by the PX330 carrier after BbsI digestion；Obtain targeting vector PX330-pAPN2 and targeting vector
PX330-pAPN3；
(2) the targeting vector PX330-pAPN2 of acquisition or targeting vector PX330-pAPN3 are transferred to porcine fetus fibroblasts
In, the positive colony that double equipotential pAPN are knocked out is obtained through monoclonal and screening；
(3) using positive cell as nuclear donor, in implantation stoning porcine oocytes, positive reconstruct Pig embryos are obtained；Pig will be reconstructed again
It is pregnant in embryo implantation replace-conceive Gilt Uterus, obtain the clone pig.
2. the system of the coronavirus resistance clone pig according to claim 1 based on CRISPR/Cas9 gene editing technology
Preparation Method, which is characterized in that target practice recognition site SEQ ID No.1 and SEQ ID No.2 is located at the downstream ATG of gene coding region
The 1-2 exon most started on.
3. the system of the coronavirus resistance clone pig according to claim 1 based on CRISPR/Cas9 gene editing technology
Preparation Method, which is characterized in that select CRISPR skeleton carrier PX330, and gene editing site sequence structure is entered into skeleton carrier and is obtained
Obtain pAPN special CRISPR targeting vector PX330-pAPN2 and PX330-pAPN3.
4. the system of the coronavirus resistance clone pig according to claim 3 based on CRISPR/Cas9 gene editing technology
Preparation Method, which is characterized in that CRISPR targeting vector PX330-pAPN2 and PX330-pAPN3 are transfected into pig fetus into fiber
In cell, by low density cell culture, pure single cell clone is obtained in the case where not needing antibiotic-screening.
5. the system of the coronavirus resistance clone pig according to claim 4 based on CRISPR/Cas9 gene editing technology
Preparation Method, which is characterized in that the single cell clone of screening, using PCR amplification and sequencing, and peak figure analysis method mirror is sequenced
Make the cell clone with different genotype, the amplification of use and sequencing primer include gRNA2 target practice Locus Analysis in Shoots primer and
GRNA3 target practice Locus Analysis in Shoots primer, the nucleotide sequence of the gRNA2 target practice Locus Analysis in Shoots primer is successively such as SEQ ID No.5-
Shown in 6；The nucleotide sequence of the gRNA3 target practice Locus Analysis in Shoots primer is successively as shown in SEQ ID No.7-8.
6. the system of the coronavirus resistance clone pig according to claim 5 based on CRISPR/Cas9 gene editing technology
Preparation Method, which is characterized in that the edit mode that the pAPN of body cell is used is introduced on its exon 2 or 3 using CRISPR
Double equipotential frameshift mutations, so that the expression of two equipotential pAPN genes lacks completely.
7. the coronavirus resistance based on CRISPR/Cas9 gene editing technology described in -6 any one according to claim 1
The preparation method of clone pig, which is characterized in that prepare gene editing pig in conjunction with somatic cell gene editor and body-cell neucleus transplanting.
8. the system of the coronavirus resistance clone pig according to claim 7 based on CRISPR/Cas9 gene editing technology
Preparation Method, which is characterized in that porcine oocytes are enucleated, the body cell of pAPN gene editing is merged into production with enucleation oocyte
The reconstruct Pig embryos of raw pAPN gene editing will reconstruct embryo implantation replace-conceive Gilt Uterus, pass through gestation birth and donorcells
The consistent clone pig of genotype.
9. a kind of coronavirus resistance clone pig based on CRISPR/Cas9 gene editing technology, which is characterized in that use right
It is required that the preparation side of the coronavirus resistance clone pig described in 1-8 any one based on CRISPR/Cas9 gene editing technology
Method obtains.
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| CN112779291A (en) * | 2021-02-22 | 2021-05-11 | 杭州合欣源生物科技有限公司 | Method for constructing high-quality pig nuclear transplantation donor cells with high lean meat percentage, fast growth, high reproductive capacity and resistance to series epidemic diseases and application thereof |
| CN113604502A (en) * | 2021-08-26 | 2021-11-05 | 中国农业科学院北京畜牧兽医研究所 | Gene editing system of pAPN gene 16 th exon and application thereof |
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| CN112779291A (en) * | 2021-02-22 | 2021-05-11 | 杭州合欣源生物科技有限公司 | Method for constructing high-quality pig nuclear transplantation donor cells with high lean meat percentage, fast growth, high reproductive capacity and resistance to series epidemic diseases and application thereof |
| CN112779292B (en) * | 2021-02-22 | 2023-03-10 | 南京启真基因工程有限公司 | Method for constructing high-quality pig nuclear transplantation donor cells with high lean meat percentage and rapid growth and capable of resisting blue ear diseases and serial diarrhea diseases and application of donor cells |
| CN112779291B (en) * | 2021-02-22 | 2023-03-28 | 南京启真基因工程有限公司 | Method for constructing high-quality pig nuclear transplantation donor cells with high lean meat percentage, fast growth, high reproductive capacity and resistance to series epidemic diseases and application thereof |
| CN113604502A (en) * | 2021-08-26 | 2021-11-05 | 中国农业科学院北京畜牧兽医研究所 | Gene editing system of pAPN gene 16 th exon and application thereof |
| CN113957093A (en) * | 2021-08-26 | 2022-01-21 | 中国农业科学院北京畜牧兽医研究所 | System for site-directed modification of pAPN gene and application thereof |
| CN114774468A (en) * | 2022-04-20 | 2022-07-22 | 温氏食品集团股份有限公司 | Novel allele molecular marker and anti-blue-ear disease pig group construction method |
| CN114774468B (en) * | 2022-04-20 | 2022-12-20 | 温氏食品集团股份有限公司 | Allele molecular marker and anti-blue-ear-disease pig group construction method |
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