Coronavirus resistance clone pig and preparation method thereofTechnical field
The present invention relates to gene technology, in particular to a kind of coronavirus based on CRISPR/Cas9 gene editing technologyResistance 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 resistingThallus 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 transformedWith it is internal.The system is guided by one with the consistent guide RNA (gRNA) of target DNA site sequence, to mediateCas9 albumen specifically cuts purpose site, to form DNA double chain fracture.And the double-strand break on genome canTo be obviously improved the efficiency of subsequent gene knockout (Knockout) and gene knock-in (Knockin), so as to efficiently rightPurpose 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 withArbitrary 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 breathingSystemic disease especially has compared with high lethality rate piglet.The pig coronavirus of current Major Epidemic includes epidemic gastroenteritis diseasePoison (Transmissible gastroenteritis virus, TGEV), Porcine epidemic diarrhea virus (PorcineEpidemic diarrhea virus, PEDV), porcine respiratory coronavirus (porcine respiratoryCoronavirus, PRCV) and fourth type coronavirus (Porcine deltacoronavirus, PDCoV) etc..Among these especially withPEDV is the most serious, is widely current in recent years, normality outburst, to the lethality of nursery-age pig up to 95% or more, provisionsPig 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 wayDiameter, 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 coronavirusBorn of the same parents' receptor, including TGEV, PRCV, PDCoV etc., the receptor that PEDV infects host may also be pAPN, but still have dispute.CellIt can become the sensitive cells of coronavirus after the horizontal non-sensitive cell importing expression pAPN of research discovery coronavirus；And it hindersThe combination of disconnected coronavirus and pAPN can inhibit its infection to host cell.Therefore as can lacking pAPN gene in pig bodyExpression, 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 technologyVirus 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 technologyThe preparation method of grand pig carries out frameshift mutation to pAPN gene using the fixed point Knockout technology of CRISPR/Cas9 System-mediated, fromAnd 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 withLower step:
(1) the gRNA sequence of selectively targeted pAPN Exon 2 and the 3rd exon, nucleotide sequence are obtainedSuccessively 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 vectorPreparation Method are as follows: PX330 carrier is subjected to digestion with restriction enzyme BbsI；It is respectively synthesized exon 2 and the 3rd exonGRNA 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 endsColumn；The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA to mix respectively, then by mixed mixing sequenceColumn, 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 mutuallyIt 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；AnnealingThe double-stranded DNA of formation is connect with by the PX330 carrier after BbsI digestion；Obtain targeting vector PX330-pAPN2 and targeting vectorPX330-pAPN3；
(2) the targeting vector PX330-pAPN2 of acquisition or targeting vector PX330-pAPN3 are transferred to pig fetus into fiber finerIn 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 againStructure 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 onThe 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 adoptedIt is transferred in porcine fetus fibroblasts with the CRISPR/Cas9 gene editing carrier of targeting pig pAPN gene, obtains pAPN and practice shootingPositive cell clone, be introduced into frameshift mutation in the 2nd or the 3rd exon of pAPN in positive colony, cause pAPN albumen withoutMethod expression；The positive colony practiced shooting using pAPN obtains reconstruct clone as nuclear transfer donor cell, by somatic cell nuclear transfer techniquePig 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 ofThe 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 pigThe 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 3Figure.
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 attachedFigure is explained.
The most critical design of the present invention is: using CRISPR/Cas9 gene editing system porcine somatic cell pAPN baseFrameshift 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 transplantingLack 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 withLower step:
(1) the gRNA sequence of selectively targeted pAPN Exon 2 and the 3rd exon, nucleotide sequence are obtainedSuccessively 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 vectorPreparation Method are as follows: PX330 carrier is subjected to digestion with restriction enzyme BbsI；It is respectively synthesized exon 2 and the 3rd exonGRNA 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 endsColumn；The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA to mix respectively, then by mixed mixing sequenceColumn, 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 mutuallyIt 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；AnnealingThe double-stranded DNA of formation is connect with by the PX330 carrier after BbsI digestion；Obtain targeting vector PX330-pAPN2 and targeting vectorPX330-pAPN3；
(2) the targeting vector PX330-pAPN2 of acquisition or targeting vector PX330-pAPN3 are transferred to pig fetus into fiber finerIn 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 againStructure 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 adoptedIt is transferred in porcine fetus fibroblasts with the CRISPR/Cas9 gene editing carrier of targeting pig pAPN gene, obtains pAPN and practice shootingPositive cell clone, be introduced into frameshift mutation in the 2nd or the 3rd exon of pAPN in positive colony, cause pAPN albumen withoutMethod expression；The positive colony practiced shooting using pAPN obtains reconstruct clone as nuclear transfer donor cell, by somatic cell nuclear transfer techniquePig 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 ofThe 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 pigThe 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.EnzymeIt 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 regionThe 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 regionOn 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 obtainedPAPN 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 finerIn 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 sequencedMake the cell clone with different genotype, the amplification of use and sequencing primer include gRNA2 target practice Locus Analysis in Shoots primer andGRNA3 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 CRISPREquipotential 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 oocyteThe reconstruct Pig embryos of raw pAPN gene editing will reconstruct embryo implantation replace-conceive Gilt Uterus, pass through gestation birth and donorcellsThe 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 ATGOn 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 chosenA 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 andGATGTTGAACGTGGCCTTCA.PAPN gene structure and the target practice site information of selection are as shown in Figure 1.According to selected target practiceSite 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 exonGRNA 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 endsColumn；The gRNA normal chain of exon 2 and the 3rd exon is complementary chain DNA to mix respectively, then by mixed mixing sequenceColumn, 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 strandsDNA 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 formedDouble-stranded DNA connect with by the PX330 carrier after BbsI digestion；Among the above, by PX330 carrier with restriction enzyme BbsI intoAfter 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 16It 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.PointIt 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 pickingIt 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 ammoniaShaken 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 carefullyDysuria 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 removalCell culture fluid, is then resuspended cell with PBS, and adjustment cell concentration is 105A/100 μ l PBS.Take 100 μ l cell suspensions withThe 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 turnsCell 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 48Hole 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 μL lysate carries out PCR detection directly as template.PAPN2 target practice site is by pAPN2-exon2-F/pAPN2-exon2-R primerIt 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 pointFor 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 surveysSequence sequence and wild type are compared occurs missing or insertion (Indel) at target practice site, and all shows as unimodal, is accredited as doubleEquipotential 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 reflectIt is set to double equipotential knockout types (double knockout types)；(5) not contour bimodal, multimodal etc. is generally multiple clone mix, therefore notConsider.
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 3Born 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 embodimentIt 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 operationGestation, every receptor move into 200 pieces or so of reconstructed embryo.30 days progress first time B ultrasound detect whether gestation after operation, hereafter fixedPhase 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 (1Monthly 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 positionIt 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 feelingsCondition 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 geneBecome, 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 crackingWhite expression.Clone pig is without pAPN protein expression as the result is shown, and wild type control pig has significant expression (as schemed3)。
In conclusion the coronavirus resistance clone pig provided by the invention based on CRISPR/Cas9 gene editing technologyPreparation method, can get the complete loss of expression of pig coronavirus receptor pAPN clone pig, the clone pig of preparation is without pAPN eggWhite 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 hairEquivalents made by bright specification and accompanying drawing content are applied directly or indirectly in relevant technical field, similarly includeIn 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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