CN110055251A - A kind of sequence and its application of regulation PCV2 virus multiplication - Google Patents
A kind of sequence and its application of regulation PCV2 virus multiplication Download PDFInfo
- Publication number
- CN110055251A CN110055251A CN201910336909.7A CN201910336909A CN110055251A CN 110055251 A CN110055251 A CN 110055251A CN 201910336909 A CN201910336909 A CN 201910336909A CN 110055251 A CN110055251 A CN 110055251A
- Authority
- CN
- China
- Prior art keywords
- grna
- pcv2
- cas9
- grnas
- orf1
- 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
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Oncology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
Abstract
The present invention provides a kind of gRNAs sequences of regulation PCV2 virus multiplication.The present invention devises 8 gRNA targeting segment relevant to PCV2 duplication: gRNA-Oc8, gRNA-O13, gRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT, targets eight dimeric structures of PCV2 replication orgin, the overlay region of ORF1 and ORF3, ORF1 respectively, ORF3 and the overlay region of ORF4, the overlay region of ORF2 and ORF6, the 23-25aa glycosylation site of ORF1, the 256-258aa glycosylation site of ORF1 and ORF1 286-288aa glycosylation site.Using pX458 as expression vector, PCV2 Cas9/gRNAs recombinant plasmid is obtained, and sequencing identification has been carried out to positive Cas9/gRNAs clone.Meanwhile the proliferation duplication for successfully obtaining with the PK-15 cell line of PCV2 Cas9/gRNAs Transfected Recombinant Plasmid, and PCV2 Cas9/gRNAs recombinant plasmid can effectively edit PCV2 genome, and inhibiting PCV2 intracellular in PK-15.
Description
Technical field
The invention belongs to molecular biology and biomedicine field, and in particular to a kind of sequence of regulation PCV2 virus multiplication
Column.
Background technique
CRISPR/Cas9 system is identified by gRNA (tracrRNA+crRNA) and target DNA PAM sequence-specific, is caused
Cas9 nuclease fixed point cuts complementation double-stranded DNA, and with preparing, simple, editing locus specificity is high and easy in terms of gene editing
In the advantages such as multiple gene locis of editing simultaneously, mammalian cell gene editing has been widely used in it.Currently, CRISPR/
Cas9 has been successfully applied to mouse, zebra fish and human cell even the genome accurate edits of bacterium.CRISPR/Cas9 is to disease
The editor of virus gene group also has been reported that in succession, and CRISPR/Cas9 technology has been applied successfully to double-stranded DNA virus and single stranded RNA disease
Poison has carried out effective editor.There has been no the reports edited using CRISPR/Cas9 technology to single-stranded DNA viruses at present.
Pig circular ring virus (Porcine circovirus, PCV) is the minimum virus for infecting mammal being currently known,
Belong to circovirus section Circovirus, is a kind of sub-thread cyclic DNA virus of no cyst membrane.1 type of pig circular ring virus (PCV1) and
Porcine circovirus 2 type (PCV2) is two main genotypes of pig circular ring virus.PCV1 is divided from pig renal epithelial cell PK-15
The non-pathogenic virus particle come is separated out, it is to cause pig circular ring virus related diseases (Porcine that PCV2, which is found in 1998,
Circovirus-associated disease, PCVAD) main pathogens.Originally PCVAD is described as weanling pig more
System failure syndrome (Post weaning multisystemic wasting syndrome, PMWS).With more and more
The appearance of clinical symptoms (such as breeding difficulty, intestines problem and respiratory symptom) relevant to PCV2 infection, just by this disease
Change and is named as pig circular ring virus related diseases (U.S.) and Porcine circovirus desease (Europe).
As a kind of single stranded circle DNA virus of only about 1.7kb size, the duplication of PCV2 is largely dependent upon
The operating of host cell.Therefore, during virus replication, PCV2 by the interaction with a variety of host cell factors come
Adjust host immune function, so as to cause cytokine imbalance, immunosupress or even disease.PCV2 genomic DNA has 11 to dive
In open reading frame.Spacer region IR contains loop-stem structure relevant to replication orgin.Since the minimum genome of PCV2 limits
Its code capacity, therefore the life cycle of PCV2 is heavily dependent on the replication capacity of its own.
On morphology, PCVs particle diameter is about 15-20nm, no cyst membrane, the capsid protein package of icosahedral symmetry
Whole gene group.The virion of PCVs is the structure comprising 60 capsid proteins copy.PCV1 and PCV2 genome includes 11
A potential ORFs.In these ORFs, ORF1 and ORF2 are two main ORFs, and along opposite direction encoding, in this way
Coding mode just derive an ambisense genome structure and 2 intergenic regions (IR).Wherein, ORF1 and ORF2 gene
IR between 3 ' ends is shorter intergenic region, and the IR between their 5 ' ends is longer intergenic region, should
It include the start position of PCV2 duplication in region.PCV2 replication initiation in one assume loop-stem structure (Stem-Loop,
SL it), and with rolling-circle replication mode is replicated.The loop-stem structure (Stem-Loop) assumed in spacer region between ORF1 and ORF2
It is related with DNA replication dna starting with 3 Hexanucleotide repetitive sequences (H1, H2, H3:CGGCAG).Loop-stem structure is inverted repeat sequence
Column, ring region contain 10 nucleotide, contain eight conservative aggressiveness (Oc8:A1x2T3A4x5T6 ↓ A7C8；Arrow representation DNA is multiple
Incision site when processed).ORF1 encodes replication protein Rep, ORF2 encoding capsid protein Cap, ORF3 encode non-structural protein by.
The Rep albumen size of PCV2 is 35.8kDa (314aa), is encoded by ORF1 overall length.Contain 3 glycosylation sites, 23-25aa
(NPS)；256-258aa (NQT) and 286-288aa (NAT).In order to indirectly control management PCVAD, the duplication Journal of Sex Research of PCV2 is extremely
It closes important.The sequence for reducing PCV2 virus multiplication can be used for the propagation or generation, development of control stategy PCVAD.
Summary of the invention
Lack porcine circovirus 2 type regulating and controlling sequence aiming at the problem that, the present invention provides a kind of regulation PCV2 virus multiplication
Sequence, can be with the proliferation of Effective Regulation PCV2.
To achieve the above object, the present invention is based on the design principle of CRISPR/Cas9 system principle and its gRNA, with
PX458 is expression vector, obtains PCV2 Cas9/gRNAs recombinant plasmid, and positive Cas9/gRNAs clone is sequenced
Identification.The present invention adopts the following technical scheme that.
A kind of gRNA sequence of regulation porcine circovirus 2 type (PCV2) virus replication, base sequence such as SEQ IDNO:1-
7 is any shown.It is described to be regulated to inhibit regulation.
The gRNA sequence, SEQ ID NO:1 target eight dimeric structures of PCV2 replication orgin, and base sequence is referred to as
gRNA-Oc8.SEQ ID NO:2 targets the overlay region of ORF1, ORF3 and ORF4, and base sequence is referred to as gRNA-O134.SEQ
ID NO:3 targets the overlay region of ORF1 and ORF3, and base sequence is referred to as gRNA-O13.SEQ ID NO:4 target ORF2 and
The overlay region of ORF6, base sequence are referred to as gRNA-O26.SEQ ID NO:5 targets the glycosylation position of ORF1 coding 23-25aa
Point NPS, base sequence are referred to as gRNA-NPS.SEQ ID NO:6 targets the glycosylation site NQT of ORF1 coding 256-258aa,
Base sequence is gRNA-NQT.SEQ ID NO.7 targets the glycosylation site NAT of ORF1 coding 286-288aa, base sequence letter
Referred to as gRNA-NAT.
The corresponding PAM sequence of above-mentioned gRNA sequence is successively are as follows: CGG, TGG, TGG, GGG, AGG, AGG, CGG.
A kind of PCV2 Cas9/gRNAs recombinant plasmid containing above-mentioned gRNA.
A kind of cell line of above-mentioned PCV2 Cas9/gRNA Transfected Recombinant Plasmid.
The transfection process are as follows: while recombinant C as9/gRNAs transfects cell, felt with the PCV2-SD virus of equivalent
Dye.
A kind of above-mentioned gRNA, recombinant plasmid and transfectional cell series treat and prevent porcine circovirus 2 type virus in preparation and draw
Play the purposes in disorder agent.
The invention has the following advantages that
GRNAs the sequence gRNA-Oc8, gRNA-O13 of regulation porcine circovirus 2 type (PCV2) virus replication provided by the invention,
GRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT target eight aggressiveness knots of PCV2 replication orgin respectively
Structure, the overlay region of ORF1 and ORF3, ORF1, ORF3 and the overlay region of ORF4, the overlay region of ORF2 and ORF6, ORF1 encode 23-
The glycosyl of glycosylation site NQT and ORF1 the coding 286-288aa of glycosylation site NPS, ORF1 coding 256-258aa of 25aa
Change site NAT.By the recombinant C as9/gRNAs of building, PCV2 genome can effectively be edited and inhibit PCV2 in PK-
15 intracellular proliferation duplications.
Detailed description of the invention
Fig. 1 is target site of the gRNAs sequence in PCV2 genome；
Fig. 2 is the comparison of gRNAs sequence-specific；
Fig. 3 is external digestion electrophoresis result.M is DL-2000DNA Marker；NC1It is the transcription product without Cas9 digestion；
NC2It is the non-transcription product through Cas9 digestion；G1 is the standard gRNA1 through Cas9 digestion；G2 is the standard through Cas9 digestion
gRNA2；GRNA-Oc8, gRNA-H3, gRNA-O13, gRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-
NAT is the gRNAs through Cas9 digestion；
Fig. 4 is that different recombinant C as9/gRNAs infect macroscopic cell proliferative conditions after PK-15 cell 48h；
Fig. 5 is the protein expression result for recombinating Cas9/gRNAs.M is albumen Marker；Oc8 is recombination PCV2 Cas9/gRNA-
Oc8；O134 is recombination PCV2 Cas9/gRNA-O134；O13 is recombination PCV2 Cas9/gRNA-O13；H3 is recombination PCV2
Cas9/gRNA-H3；O26 is recombination PCV2 Cas9/gRNA-O26；NAT is recombination PCV2 Cas9/gRNA-NAT；NQT is weight
Group PCV2 Cas9/gRNA-NQT；NPS is recombination PCV2 Cas9/gRNA-NPS；NC is that PCV2 pX458 cotransfection work is negative right
According to；
Fig. 6 is recombination Cas9/gRNAs cytotoxicity proliferation experiment result.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
Embodiment 1 targets the gRNAs design of PCV2 genome
The gRNAs design of 1 targeting PCV2 genome
According to the PCV2SD pnca gene sequence (Genbank Accession Number JQ653449) in NCBI on GenBank
And PCV2 full-length genome structure, it designs 8 gRNA relevant to PCV2 duplication and targets segment: gRNA-Oc8, gRNA-H3, gRNA-
O13, gRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT, target PCV2 replication orgin respectively eight are poly-
The overlay region of body structure, six aggressiveness of loop-stem structure side arm, the overlay region of ORF1 and ORF3, ORF1, ORF3 and ORF4, ORF2 and
The overlay region of ORF6, the segment of ORF1 coding 23-25aa glycosylation site NPS, ORF1 encode 256-258aa glycosylation site
The segment of segment and ORF1 coding the 286-288aa glycosylation site NAT of NQT, as shown in Figure 1.
It is carried out using CRISPR/Cas9gRNA design website (http://crispr.mit.edu/) of the offers such as peak
The design of gRNAs.The gRNA of acquisition is subjected to BLAST and compares screening, should be had the feature that by the gRNAs that screening obtains
1) in the genome consensus sequence of PCV2 different genotype, identical base constitutes, it is ensured that gRNA has targeting each base of PCV2
Because of the ability of type conserved sequence；2) 3 ' ends are " NGG ", it is ensured that precisely identification PAM sequence, and then ensure that PCV2 sequence can be by Cas9
Restriction endonuclease identification and cutting.GRNAs sequence such as table 1 after screening.
1 gRNAs sequence information of table
| Name | gRNA sequence(5'-3') | PAM | Nucleotide position |
| gRNA-Oc8 | TTACCAGCGCACTTCGGCAG | CGG | 1766-21 |
| gRNA-H3 | TTGCTGCTGAGGTGCTGCCG | ATG | 30-53 |
| gRNA-O134 | TGTCAGAAATTTCCGCGGGC | TGG | 479-502 |
| gRNA-O13 | GGCAACTTACTGATAGAATG | TGG | 351-374 |
| gRNA-O26 | GACAGTATAACCGATGGTGC | GGG | 1430-1407 |
| gRNA-NPS | GCGGACCCCAACCACATAAA | AGG | 76-99 |
| gRNA-NQT | AGAATACTGCGGGCCAAAAA | AGG | 750-773 |
| gRNA-NAT | CAGCTGTAGAAGCTCTCTAT | CGG | 855-878 |
By NCBI BLAST sequence alignment, Fig. 2 is as a result seen.As shown in Figure 2, gRNA-Oc8, gRNA-H3, gRNA-O13,
GRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT all have preferable specificity to PCV2.
2 are transcribed in vitro gRNAs
The acquisition of 2.1gRNAs PCR product
According to target site (thickened portion is free of PAM sequence) synthesis different primers F and primer R of gRNAs, sequence such as 2 institute of table
Show.
2 gRNAs transcription primers sequence information of table
With gRNA-Oc8-F and R, gRNA-H3-F and R, gRNA-O134-F and R, gRNA-O13-F and R, gRNA-O26-F and R,
GRNA-NPS-F and R, gRNA-NQT-F and R, gRNA-NAT-F and R eight do primer pair using standard gRNA segment as template
PCR, while (Beijing only Shang Lide Biotechnology Co., Ltd, product are compiled according to spCas9-gRNA target spot Efficiency testing kit
Number: NO.VK007) prepare the PCR product of standard gRNA1 (g1) and standard gRNA2 (g2).Pass through the external digestion target of Cas9/gRNA
Point DNA fragmentation, carries out the assessment of gRNA target active.PCR reaction system is as follows:
| Component | Dosage |
| gRNAs | 10ng |
| gRNAs-F(10μM) | 1.5μL |
| R(10μM) | 1.5μL |
| 2×Pfu Mix | 25μL |
| dd H2O | It mends to 50 μ L |
Response procedures are as follows: 95 DEG C of 3min of initial denaturation are denaturalized 94 DEG C of 30s, and anneal 58 DEG C of 30s, extend 72 DEG C of 30s, totally 35 circulations；
Last 72 DEG C of extensions 10min, later 16 DEG C of 10min.
PCR product carries out gel electrophoresis, the purpose band of recycling 120bp or so, the DNA mould as subsequent in vitro transcription
Plate.GRNAs target spot (sequence containing PAM) is constructed in above-mentioned DNA profiling by way of bridging PCR.
The external endonuclease reaction of 2.2Cas9/gRNA
Digestion presequence are as follows: NNNNNNNNN (270bp) MMMMMMMMMMM (gRNA target site position, sequence containing PAM)
NNNNNNNNNNN (450bp), clip size is 740bp before digestion, and purpose band size should be respectively as follows: 280bp after digestion
+ 460bp or so.Reaction system is as follows:
| Component | Dosage |
| Cas9 enzyme | 1U |
| 10×Cas9 buffer | 2μL |
| GRNA (in-vitro transcription) | 50ng(gRNAs) |
| ddH2O | It mends to 20 μ L |
| The dsDNA of digestion | 50ng |
After being sufficiently mixed, 37 DEG C of reaction 0.5h, 65 DEG C are boiled product progress agarose gel electrophoresis after 5min, test and analyze digestion knot
Fruit, external digestion electrophoresis result such as Fig. 3, with standard gRNA1 and standard gRNA2 comparing calculation activity.According to the ash of digestion band
Degree conversion gRNA activity, such as table 3.
For standard gRNA1 (g1) and standard gRNA2 (g2) by SSA luciferase kit detection activity, SSA is living
Property is respectively as follows: standard gRNA1 (g1)=3, standard gRNA2 (g2)=10.
Therefore, if the digesting efficiency of sample gRNA < standard gRNA1, show that sample gRNA target active is poor；If
40%-50% >=sample gRNA digesting efficiency >=standard gRNA1 shows that sample gRNA target active is qualified；If standard gRNA2
Digesting efficiency >=40%-50% of >=sample gRNA shows that sample gRNA target active is good；If the digesting efficiency of sample gRNA
>=standard gRNA2 (g2) shows that sample gRNA target active is very high.
3 gRNAs external activity testing result of table
| Title | Target sequence | External digestion activity | With standard control |
| gRNA-Oc8 | TTACCAGCGCACTTCGGCAGCGG | 70% | Equal to gRNA2 |
| gRNA-H3 | TTGCTGCTGAGGTGCTGCCGAGG | 1% | Less than gRNA1 |
| gRNA-O134 | TGTCAGAAATTTCCGCGGGCTGG | 73% | Greater than gRNA2 |
| gRNA-O13 | GGCAACTTACTGATAGAATGTGG | 98% | Greater than gRNA2 |
| gRNA-O26 | GACAGTATAACCGATGGTGCGGG | 98% | Greater than gRNA2 |
| gRNA-NPS | GCGGACCCCAACCACATAAAAGG | 95% | Greater than gRNA2 |
| gRNA-NQT | AGAATACTGCGGGCCAAAAAAGG | 94% | Greater than gRNA2 |
| gRNA-NAT | CAGCTGTAGAAGCTCTCTATCGG | 70% | Equal to gRNA2 |
Seen from table 3, all gRNAs in addition to gRNA-H3 have good activity.
Embodiment 2 is suitable for editing the foundation of the CRISPR/Cas9 system of PCV2 genome
1PCV2 Cas9/gRNAs construction of recombinant plasmid
1.1 synthesis gRNAs dimers
Its reverse complemental primer pair is synthesized according to the designed gRNAs of embodiment 1, as shown in table 4:
4 CRISPR gRNAs complementary primer of table is to sequence information
| Title | Positive-sense strand Sense (5 ' -3 ') | Antisense strand Anti (5 ' -3 ') |
| gRNA-Oc8 | TTACCAGCGCACTTCGGCAG | CTGCCGAAGTGCGCTGGTAA |
| gRNA-H3 | TTGCTGCTGAGGTGCTGCCG | CGGCAGCACCTCAGCAGCAA |
| gRNA-O134 | TGTCAGAAATTTCCGCGGGC | GCCCGCGGAAATTTCTGACA |
| gRNA-O13 | GGCAACTTACTGATAGAATG | CATTCTATCAGTAAGTTGCC |
| gRNA-O26 | GACAGTATAACCGATGGTGC | GCACCATCGGTTATACTGTC |
| gRNA-NPS | GCGGACCCCAACCACATAAA | TTTATGTGGTTGGGGTCCGC |
| gRNA-NQT | AGAATACTGCGGGCCAAAAA | TTTTTGGCCCGCAGTATTCT |
| gRNA-NAT | CAGCTGTAGAAGCTCTCTAT | ATAGAGAGCTTCTACAGCTG |
The gRNAs primer pair of synthesis is diluted to 10 μM, is mixed in following ratio:
| Component | Dosage |
| gRNA-Sense | 1μL |
| gRNA-Anti | 1μL |
| T4PNK | 5μL |
| ddH2O | 3μL |
After mixing, handles: PCR pipe being placed in 95 DEG C of water, cooled to room temperature by following procedure.
The building of 1.2 recombinant C as9/gRNAs
Step 1.1 products therefrom gRNAs dimer in the present embodiment is added in following system:
| Component | Dosage |
| PX458 plasmid | 1μL |
| GRNAs dimer | 2μL |
| ddH2O | 7μL |
After mixing well, 25 DEG C of standing 5min.
1.3 conversion
The product of step 1.2 in the present embodiment is transformed into Stbl3 competent cell.
The extraction of 1.4 plasmids
5 single colonies of random picking on cultivating good LB plate are respectively put into the LB liquid that Amp resistance is had containing 5mL
In culture medium, then shaken cultivation 16h on 37 DEG C of 200rpm shaking tables extracts plasmid, is placed in -20 DEG C of preservations.
The identification of 1.5 positive Cas9/gRNAs clone
Step 1.4 gained recombinant plasmid dna in the present embodiment is subjected to sequencing identification, sequencing primer:
TGAGCGTCGATTTTTGTGATGCTCGTCAG。
2.Cas9/gRNAs recombinant vector functional verification
The preparation of 2.1 transfection cells
The cryopreservation tube equipped with PK-15 cell is taken, after recovery, in the DMEM cell culture fluid culture containing 10% fetal calf serum, is used
Pancreatin processing after-blow breaks into individual cells, and cell culture fluid is transferred in 6 porocyte culture plates, and 2mL is added in the every hole of 6 orifice plates,
Its density is 0.25 × 106~1 × 106；Set CO2In incubator, 37 DEG C of cultures are spare.
2.2 transfections and Transfected cells growing state
Transfection procedure is carried out according to 3000 kit specification of LipofectamineTM.Using two ways:
(1) it while recombinant C as9/gRNAs transfects cell, is infected with the PCV2-SD virus of equivalent, control group is added etc.
Cell plates, are put into CO by the empty pX458 plasmid of amount2Continue culture in incubator to 48h；
(2) it after recombinant C as9/gRNAs transfects cell for 24 hours, is infected with the PCV2-SD virus of equivalent, equivalent is added in control group
Empty pX458 plasmid, cell plates are put into CO2Continue culture in incubator to 48h.
It digests every hole cell respectively with pancreatin, prepares cell suspension, carry out cell count, cell suspension cell number/mL=4
A big lattice total number of cells/4 × 10000, obtained experimental data carry out significance difference analysis through SPSS software.To two different
Virus infection mode is found after comparing, while recombinant C as9/gRNAs transfects cell, mode (1): with equivalent
The ratio mode (2) that PCV2-SD virus is infected: after recombinant C as9/gRNAs transfects cell for 24 hours, with the PCV2-SD of equivalent
Virus carries out the variation that infection is more prone to produce in macroscopic cell growth.
Recombinant plasmid and virus transfect the cell growth status after 48h under the same visual field simultaneously, see Fig. 4.PCV2 can feel
The growth rate of PK-15 cell can be influenced although cytopathy cannot be formed by contaminating PK-15 cell.From fig. 4, it can be seen that with a batch
For PK-15 cell under identical growth conditions, control group compares nature growth with the PCV2-SD cell infected with sky pX458
Pure PK-15 cell slow growth；Infect PCV2-SD and recombinant C as9/gRNA-Oc8, Cas9/gRNA-O134, Cas9/gRNA-
The cell of O13, Cas9/gRNA-O26, Cas9/gRNA-NPS and Cas9/gRNA-NQT are obviously faster than control group proliferation；Recombination
It is slow that other recombinant Cs as9/gRNAs compare in the cell growth that Cas9/gRNA-NAT and PCV2-SD infect, but than control group growth
Comparatively fast；The cell Proliferation and control group that recombinant C as9/gRNA-H3 and PCV2-SD infects simultaneously are not much different.
After recombinant C as9/gRNAs and PCV2 cotransfection PK-15 cell 48h that method (1) obtains, to the shadow of cell quantity
Sound is shown in Table 5:
Influence of the different recombination PCV2 Cas9/gRNAs of table 5 to PK-15 proliferation quantity
| Group | Cell quantity |
| Oc8 | 35.742±1.855b |
| H3 | 24.188±0.964a |
| O134 | 33.232±0.753b |
| O13 | 33.745±0.677a |
| O26 | 31.223±0.875b |
| NPS | 33.344±1.853b |
| NQT | 33.875±0.289b |
| NAT | 28.098±1.584c |
| Control | 23.542±0.875a |
Note: the expression difference for indicating same letter is not significant (P > 0.05)；Indicate different letters expression significant difference (p <
0.05)。
Cell count statistical analysis is shown, after different recombination PCV2 Cas9/gRNAs transfection PK-15 cell 48h, weight
The cell quantity of group PCV2 Cas9/gRNA-H3 and control group transfection PCV2 pX458 empty plasmid difference is not significant；Recombinate PCV2
Cas9/gRNA-Oc8、PCV2 Cas9/gRNA-O134、PCV2 Cas9/gRNA-O13、PCV2 Cas9/gRNA-O26、PCV2
The cell quantity and control group significant difference of Cas9/gRNA-NPS and PCV2 Cas9/gRNA-NQT；Recombinate PCV2 Cas9/
The cell quantity and control group significant difference of gRNA-NAT simultaneously recombinate PCV2 Cas9/gRNAs significant difference with remaining.
The detection of 2.3 protein expressions
When transfecting 48h, tissue culture plate is taken out, nutrient solution is discarded, is cracked, is centrifuged, take supernatant into sterile EP pipe,
SDS-PAGE sample-loading buffer is added, carries out SDS-PAGE electrophoresis.The careful taking-up of the glue of acquisition, excision upper layer are concentrated glue, cut
Except edge be more than part, successively through transfer, close, wash film, primary antibody be incubated for, wash film, secondary antibody be incubated for, wash film, development and etc., use
The luminous colour reagent box of ECL ultra-sensitive chemical develops the color, and observation is exposed on ChemiDoc MP Almightiness type gel imaging system.
Protein immunoblot result is shown in Fig. 5.As seen from Figure 5, recombination PCV2 Cas9/gRNA-H3 and control group are expressed
Rep protein content is not much different；Recombination PCV2 Cas9/gRNA-NAT can express a certain amount of albumen, but expressing quantity is than control
Group is few；Recombination PCV2 Cas9/gRNA-NPS, PCV2 Cas9/gRNA-NQT and PCV2 Cas9/gRNA-O26 are also shown
The expression of albumen, but expression quantity even lower than recombinates the protein expression of PCV2 Cas9/gRNA-NAT well below control group
Amount；The expressing quantity for recombinating PCV2 Cas9/gRNA-O134 and PCV2 Cas9/gRNA-O13 is micro- and micro-；Recombinate PCV2
Cas9/gRNA-Oc8 is almost beyond expression Rep albumen.
2.4PCV2 Cas9/gRNAs CCK-8 virulence test
Cell Proliferation and oxicity analysis are carried out using CCK-8 method: after 96 orifice plates transfect 48h, it is molten that 10 μ L CCK-8 are added to every hole
Liquid (avoids generating bubble in hole), and culture plate is incubated for 1-3h in incubator, respectively after the addition 0h, 1h, 2h when enzyme
Mark absorbance of the instrument measurement at 450nm.Absorbance is measured as reference when 0h after addition CCK-8.As a result see Fig. 6, it can by Fig. 6
Know, except recombination PCV2 Cas9/gRNA-Oc8 and PCV2 Cas9/gRNA-H3 in addition to 0h dulling luminosity ratio control group is low, other
The absorbance of PCV2 Cas9/gRNAs is above control group；Absorbance of all recombination PCV2 Cas9/gRNAs in 1h is high
It is close with control group in the absorbance of control group, PCV2 Cas9/gRNA-H3；Except recombination PCV2 Cas9/gRNA-H3 is inhaled in 2h
Except luminosity is more slightly lower than control group, the absorbance of other PCV2 Cas9/gRNAs is above control group.
It is a large amount of studies have shown that CRISPR/Cas9 technology there are a degree of undershooting-effects.Influence gene editing efficiency
It is varied with the factor of specificity.Scientists are to the genome editorial efficiency of CRISPR/Cas9 System-mediated and de- at present
The research of targeted effect is not still deep enough, viewpoint still disunity.Design and missed the target in forecasting software in various gRNA, gRNA activity and
The evaluation criteria of specificity is also inconsistent.Although presently, there are a variety of CRISPR sequence design tools, many software programs are complete
It is complete to be analyzed dependent on calculating, without true visible verifying.Experiment sieving identifies that effective gRNA is to influence CRISPR/Cas9 to compile
Collect the key factor of efficiency.
Several gRNAs are respectively devised to this 8 different target sites respectively using online website in this experiment, it is then right
Select the 8 high gRNAs that score again from these gRNAs, by testing these gRNAs Validation in vitro, the results show that in addition to
For on spacer region loop-stem structure side arm six aggressiveness synthesis gRNA-H3 external activity it is low except, other 7 gRNAs have
There is higher shear active.This also illustrates experiment sieving identification is the only resource of determining gRNA validity.
Sequence table
<110>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricul
<120>a kind of sequence and its application of regulation PCV2 virus multiplication
<130> 20190423
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 1
ttaccagcgc acttcggcag 20
<210> 2
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 2
tgtcagaaat ttccgcgggc 20
<210> 3
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 3
ggcaacttac tgatagaatg 20
<210> 4
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 4
gacagtataa ccgatggtgc 20
<210> 5
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 5
gcggacccca accacataaa 20
<210> 6
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 6
agaatactgc gggccaaaaa 20
<210> 7
<211> 20
<212> DNA
<213> Porcine circovirus
<400> 7
cagctgtaga agctctctat 20
Claims (6)
1. a kind of gRNA sequence of regulation porcine circovirus 2 type virus replication, base sequence such as SEQ ID NO:1-7 are any
It is shown.
2. the corresponding PAM sequence of gRNA as described in claim 1 a kind of, which is characterized in that SEQ ID NO:1-7 is successively
Corresponding PAM sequence are as follows: CGG, TGG, TGG, GGG, AGG, AGG, CGG.
3. a kind of PCV2 Cas9/gRNAs recombinant plasmid containing gRNA described in claim 1.
4. a kind of cell line of PCV2 Cas9/gRNA Transfected Recombinant Plasmid as claimed in claim 2.
5. a kind of cell line as claimed in claim 4, which is characterized in that the transfection process is the following steps are included: recombination
While Cas9/gRNAs transfects cell, infected with the PCV2-SD virus of equivalent.
6. a kind of gRNA as described in claim 1-4, PCV2 Cas9/gRNAs recombinant plasmid and PCV2 Cas9/gRNA recombination
The cell line of plasmid transfection treats and prevents porcine circovirus 2 type virus in preparation and causes the purposes in disorder agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910336909.7A CN110055251B (en) | 2019-04-25 | 2019-04-25 | Sequence for regulating and controlling PCV2 virus proliferation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910336909.7A CN110055251B (en) | 2019-04-25 | 2019-04-25 | Sequence for regulating and controlling PCV2 virus proliferation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110055251A true CN110055251A (en) | 2019-07-26 |
| CN110055251B CN110055251B (en) | 2022-05-27 |
Family
ID=67320725
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910336909.7A Active CN110055251B (en) | 2019-04-25 | 2019-04-25 | Sequence for regulating and controlling PCV2 virus proliferation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110055251B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607356A (en) * | 2019-06-14 | 2019-12-24 | 山东大学 | Genome editing detection method, kit and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200738880A (en) * | 2005-12-01 | 2007-10-16 | Consejo Superior Investigacion | Nucleic acids encoding TGEV and PRRSV sequences for improved expression of PRRSV sequences |
| CN105688201A (en) * | 2012-05-17 | 2016-06-22 | 佐蒂斯有限责任公司 | Effective vaccination against porcine reproductive and respiratory syndrome (prrs) virus prior to weaning |
| CN108690844A (en) * | 2018-05-25 | 2018-10-23 | 西南大学 | CRISPR/Cas9-gRNA target practices sequence pair, plasmid and the HD cell models of HTT |
-
2019
- 2019-04-25 CN CN201910336909.7A patent/CN110055251B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200738880A (en) * | 2005-12-01 | 2007-10-16 | Consejo Superior Investigacion | Nucleic acids encoding TGEV and PRRSV sequences for improved expression of PRRSV sequences |
| CN105688201A (en) * | 2012-05-17 | 2016-06-22 | 佐蒂斯有限责任公司 | Effective vaccination against porcine reproductive and respiratory syndrome (prrs) virus prior to weaning |
| CN108690844A (en) * | 2018-05-25 | 2018-10-23 | 西南大学 | CRISPR/Cas9-gRNA target practices sequence pair, plasmid and the HD cell models of HTT |
Non-Patent Citations (2)
| Title |
|---|
| XU DAN等: "P53 signaling modulation of cell cycle arrest and viral replication in porcine circovirus type 2 infection cells", 《VETERINARY RESEARCH》 * |
| 李俊等: "猪圆环病毒2型双拷贝感染性DNA的构建及体外拯救", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607356A (en) * | 2019-06-14 | 2019-12-24 | 山东大学 | Genome editing detection method, kit and application |
| CN110607356B (en) * | 2019-06-14 | 2021-02-02 | 山东大学 | Genome editing detection method, kit and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110055251B (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Decaro et al. | Hobi-like pestivirus: both biotypes isolated from a diseased animal | |
| CN108602859A (en) | Enhance the modification capsid protein of parvovirus vectors delivering | |
| Li et al. | Genetic analysis of porcine circovirus type 2 (PCV2) strains isolated between 2001 and 2009: genotype PCV2b predominate in postweaning multisystemic wasting syndrome occurrences in eastern China | |
| Dong et al. | Emergence of fatal European genotype CyHV-3/KHV in mainland China | |
| CN110592172B (en) | Method and target for screening JEV resistance gene by using CRISPR/Cas9 knockout library technology | |
| WO2019119521A1 (en) | Marc-145 cell line against porcine reproductive and respiratory syndrome and preparation method and use thereof | |
| CN107955839A (en) | For detecting double PCR primer, detection method and the kit of 3 type of porcine circovirus 2 type and circovirus | |
| Kim et al. | Prevalence of porcine parvovirus 1 through 7 (PPV1-PPV7) and co-factor association with PCV2 and PRRSV in Korea | |
| CN106399313B (en) | A kind of microRNA sample virus tiny RNA sequence of anti-PRRSV and application thereof and detection method | |
| Zhao et al. | Recent advances in the study of NADC34-like porcine reproductive and respiratory syndrome virus in China | |
| Jiang et al. | A novel technique for constructing infectious cloning of type 3 porcine circovirus | |
| CN113736750B (en) | Gata virus strain and application thereof | |
| CN110055251A (en) | A kind of sequence and its application of regulation PCV2 virus multiplication | |
| Xu et al. | First molecular detection and genetic analysis of porcine circovirus 4 in the Southwest of China during 2021–2022 | |
| CN114107311A (en) | Target participating in porcine transmissible gastroenteritis virus infection and application thereof | |
| CN108315306A (en) | One plant height fertility swine fever virus and its construction method | |
| Chowdhury et al. | Pathogenicity, sequence and phylogenetic analysis of Malaysian Chicken anaemia virus obtained after low and high passages in MSB-1 cells | |
| CN101089177A (en) | Type II genetic marker strain of porcine circovirus and its application | |
| CN116064417A (en) | Cat panleukopenia virus strain and application thereof | |
| Chang et al. | Isolation and characterization of a Sagiyama virus from domestic pigs | |
| CN110904056B (en) | Infectious bronchitis virus rH120-YZS1 delta 5a and construction method and application thereof | |
| CN104878043B (en) | Sheep of virus virulence gene VIR deletion mutation strains and its preparation method and application | |
| CN107018955A (en) | A kind of transgene pig of the type of resisting porcine circovirus 2 | |
| CN109679952B (en) | Porcine-derived miR-c89 for resisting PRRSV infection and application thereof | |
| CN106497951B (en) | A kind of VP2 fusion, rHEP-rVP2 plants of recombinant rabies virus, construction method and its application |
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 |