CN109486773A - The seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T and its construction method - Google Patents
The seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T and its construction method Download PDFInfo
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Abstract
The present invention provides a kind of seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T and its construction methods.The present invention utilizes GS1783 Escherichia coli and pEPkan-S plasmid, the region duck plague virus gE gene C T is lacked through homologous recombination in bacterial artificial chromosome recombination duck plague virus rescue system platform, completes the building of the remaining seamless gene-deleted strain in the region duck plague virus gE gene C T of no external source base for the first time.Base is remained in deletion segment when technical solution of the present invention solves the problems, such as to lack duck plague virus gene, provides sufficient technical support accurately to probe into the building of duck plague virus gene function and attenuated live vaccine.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of seamless gene-deleted strain in the region duck plague virus gE gene C T
CHv-BAC-G-gE Δ CT and its construction method.
Background technique
A kind of bacterial artificial chromosome (bacterial artificial chromosome, BAC) new development is got up
DNA vector system, it has many advantages, such as, and capacity is big, hereditary capacity is stable, easily operated, in construction of gene library and gene function
Analysis etc. is widely used.Whole viral genes group DNA molecular is inserted into BAC carrier, most using the vector encoded
Small fertility factor replicon (Minimal fertility factor replicon, Mini-F) obtains molecular cloning disease
Poison, and assignment of genes gene mapping modification technique mature in Escherichia coli is combined, to realize the missing of the viral gene in prokaryotic system
And the insertion of foreign gene.Current more common bacillus coli gene locator qualification technology mainly includes the same of Red/ET mediation
The homologous recombination technique and Tn transposons of source recombinant technique, RecA protein mediated homologous recombination technique, Cre/loxP mediation
The radom insertion and mutating technology of mediation.Mature bacterial artificial chromosome duck has been obtained using molecular cloning technological means
Pestivirus saves system platform, while can be thin using the homologous recombination technique that bacillus coli gene locator qualification Red/ET is mediated
Bacterium artificial chromosome duck plague virus save system platform on carry out duck plague virus gene missing and foreign gene insertion, this at
Fruit has greatly pushed the flow of research of duck plague virus gene function.
The homologous recombination technique that Red/ET is mediated is based on lambda phage Red operon (Red α/Red β/Red γ) and Rac
The homologous recombination Knockout technology of bacteriophage RecE/RecT homologous recombination enzyme.The technical operation is simple, quick, efficient, is widely applied
In gene delection, mutation work.But gene delection is carried out using the operating technology, is mutated and can be remained in missing or mutational site
About 80bp or so external source base sequence (site FRT), the residual in the site will affect the accurate analysis of gene function.
Duck plague (Duck Plague, DP) be by duck plague virus in Alphaherpesviridae (Duck Plague virus,
The acute contact height lethal infectious diseases of the aquatic birds such as duck caused by DPV), goose.The disease is reported by Holland first, immediately at me
The more flourishing area of the duck culturing industries such as state south China, Central China and East China is popular, causes serious economic damage to the duck culturing industry in China
It loses.Therefore it understands duck plague virus gene function in depth, reinforce to the research of duck plague epidemic disease to ensuring China's duck culturing industry health, can hold
Supervention exhibition is particularly important.
Duck plague virus DPV-CHv pnca gene group DNA overall length 162175bp, includes 78 open reading frame, and codified participates in
The structural proteins and non-structural protein of duck plague virus life cycle, wherein structural proteins mainly include capsid protein, cortex albumen
And envelope protein.Envelope protein is glycosylation albumen, including gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM, gN 12
Kind.Glycoprotein has the function of that mediate retroviral adsorbs, is viral in spread between cells into sensitive cells and promotion, carries simultaneously
Antigenic determinant can induce animal body immune system to the identification of virus and cause the pathology damage of tissue, therefore probes into capsule
Effect of the membrane glycoprotein in duck plague virus life cycle is to deeply probing into duck plague virus gene function and to carry out duck plague epidemic disease anti-
It is most important to control work.
It is utilized in the prior art using BAC as the technology of plateform molecules cloning virus, duck plague virus genome recombination is arrived
In Baculovirus transfer vector containing BAC, building bacterial artificial chromosome recombination duck plague virus saves system platform DPV CHv-
BAC-G.In combination with Red/ET modification technique, duck plague virus base is completed by mature genetic manipulation means in prokaryotic system
Because of missing and foreign gene insertion.But system is saved in bacterial artificial chromosome recombination duck plague virus using Red/ET modification technique
On platform to duck plague virus gene delection after, can at the missing gene site FRT at remnants two.The residual pair in FRT external source site
The probing into of gene function, the exploitation of attenuated live vaccine and license exist and influence.
Summary of the invention
For the above-mentioned problems in the prior art, it is seamless scarce that the present invention provides a kind of region duck plague virus gE gene C T
Strain CHv-BAC-G-gE Δ CT and its construction method are lost, which can effectively solve lacking when missing duck plague virus gene
Site remains the problem of base.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
The seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T, construction method includes following step
It is rapid:
(1) pBAC-DPV plasmid is transformed into GS1783 E. coli competent, obtains GS1783-pBAC-DPV bacterium
Strain, then prepares GS1783-pBAC-DPV competence;
(2) using pEPkan-S as template, using GS1783-BAC-gE Δ CT-F and GS1783-BAC-gE Δ CT-R as drawing
Object includes the base fragment and gE gene C T region upstream of I_SceI restriction enzyme site and Kana element by PCR method amplification
With the target practice segment I_SceI-Kana-CT of each 40bp homology arm in downstream, gel extraction obtains I_SceI-Kana-CT segment;
(3) I_SceI-Kana-CT segment is transformed into GS1783-pBAC-DPV competence, is screened, obtain positive gram
Longzi GS1783-pBAC-DPV-CT-Kana;
(4) the I_SceI-Kana segment in positive clone molecule GS1783-pBAC-DPV-CT-Kana is removed, through antibiosis
Element screening and PCR identification, obtain positive clone molecule GS1783-pBAC-DPV-gE Δ CT;
(5) pBAC-DPV-gE Δ CT plasmid is extracted from positive clone molecule GS1783-pBAC-DPV-gE Δ CT, it will
PBAC-DPV-gE Δ CT plasmid transfection DEF cell obtains the seamless gene-deleted strain CHv- in the region gE gene C T by colony screening
BAC-G-gEΔCT。
Further, primer sequence in step (2) are as follows:
GS1783-BAC-gEΔCT-F:5’-CGTGACTATTATAATCCTGGCTGTTTCATCCATCTTTTTAtaggg
ataacagggtaatcgattt-3';
GS1783-BAC-gEΔCT-R:5’-CTATTTCACTAGTGAGTCATTAGTTCAACATCCATGATCATAAAA
AGATGGATGAAACAGCCAGGATTATAATAGTCACGgccagtgttacaaccaat-3’。
Further, PCR amplification system in step (2) are as follows: ddH2O 22μl、MaxDNA
25 μ l of Polymerase, 1 μ l of upstream primer, 1 μ l of downstream primer, 1 μ l of template;PCR amplification condition are as follows: 98 DEG C of initial denaturation 2min,
98 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 72 DEG C of extension 5s, totally 30 recycle, last 72 DEG C of extensions 10min.
The seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T provided by the invention and its building side
Method has the advantages that
To obtain without the remaining duck plague virus gene-deleted strain of external source base, the present invention recombinates duck in bacterial artificial chromosome
Pestivirus is saved on the basis of system platform, using Red-based modification technique, i.e., using containing codified Red operon and
The GS1783 coli strain of I_SceI enzyme gene sequence and the plasmid of resistance and I_SceI restriction enzyme site is received containing coding card
PEPkan-S lacks duck plague virus gE base through homologous recombination in bacterial artificial chromosome recombination duck plague virus rescue system platform
Because of the region CT, the building of the seamless gene-deleted strain of no remaining duck plague virus of external source base is completed for the first time.Technical solution of the present invention solution
When missing of having determined duck plague virus gene the problem of deletion segment remains base, accurately to probe into duck plague virus gene function and subtracting
The building of virus live vaccine provides sufficient technical support.
Detailed description of the invention
Fig. 1 is pEPkan-S plasmid map.
Fig. 2 is to recombinate duck plague virus in bacterial artificial chromosome using Red-Based modification technique to save in system platform
Carry out the operational flowchart of gene delection.
Fig. 3 is fluorescent spot picture after the seamless deleted virus strain virus rescue of CHv-BAC-G-gE Δ CT.
Fig. 4 is the seamless deleted virus strain plaque test picture of CHv-BAC-G-gE Δ CT.
Specific embodiment
The seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T, building process material therefor and reagent
It is as follows:
1, experimental material
(1) cell, bacterial strain, virus stain, plasmid
Primary duck embryo fibroblasts are prepared according to a conventional method by the nonimmune fertilization duck embryos of 10-11 age in days;GS1783 bacterial strain
It is saved by Sichuan Agricultural University laboratory;PBAC-DPV plasmid is constructed and is saved by Sichuan Agricultural University laboratory;pEPkan-S
Plasmid is saved by Sichuan Agricultural University laboratory.
2, molecular biology reagents
The small extraction reagent kit of plasmid is purchased from TIANGEN company;QIAGEN Plasmid Midi Kit is purchased from QIAGEN company;
Plain agar sugar gel DNA QIAquick Gel Extraction Kit is purchased from TIANGEN company;Max DNA Polymerase purchase
From Takara company;TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 is public purchased from TaKaRa
Department;Lipofectamine 3000 is purchased from Invitrogen company;Instant SABC immunohistochemical staining kit (rabbit igg) purchase
From doctor's moral company;DAB colour reagent box (Huang) is purchased from doctor's moral company.
3, solution used and its preparation are tested
LB liquid medium: weigh Tryptone 10g, Yeast Extract 5g, sodium chloride 10g be dissolved in 800mL go from
It in sub- water, is sufficiently stirred, is settled to lL, autoclave sterilization.
LB solid medium: being added 15g agar powder in the LB liquid medium for being settled to 1L, after autoclave sterilization,
60 DEG C or so are cooled to, 1.5mL chloramphenicol (storage concentration 25mg/ml) or 1.5mL kanamycins (storage concentration 50mg/ is added
ML), paved plate, after to be solidified, 4 DEG C of preservations.
MEM: being dissolved in 800mL deionized water for 9.6g MEM dry powder and 2.2g sodium bicarbonate, be sufficiently stirred, adjust pH value to
7.4, it is settled to lL, filtration sterilization, 4 DEG C of preservations.
Embodiment 1 prepares the seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T
The seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T, construction method includes following step
It is rapid:
1, preparation GS1783 electricity turns competence, electrotransformation pBAC-DPV plasmid
(1) Escherichia coli of the recovery with pBAC-DPV plasmid are in the LB solid medium containing chloramphenicol, 37 DEG C of cultures
Overnight;It chooses single colonie to be inoculated in the LB liquid medium containing chloramphenicol, 37 DEG C of overnight incubations;
(2) pBAC-DPV plasmid is extracted according to QIAGEN Plasmid Midi Kit operating instruction;
(3) recovery GS1783 freezes bacterium in LB solid medium, 30 DEG C of overnight incubations;
(4) picking GS1783 single colonie is inoculated in 5mL LB liquid medium, 30 DEG C of overnight incubations, obtains seed liquor;
(5) 5mL seed liquor is added in 100mL LB liquid medium, 30 DEG C are shaken to OD600Value is between 0.5~0.7;
(6) bacterium solution obtained by step (5) is immediately placed in mixture of ice and water cooling 20min;
(7) take bacterium solution obtained by step (6), 4 DEG C, 4500 × g centrifugation 10min remove supernatant;
(8) with pre-cooling ultrapure water cleaning step (7) bacterial sediment repeatedly on ice;
(9) ultrapure water is added into thallus obtained by step (8), bacterium solution is settled to 500 μ l, every 100 μ l of pipe is dispensed to pre-cooling
EP pipe obtains GS1783 electricity and turns competence;
(10) turn that 20ng pBAC-DPV plasmid is added in competence in 100 μ l GS1783 electricity, after mixing by competence and
The electric shock bottom of a cup portion of 2mm pre-cooling is added in plasmid together, shocks by electricity under the conditions of 15kV/cm;
(11) take 100 μ l LB liquid mediums that thallus after electric shock is resuspended, after 30 DEG C are shaken bacterium 1h, 4500 × g is centrifuged thallus
2min abandons supernatant, is suspended and is precipitated using 200 μ l LB liquid mediums, is coated on the LB solid medium containing chloramphenicol,
30 DEG C of cultures for 24 hours, obtain GS1783-pBAC-DPV bacterial strain.
2, I_SceI-Kana-CT target practice segment is expanded
(1) Escherichia coli of the recovery with pEPkan-S plasmid are in LB solid medium containing kanamycin, and 37 DEG C
Overnight incubation;It chooses single colonie to be inoculated in LB liquid medium containing kanamycin, 37 DEG C of overnight incubations are mentioned using plasmid is small
Kit extracts pEPkan-S plasmid (pEPkan-S plasmid map is shown in Fig. 1);
(2) using pEPkan-S plasmid as template, GS1783-BAC-gE Δ CT-F and GS1783-BAC-gE Δ CT-R is to draw
Object expands I_SceI-Kana-CT target practice segment, recycles amplified fragments using plain agar sugar gel DNA QIAquick Gel Extraction Kit;
GS1783-BAC-gEΔCT-F:5’-CGTGACTATTATAATCCTGGCTGTTTCATCCATCTTTTTAtaggg
Ataacagggtaatcgattt-3 ' (SEQ ID NO:1)
GS1783-BAC-gEΔCT-R:5’-CTATTTCACTAGTGAGTCATTAGTTCAACATCCATGATCATAAAA
AGATGGATGAAACAGCCAGGATTATAATAGTCACGgccagtgttacaaccaat-3 ' (SEQ ID NO:2)
PCR amplification system are as follows: ddH2O 22μl、25 μ l of Max DNA Polymerase, upstream are drawn
1 μ l of object GS1783-BAC-gE Δ CT-F, 1 μ l of downstream primer GS1783-BAC-gE Δ CT-R, 1 μ of template pEPkan-S plasmid
l;
PCR amplification condition are as follows: 98 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 72 DEG C of extension 5s, totally 30
A circulation, last 72 DEG C of extensions 10min are saved in 16 DEG C.
Base is carried out in bacterial artificial chromosome recombination duck plague virus rescue system platform using Red-Based modification technique
Because the operational flowchart of missing is shown in Fig. 2, detailed process includes 3 and 4 two following processes.
3, preparation GS1783-pBAC-DPV electricity turns competence and carries out target practice segment target practice
(1) recovery GS1783-pBAC-DPV freezes bacterium in the LB solid medium containing chloramphenicol, and 30 DEG C were cultivated
Night;
(2) picking GS1783-pBAC-DPV single colonie is inoculated in LB liquid medium of the 5mL containing chloramphenicol, 30 DEG C of trainings
It supports overnight, obtains seed liquor;
(3) 5mL seed liquor is added in LB liquid medium of the 100mL containing chloramphenicol, is placed in 30 DEG C and shakes to OD600Value exists
Between 0.5~0.7;
(4) bacterium solution obtained by step (3) is immediately placed in mixture of ice and water cooling 20min after 42 DEG C of culture 15min;
(5) bacterium solution obtained by 50mL step (4) is taken, 10min is centrifuged in 4 DEG C of 4500 × g, removes supernatant;
(6) with pre-cooling ultrapure water cleaning step (5) bacterial sediment repeatedly on ice;
(7) ultrapure water is added into thallus obtained by step (6), bacterium solution is settled to 500 μ l, every 100 μ l of pipe is dispensed to pre-cooling
EP pipe obtains GS1783-pBAC-DPV electricity and turns competence;
(8) turn that 200ng I_SceI-Kana-CT target practice segment is added in competence in 100 μ l electricity, by competence after mixing
The electric shock bottom of a cup portion of 2mm pre-cooling is added together with target practice segment, shocks by electricity under the conditions of 15kV/cm;
(9) it takes 100 μ l LB liquid mediums that thallus after electric shock is resuspended, after 30 DEG C are shaken bacterium 1h, is centrifuged thallus in 4500 × g
2min abandons supernatant, and 200 μ l LB liquid mediums, which suspend, to be precipitated, and is coated on containing kanamycin and chloramphenicol twin antibiotic resistance
LB solid medium, in 30 DEG C of culture 48h;
(10) single colonie obtained by PCR authentication step (9) obtains positive bacterium colony GS178-pBAC-DPV-CT-Kana, with step
Suddenly the single colonie re-suspension liquid grown in (9) is template, utilizes amplification I_SceI-Kana-CT target practice segment upstream primer GS1783-
BAC-gE Δ CT-F and identification gE gene C T region downstream primer gE-R identify positive bacterium colony, obtain positive clone molecule GS1783-
pBAC-DPV-gE-Kana;
GS1783-BAC-gEΔCT-F:5’-CGTGACTATTATAATCCTGGCTGTTTCATCCATCTTTTTAtaggg
Ataacagggtaatcgattt-3 ' (SEQ ID NO:1)
GE-R:5 '-AGCGAGTACTTCTCTGCGTC-3 ' (SEQ ID NO:3)
PCR amplification system are as follows: ddH2O 22μl、25 μ l of Max DNA Polymerase, upstream are drawn
1 μ l of object GS1783-BAC-gE Δ CT-F, 1 μ l of downstream primer gE-R, template are 1 μ l of step (9) single colonie re-suspension liquid;
PCR amplification condition are as follows: 98 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 72 DEG C of extension 5s, totally 30
A circulation, last 72 DEG C of extensions 10min are saved in 16 DEG C.
4, remove I_SceI-Kana segment
(1) picking GS1783-pBAC-DPV-gE-Kana single colonie is inoculated in LB liquid medium of the 2mL containing chloramphenicol
In, 30 DEG C of overnight incubations obtain seed liquor;
(2) seed liquor obtained by 10 μ l steps (1) is taken to be inoculated in LB liquid medium of the 2mL containing chloramphenicol, 30 DEG C of cultures
Slight cloud is presented to bacterium solution in 2h;
(3) LB liquid medium and 5M final concentration of 2% of the 1mL containing chloramphenicol is added into bacterium solution obtained by step (2)
L-arabinose, 30 DEG C of culture 1h;
(4) bacterium solution obtained by step (3) is immediately placed in 42 DEG C of water-baths and cultivates 30min;
(5) after bacterium solution obtained by step (4) being placed in 30 DEG C of culture 2h, take 1 μ l bacterium solution that 200 μ l LB liquid mediums are added
The LB solid medium containing chloramphenicol, 30 DEG C of culture for 24 hours~48h are applied to after middle mixing;
(6) gained single colonie is containing chloramphenicol and the Double LB solid medium of Ka Na mycin and chlorine in picking step (5)
Parllel screening is carried out on mycin monoclonal antibody LB solid medium, chloramphenicol and the Double LB solid medium of Ka Na mycin are not given birth to
Long, the bacterium colony of chloramphenicol monoclonal antibody LB solid culture basal growth identifies that primer is identified using the region gE gene C T by PCR method,
Obtain positive clone molecule GS1783-pBAC-DPV-gE Δ CT.
GE-F:5 '-TCTCAAGACGCTCTGGAATC-3 ' (SEQ ID NO:4)
GE-R:5 '-AGCGAGTACTTCTCTGCGTC-3 ' (SEQ ID NO:3)
PCR amplification system are as follows: ddH2O 22μl、25 μ l of Max DNA Polymerase, upstream are drawn
1 μ l of object gE-F, 1 μ l of downstream primer gE-R, template are 1 μ l of step (6) single colonie re-suspension liquid;
PCR amplification condition are as follows: 98 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 72 DEG C of extension 5s, totally 30
A circulation, last 72 DEG C of extensions 10min are saved in 16 DEG C.
5, Revive virus
(1) recovery GS1783-pBAC-DPV-gE Δ CT freezes bacterium in the LB solid medium containing chloramphenicol, and 30 DEG C
Overnight incubation;
(2) pBAC-DPV-gE Δ CT plasmid is extracted according to QIAGEN Plasmid Midi Kit operating instruction;
(3) it prepares duck embryo fibroblasts (DEF) and is inoculated in 12 orifice plates, 37 DEG C, 5%CO2After culture for 24 hours, according to
3000 operating instruction of Lipofectamine transfects pBAC-DPV-gE Δ CT plasmid, and fluorescent spot is observed after 96h, collects virus, instead
Multiple freeze thawing is inoculated in the 6 orifice plates for covering with DEF afterwards three times, and fluorescent spot is observed after 96h, obtains the seamless gene-deleted strain in the region gE gene C T
CHv-BAC-G-gE Δ CT (see Fig. 3).
The measurement of the seamless gene-deleted strain CHv-BAC-G-gE Δ CT growth curve of embodiment 2
1, the measurement of one step growth curve
By the parental virus DPV CHv-BAC-G and gE gene C T seamless gene-deleted strain CHv-BAC-G-gE Δ CT in region respectively with
2MOI is inoculated with DEF cell, meets 6h, 12h, 18h, for 24 hours collection supernatant cell after poison, each time point is done to be repeated three times.It is due-in
After collection is complete, multigelation 2 times, virus titer is detected in 96 orifice plates.The result shows that the missing in the region gE gene C T will not be significant
Influence the duplication of DPV CHv virus.
2, the measurement of multistep growth curve
By the parental virus DPV CHv-BAC-G and gE gene C T seamless gene-deleted strain CHv-BAC-G-gE Δ CT in region respectively with
0.01MOI is inoculated with DEF cell, connect 12h after poison, for 24 hours, 48h, 72h collect supernatant cell, each time point is done to be repeated three times.
After collecting completely, multigelation 2 times, virus titer is detected in 96 orifice plates.The result shows that the missing in the region gE gene C T will not
Significantly affect the proliferative conditions of DPV CHv virus.
The experiment of the seamless gene-deleted strain CHv-BAC-G-gE Δ CT plaque test of embodiment 3
By the parental virus DPV CHv-BAC-G and gE gene C T seamless gene-deleted strain CHv-BAC-G-gE Δ CT in region respectively with
0.01MOI is inoculated with the 6 orifice plates for being covered with DEF cell, 37 DEG C, 5%CO2After adsorbing 2h, supernatant is removed, 2mL1% Methyl cellulose is added
Element, 37 DEG C, 5%CO2After cultivating 48h, 1% methylcellulose is removed, PBS is washed 3 times, and 4% 4 DEG C of paraformaldehyde fixation is stayed overnight, PBS
H is added in washing 3 times2O2The mixed liquor mixed with methanol with volume ratio for 1:50 is incubated at room temperature 30min, distills water washing 3 times,
5%BSA confining liquid is added, is incubated at room temperature 30min, rabbit-anti DPV, 4 DEG C of overnight incubations is added, PBS is washed 3 times, and biotin is added
Change goat anti-rabbit igg, 37 DEG C of incubations 30min, PBS are washed 3 times, be added dropwise SABC substrate, 37 DEG C incubation 30min, PBS washing 3 times,
DAB developing solution is protected from light colour developing, mounting.Plaque test experimental result is as shown in Figure 4.The result shows that the missing in the region gE gene C T can
DPV CHv virus can be influenced to be proliferated in the cell and propagation condition.
In addition, the present invention has also done heredity to the duck plague virus gE gene C T seamless gene-deleted strain CHv-BAC-G-gE Δ CT in region
Stability experiment.
Genetic stability: the duck plague virus gE gene C T seamless gene-deleted strain CHv-BAC-G-gE Δ CT in region is on DEF cell
It passed on for 20 generations, plaque occurs, show the seamless gene-deleted strain CHv-BAC-G-gE Δ in the region duck plague virus gE gene C T obtained
CT stablizes heredity in DEF.
Sequence table
<110>Sichuan Agricultural University
<120>the seamless gene-deleted strain CHv-BAC-G-gE CT in the region duck plague virus gE gene C T and its construction method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 64
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cgtgactatt ataatcctgg ctgtttcatc catcttttta tagggataac agggtaatcg 60
attt 64
<210> 2
<211> 98
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ctatttcact agtgagtcat tagttcaaca tccatgatca taaaaagatg gatgaaacag 60
ccaggattat aatagtcacg gccagtgtta caaccaat 98
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agcgagtact tctctgcgtc 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tctcaagacg ctctggaatc 20
Claims (5)
1. the construction method of the duck plague virus gE gene C T seamless gene-deleted strain CHv-BAC-G-gE Δ CT in region, which is characterized in that packet
Include following steps:
(1) pBAC-DPV plasmid is transformed into GS1783 E. coli competent, obtains GS1783-pBAC-DPV bacterial strain, so
After prepare GS1783-pBAC-DPV competence;
(2) using pEPkan-S as template, using GS1783-BAC-gE Δ CT-F and GS1783-BAC-gE Δ CT-R as primer, lead to
Cross base fragment and gE gene C T region upstream and the downstream that PCR method amplification includes I_SceI restriction enzyme site and Kana element
The target practice segment I_SceI-Kana-CT of each 40bp homology arm, gel extraction obtain I_SceI-Kana-CT segment;
(3) I_SceI-Kana-CT segment is transformed into GS1783-pBAC-DPV competence, is screened, obtain positive clone molecule
GS1783-pBAC-DPV-CT-Kana;
(4) the I_SceI-Kana segment in positive clone molecule GS1783-pBAC-DPV-CT-Kana is removed, is sieved through antibiotic
Choosing and PCR identification, obtain positive clone molecule GS1783-pBAC-DPV-gE Δ CT;
(5) pBAC-DPV-gE Δ CT plasmid is extracted from positive clone molecule GS1783-pBAC-DPV-gE Δ CT, by pBAC-
DPV-gE Δ CT plasmid transfection DEF cell obtains the seamless gene-deleted strain CHv-BAC-G-gE in the region gE gene C T by colony screening
ΔCT。
2. the building of the seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T according to claim 1
Method, which is characterized in that primer sequence in step (2) are as follows:
GS1783-BAC-gEΔCT-F:5’-CGTGACTATTATAATCCTGGCTGTTTCATCCATCTTTTTAtagggataa
cagggtaatcgattt-3';
GS1783-BAC-gEΔCT-R:5’-CTATTTCACTAGTGAGTCATTAGTTCAACATCCATGATCATAAAAAGAT
GGATGAAACAGCCAGGATTATAATAGTCACGgccagtgttacaaccaat-3’。
3. the building of the seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T according to claim 1
Method, which is characterized in that PCR amplification system in step (2) are as follows: ddH2O 22μl、Max DNA
25 μ l of Polymerase, 1 μ l of upstream primer, 1 μ l of downstream primer, 1 μ l of template.
4. the building of the seamless gene-deleted strain CHv-BAC-G-gE Δ CT in the region duck plague virus gE gene C T according to claim 1
Method, which is characterized in that PCR amplification condition in step (2) are as follows: 98 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10s, 55 DEG C of annealing
15s, 72 DEG C of extension 5s, totally 30 recycle, last 72 DEG C of extensions 10min.
5. the seamless gene-deleted strain in the duck plague virus region gE gene C T constructed using the described in any item methods of claim 1-4
CHv-BAC-G-gEΔCT。
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