CN109593731A - The seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene and its construction method - Google Patents

The seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene and its construction method Download PDF

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CN109593731A
CN109593731A CN201811597323.8A CN201811597323A CN109593731A CN 109593731 A CN109593731 A CN 109593731A CN 201811597323 A CN201811597323 A CN 201811597323A CN 109593731 A CN109593731 A CN 109593731A
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gene
bac
dpv
pbac
duck plague
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汪铭书
刘田
程安春
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Sichuan Agricultural University
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Abstract

The present invention provides a kind of seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene and its construction methods.The present invention utilizes GS1783 Escherichia coli and pEPkan-S plasmid, duck plague virus gI gene 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 of duck plague virus gI gene 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

The seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene and its construction method
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of seamless gene-deleted strain CHv- of duck plague virus gI gene BAC-G- Δ gI 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 the duck caused by duck plague virus in Alphaherpesviridae (Duck Plague virus, DPV), The acute contact height lethal infectious diseases of the aquatic birds such as goose.The disease first by Holland report, immediately China south China, Central China and The more flourishing area of the duck culturing industries such as East China is popular, causes serious economic loss to the duck culturing industry in China.Therefore deeply Solution duck plague virus gene function is reinforced to the research of duck plague epidemic disease to ensuring that China's duck culturing industry health, sustainable development are especially heavy It wants.
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, the present invention provides a kind of seamless gene-deleted strain of duck plague virus gI gene CHv-BAC-G- Δ gI and its construction method, the construction method can effectively solve residual in deletion segment when missing duck plague virus gene The problem of staying 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- Δ gI of duck plague virus gI gene, construction method the following steps are included:
(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- Δ gI-F and GS1783-BAC- Δ gI-R as primer, lead to Cross the base fragment and gI upstream region of gene and each 40bp in downstream that PCR method amplification includes I_SceI restriction enzyme site and Kana element The target practice segment I_SceI-Kana-gI of homology arm, gel extraction obtain I_SceI-Kana-gI segment；
(3) I_SceI-Kana-gI segment is transformed into GS1783-pBAC-DPV competence, is screened, obtain positive gram Longzi GS1783-pBAC-DPV-gI-Kana；
(4) the I_SceI-Kana segment in positive clone molecule GS1783-pBAC-DPV-gI-Kana is removed, through antibiosis Element screening and PCR identification, obtain positive clone molecule GS1783-pBAC-DPV- Δ gI；
(5) pBAC-DPV- Δ gI plasmid is extracted from positive clone molecule GS1783-pBAC-DPV- Δ gI, by pBAC- DPV- Δ gI plasmid transfection DEF cell obtains the seamless gene-deleted strain CHv-BAC-G- Δ gI of gI gene by colony screening.
Further, primer sequence in step (2) are as follows:
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggat aacagggtaatcgattt-3'；
GS1783-BAC-ΔgI-R:5’-TCATAACAAAAACATTTACTTTTAGTCATACTGATGTGAATTCTATT TTTGAAACTCAATATATCGTCTATATGGCGCACgccagtgttacaaccaat-3’。
Further, 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；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- Δ gI of duck plague virus gI gene provided by the invention and its construction method, have with It is lower the utility model 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 gI base through homologous recombination in bacterial artificial chromosome recombination duck plague virus rescue system platform Cause completes the building of the seamless gene-deleted strain of no remaining duck plague virus of external source base for the first time.Technical solution of the present invention solves scarce When losing duck plague virus gene the problem of deletion segment remains base, accurately to probe into duck plague virus gene function and attenuated live epidemic disease The building of seedling 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- Δ gI.
Fig. 4 is the one step growth curve figure and multistep growth curve chart of the seamless deleted virus strain of CHv-BAC-G- Δ gI；Its Middle A is one step growth curve, and B is multistep growth curve.
Fig. 5 is the seamless deleted virus strain plaque test picture of CHv-BAC-G- Δ gI.
Specific embodiment
The seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene, building process material therefor and reagent are 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- Δ gI of duck plague virus gI gene
The seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene, construction method the following steps are included:
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-gI 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- Δ gI-F and GS1783-BAC- Δ gI-R is primer, is expanded Increase I_SceI-Kana-gI target practice segment, amplified fragments are recycled using plain agar sugar gel DNA QIAquick Gel Extraction Kit；
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggat Aacagggtaatcgattt-3 ' (SEQ ID NO:1)
GS1783-BAC-ΔgI-R:5’-TCATAACAAAAACATTTACTTTTAGTCATACTGATGTGAATTCTATT TTTGAAACTCAATATATCGTCTATATGGCGCACgccagtgttacaaccaat-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- Δ gI-F, 1 μ l of downstream primer GS1783-BAC- Δ gI-R, 1 μ l of template pEPkan-S plasmid；
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-gI 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-gI-Kana, with step Suddenly the single colonie re-suspension liquid grown in (9) is template, utilizes amplification I_SceI-Kana-gI target practice segment upstream primer GS1783- BAC- Δ gI-F and identification gI downstream of gene primer gI-R identify positive bacterium colony, obtain positive clone molecule GS1783-pBAC-DPV- gI-Kana；
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggat Aacagggtaatcgattt-3 ' (SEQ ID NO:1)
GI-R:5 '-GACCGGTAGTTCCAATCACT-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- Δ gI-F, 1 μ l of downstream primer gI-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-gI-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 is identified by PCR method using gI identified for genes primer, obtains sun Property clone GS1783-pBAC-DPV- Δ gI.
GI-F:5 '-TGTGGGTGGGTCATCTACAT-3 ' (SEQ ID NO:4)
GI-R:5 '-GACCGGTAGTTCCAATCACT-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 gI-F, 1 μ l of downstream primer gI-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- Δ gI freezes bacterium in the LB solid medium containing chloramphenicol, 30 DEG C of trainings It supports overnight；
(2) pBAC-DPV- Δ gI 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- Δ gI plasmid, and fluorescent spot is observed after 96h, collects virus, repeatedly 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 CHv-BAC-G- of gI gene Δ gI (see Fig. 3).
The measurement of the seamless gene-deleted strain CHv-BAC-G- Δ gI growth curve of embodiment 2
1, the measurement of one step growth curve
The seamless gene-deleted strain CHv-BAC-G- Δ gI of parental virus DPV CHv-BAC-G and gI gene is inoculated with respectively with 2MOI DEF cell meets 6h, 12h, 18h, for 24 hours collection supernatant cell after poison, and each time point is done to be repeated three times.After collecting completely, Multigelation 2 times, virus titer is detected in 96 orifice plates, one step growth curve is as shown in Figure 4 A.The result shows that the missing of gI gene It is not significantly affected by the duplication of DPV CHv virus.
2, the measurement of multistep growth curve
By the seamless gene-deleted strain CHv-BAC-G- Δ gI of parental virus DPV CHv-BAC-G and gI gene respectively with 0.01MOI Be 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.Wait collect Quan Hou multigelation 2 times, detects virus titer in 96 orifice plates, multistep growth curve is as shown in Figure 4 B.The result shows that gI gene Missing is not significantly affected by the proliferative conditions of DPV CHv virus.
The experiment of the seamless gene-deleted strain CHv-BAC-G- Δ gI plaque test of embodiment 3
By the seamless gene-deleted strain CHv-BAC-G- Δ gI of parental virus DPV CHv-BAC-G and gI gene respectively with 0.01MOI Inoculation is covered with the 6 orifice plates of DEF cell, 37 DEG C, 5%CO2After adsorbing 2h, supernatant is removed, be added 2mL1% methylcellulose, 37 DEG C, 5%CO2After cultivating 48h, 1% methylcellulose is removed, PBS is washed 3 times, and overnight, PBS is washed 3 times for 4% 4 DEG C of paraformaldehyde fixation, H is added2O2The mixed liquor mixed with methanol with volume ratio for 1:50 is incubated at room temperature 30min, distills water washing 3 times, is added 5% BSA confining liquid is incubated at room temperature 30min, and rabbit-anti DPV, 4 DEG C of overnight incubations is added, and PBS is washed 3 times, and it is anti-that biotinylated goat is added Rabbit igg, 37 DEG C of incubation 30min, PBS are washed 3 times, are added dropwise SABC substrate, and 37 DEG C of incubations 30min, PBS are washed 3 times, and DAB develops the color Liquid is protected from light colour developing, mounting.Plaque test experimental result is as shown in Figure 5.The result shows that the missing of gI gene may influence DPV CHv Virus is proliferated in the cell and propagation condition.
In addition, present invention gene-deleted strain CHv-BAC-G- Δ gI also seamless to duck plague virus gI gene has done genetic stability reality It tests.
Genetic stability: the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene passed on for 20 generations on DEF cell, There is plaque, shows that the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene obtained stablizes heredity in DEF.
Sequence table
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<120>the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene and its construction method
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gtgcgccata tagacgatat attgagtttc aaaaatagaa tagggataac agggtaatcg 60
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tcataacaaa aacatttact tttagtcata ctgatgtgaa ttctattttt gaaactcaat 60
atatcgtcta tatggcgcac gccagtgtta caaccaat 98
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tgtgggtggg tcatctacat 20

Claims (5)

1. the construction method of the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene, which is characterized in that including following step It is rapid:
(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- Δ gI-F and GS1783-BAC- Δ gI-R as primer, pass through Base fragment and gI upstream region of gene and downstream each 40bp of the PCR method amplification comprising I_SceI restriction enzyme site and Kana element are same The target practice segment I_SceI-Kana-gI of source arm, gel extraction obtain I_SceI-Kana-gI segment；
(3) I_SceI-Kana-gI segment is transformed into GS1783-pBAC-DPV competence, is screened, obtain positive clone molecule GS1783-pBAC-DPV-gI-Kana；
(4) the I_SceI-Kana segment in positive clone molecule GS1783-pBAC-DPV-gI-Kana is removed, is sieved through antibiotic Choosing and PCR identification, obtain positive clone molecule GS1783-pBAC-DPV- Δ gI；
(5) pBAC-DPV- Δ gI plasmid is extracted from positive clone molecule GS1783-pBAC-DPV- Δ gI, by pBAC-DPV- Δ GI plasmid transfection DEF cell obtains the seamless gene-deleted strain CHv-BAC-G- Δ gI of gI gene by colony screening.
2. the construction method of the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene according to claim 1, It is characterized in that, primer sequence in step (2) are as follows:
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggataaca gggtaatcgattt-3'；
GS1783-BAC-ΔgI-R:5’-TCATAACAAAAACATTTACTTTTAGTCATACTGATGTGAATTCTATTTTTG AAACTCAATATATCGTCTATATGGCGCACgccagtgttacaaccaat-3’。
3. the construction method of the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene according to claim 1, It is characterized in that, PCR amplification system in step (2) are as follows: ddH2O 22μl、Max DNA Polymerase 25 μ l, 1 μ l of upstream primer, 1 μ l of downstream primer, 1 μ l of template.
4. the construction method of the seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene according to claim 1, Be 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 prolong 5s is stretched, totally 30 circulations, last 72 DEG C of extensions 10min.
5. the seamless gene-deleted strain CHv- of duck plague virus gI gene constructed using the described in any item methods of claim 1-4 BAC-G-ΔgI。
CN201811597323.8A 2018-12-26 2018-12-26 The seamless gene-deleted strain CHv-BAC-G- Δ gI of duck plague virus gI gene and its construction method Pending CN109593731A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174091A (en) * 2011-01-25 2011-09-07 四川农业大学 Truncated and expressed duck plague virus (DPV) recombinant envelope gI protein and preparation method and application thereof
CN102174104A (en) * 2011-01-25 2011-09-07 四川农业大学 Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof
CN102732545A (en) * 2012-05-08 2012-10-17 四川农业大学 Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP
CN104480142A (en) * 2014-12-16 2015-04-01 青岛蔚蓝生物股份有限公司 Duck plague virus gene deletion transfer vector and application thereof
CN105385663A (en) * 2015-11-26 2016-03-09 中国兽医药品监察所 Establishment of duck enteritis virus gE and gI double gene deletion virus strain and application of establishment
CN105802922A (en) * 2016-04-19 2016-07-27 四川农业大学 Establishing method of bacterial artificial chromosome recombinant duck plague virus rescue system platform and application

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174091A (en) * 2011-01-25 2011-09-07 四川农业大学 Truncated and expressed duck plague virus (DPV) recombinant envelope gI protein and preparation method and application thereof
CN102174104A (en) * 2011-01-25 2011-09-07 四川农业大学 Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof
CN102732545A (en) * 2012-05-08 2012-10-17 四川农业大学 Duck plague virus transfer vector pUC-deltagC-EGFP, and gC-deleted recombinant strain DPV-deltagC-EGFP
CN104480142A (en) * 2014-12-16 2015-04-01 青岛蔚蓝生物股份有限公司 Duck plague virus gene deletion transfer vector and application thereof
CN105385663A (en) * 2015-11-26 2016-03-09 中国兽医药品监察所 Establishment of duck enteritis virus gE and gI double gene deletion virus strain and application of establishment
CN105802922A (en) * 2016-04-19 2016-07-27 四川农业大学 Establishing method of bacterial artificial chromosome recombinant duck plague virus rescue system platform and application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
B. KARSTEN TISCHER等: "En Passant Mutagenesis: A Two Step Markerless Red Recombination System", 《IN VITRO MUTAGENESIS PROTOCOLS: THIRD EDITION, METHODS IN MOLECULAR BIOLOGY》 *
TIAN LIU等: "Duck plague virus gE serves essential functions during the virion final envelopment through influence capsids budding into the cytoplasmic vesicles", 《SCIENTIFIC REPORTS》 *
陈柳 等: "DEV囊膜糖蛋白gI基因缺失病毒生物学特性研究", 《浙江畜牧兽医》 *

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Application publication date: 20190409