CN109536463B - Duck plague virus gE and gI double-gene traceless deleted strain DPV CHv-delta gE + delta gI and construction method thereof - Google Patents
Duck plague virus gE and gI double-gene traceless deleted strain DPV CHv-delta gE + delta gI and construction method thereof Download PDFInfo
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Abstract
The invention provides a duck plague virus gE and gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI and a construction method thereof. The invention utilizes GS1783 escherichia coli strain and pEPkan-S plasmid to delete the gE gene and the gI gene of the duck plague virus through two homologous recombinations on a bacterial artificial chromosome recombinant duck plague virus rescue system platform, and deletes the MiniF element through an intracellular spontaneous homologous recombination method, thereby completing the construction of the double-gene traceless deleted strain of the duck plague virus without exogenous base and MiniF element residue for the first time. The technical scheme of the invention solves the problem of residual basic groups at the deleted sites when the duck plague virus gene is deleted, and deletes the MiniF element, thereby providing sufficient technical support for accurately researching the functions of the duck plague virus gene and constructing attenuated live vaccines.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a duck plague virus gE and gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI and a construction method thereof.
Background
Bacterial Artificial Chromosome (BAC) is a newly developed DNA vector system, which has the advantages of large capacity, stable genetic characteristics, easy operation, and the like, and has wide applications in gene library construction, gene function analysis, and the like. The complete virus genome DNA molecule is inserted into a BAC vector, a molecular cloned virus is obtained by utilizing a Minimal fertility factor replicon (Mini-F) coded by the vector, and the deletion of a virus gene and the insertion of an exogenous gene in a prokaryotic system are realized by combining a mature gene positioning modification technology in escherichia coli. The current common Escherichia coli gene localization modification technologies mainly comprise a Red/ET mediated homologous recombination technology, a RecA protein mediated homologous recombination technology, a Cre/loxP mediated homologous recombination technology and a Tn transposon mediated random insertion and mutation technology. Mature bacterial artificial chromosome duck plague virus rescue system platform is obtained by utilizing molecular cloning technical means, and simultaneously, duck plague virus gene deletion and foreign gene insertion can be carried out on the bacterial artificial chromosome duck plague virus rescue system platform by utilizing escherichia coli gene localization modification Red/ET mediated homologous recombination technology, so that the result greatly promotes the research process of the duck plague virus gene function.
The Red/ET mediated homologous recombination technology is based on lambda-phage Red operon (Red alpha/Red beta/Red gamma) and Rac phage RecE/RecT homologous recombination enzyme homologous recombination targeting technology. The technology is simple, rapid and efficient to operate, and is widely applied to gene deletion and mutation work. However, when the operation technique is used for gene deletion and mutation, about 80bp of exogenous base sequence (FRT site) remains at the deletion or mutation site, and the residue of the site affects the accurate analysis of gene function. The MiniF element, as a minimal fertility factor replicon that maintains replication of BAC vectors, is mainly composed of repE, repF gene that regulates BAC origin of replication (oriS), sopA, sopB gene that regulate replicon distribution, and sopC gene that encodes centromeric region. In order to complete the bacterial resistance screening and BAC marker screening, a resistance screening gene and a fluorescent marker gene are added into the MiniF element. The MiniF element is inserted into the viral genome, increasing the length of the genome and producing an uncertain effect on viral replication. Meanwhile, the MiniF element as a bacterial sequence remains on the viral genome, which is not beneficial to the development and permission of attenuated live vaccines. Therefore, solving the base residue problem and removing the MiniF element to obtain a traceless gene deletion strain become the key point for researching the duck plague virus gene deletion method.
Duck Plague (Duck virus, DP) is an acute, highly lethal infectious disease of waterfowl such as ducks, geese and the like caused by Duck Plague Virus (DPV) in the sub-family of α -herpesvirus. The disease is firstly reported by the Netherlands, is immediately popular in areas with developed duck breeding industries such as south China, China and east China, and causes serious economic loss to the duck breeding industry in China. Therefore, the deep understanding of the gene function of the duck plague virus and the strengthening of the research on the duck plague are particularly important for ensuring the healthy and sustainable development of the duck breeding industry in China.
The total length of the genome DNA of the duck plague virus DPV-CHv strain is 162175bp, the genome DNA comprises 78 open reading frames and can encode structural proteins and non-structural proteins participating in the life cycle of the duck plague virus, wherein the structural proteins mainly comprise capsid proteins, cortical proteins and envelope proteins. The envelope protein is glycosylated protein, and comprises twelve gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM and gN. The glycoprotein has the functions of mediating virus adsorption, entering sensitive cells and promoting virus to spread among the cells, and simultaneously carries antigenic determinants, so that the identification of an immune system of an animal body on the virus can be induced, and the pathological damage of tissues can be caused, therefore, the exploration of the function of the envelope glycoprotein in the life cycle of the duck plague virus is of great importance for the deep exploration of the gene function of the duck plague virus and the development of the prevention and treatment of the duck plague epidemic disease.
In the prior art, a technique of cloning a virus by using BAC as a platform molecule is utilized to recombine a duck plague virus genome into a virus transfer vector containing BAC, and a bacterial artificial chromosome recombinant duck plague virus rescue system platform DPV CHv-BAC-G is constructed. Meanwhile, Red/ET modification technology is combined, and duck plague virus gene deletion and foreign gene insertion are completed in a prokaryotic system by a mature gene operation means. However, after the duck plague virus gene is deleted on a bacterial artificial chromosome recombinant duck plague virus rescue system platform by utilizing the Red/ET modification technology, two FRT sites are remained at the deleted gene, and a MiniF element is remained at the same time. The FRT exogenous site and the residual MiniF element have influence on the exploration of gene function and the development and permission of attenuated live vaccines.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a duck plague virus gE and gI double-gene traceless deleted strain DPV CHv-delta gE + delta gI and a construction method thereof, and the construction method can effectively solve the problems that basic groups are remained at deleted sites and MiniF elements are remained when the duck plague virus genes are deleted.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a double-gene traceless deletion strain of duck plague virus gE and gI is Mardivrus belonging to Mardivirus, is named as a double-deletion virus strain DPV CHv-delta gE + delta gI of duck plague virus gE and gI, is preserved in a China center for type culture collection (address: Wuhan university in Wuhan district, Wuhan city, Hubei province) in 2018, and has a preservation number of CCTCC NO: and V201841.
The construction method of the double-gene traceless deletion strain DPV CHv-delta gE + delta gI (namely the double-deletion strain DPV CHv-delta gE + delta gI of the duck plague virus gE and gI) comprises the following steps:
(1) transforming the pBAC-DPV plasmid into GS1783 escherichia coli competence to obtain a GS1783-pBAC-DPV strain, and then preparing the GS1783-pBAC-DPV competence;
(2) using pEPkan-S as a template, using GS 1783-BAC-delta gE-F and GS 1783-BAC-delta gE-R as primers, amplifying a base fragment containing an I _ SceI enzyme cutting site and a Kana element and a targeting fragment I _ SceI-Kana-gE of each 40bp homologous arm at the upstream and downstream of the gE gene by a PCR method, cutting glue and recovering to obtain an I _ SceI-Kana-gE fragment;
(3) transforming the I _ SceI-Kana-gE fragment into GS1783-pBAC-DPV competence, and screening to obtain a positive clone GS 1783-pBAC-DPV-gE-Kana;
(4) removing the I _ SceI-Kana fragment in the positive clone GS1783-pBAC-DPV-gE-Kana to prepare GS 1783-pBAC-DPV-delta gE competence;
(5) using pEPkan-S as a template, using GS 1783-BAC-delta gI-F and GS 1783-BAC-delta gI-R as primers, amplifying a base fragment containing an I _ SceI enzyme cutting site and a Kana element and a targeting fragment I _ SceI-Kana-gI of each 40bp homology arm at the upstream and downstream of a gI gene by a PCR method, cutting glue and recycling to obtain an I _ SceI-Kana-gI fragment;
(6) converting the I _ SceI-Kana-gI fragment into GS 1783-pBAC-DPV-delta gE competence, and obtaining a positive clone GS 1783-pBAC-DPV-delta gE-gI-Kana through antibiotic screening and PCR identification;
(7) removing the I _ SceI-Kana fragment in the positive clone GS 1783-pBAC-DPV-delta gE-gI-Kana to prepare GS 1783-pBAC-DPV-delta gE + delta gI competence;
(8) using pEPkan-S as a template, using GS1783-MiniF-F and GS1783-MiniF-R as primers, amplifying a base fragment containing an I _ SceI enzyme cutting site and a Kana element, a homologous arm fragment I _ SceI-Kana-MiniF located at 240bp downstream of a MiniF element ori2 gene and at 290bp downstream of a MiniF element ori2 gene by a PCR method, cutting glue and recovering to obtain an I _ SceI-Kana-MiniF fragment;
(9) using a CHv genome as a template, using CHv-UL23-F and CHv-UL23-R as primers, amplifying a homologous arm fragment which comprises a UL23 gene, an I _ SceI-Kana-MiniF downstream homologous arm overlapping 25bp and is positioned at 180bp downstream of a MiniF element ori2 gene by a PCR method, cutting glue and recovering to obtain a UL23-MiniF fragment;
(10) carrying out PCR fusion reaction by taking the I _ SceI-Kana-MiniF fragment and the UL23-MiniF fragment as templates, and then carrying out PCR amplification by taking the fusion fragment as a template and GS1783-MiniF-F and CHv-UL23-R as primers to obtain an I _ SceI-Kana-MiniF-UL23 targeting fragment;
(11) converting the I _ SceI-Kana-MiniF-UL23 targeting fragment into GS 1783-pBAC-DPV-delta gE + delta gI competence, and obtaining a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana through antibiotic screening and PCR identification;
(12) removing the I _ SceI-Kana fragment in the positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana to obtain a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL 23;
(13) extracting pBAC-DPV-delta gE + delta gI-UL23 plasmid from a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23, transfecting DEF cells with the pBAC-DPV-delta gE + delta gI-UL23 plasmid, and obtaining a gE and gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI by clone screening.
Further, the PCR amplification system in the step (2), the step (5), the step (8) and the step (9) is as follows: ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, template 1. mu.l; the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10 min.
Further, the primer sequence in the step (2) is as follows:
GS1783-BAC-ΔgE-F:5’-ATACTGCCGGCCAGACTACGGAACCTCAACAATTGGTACGtagggataacagggtaatcgattt-3’;
GS1783-BAC-ΔgE-R:5’-TAACTATTTCACTAGTGAGTCATTAGTTCAACATCCATGACGTACCAATTGTTGAGGTTCCGTAGTCTGGCCGGCAGTATgccagtgttacaaccaat-3’。
further, the primer sequence in step (5) is:
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggataacagggtaatcgattt-3’;
GS1783-BAC-ΔgI-R:5’-TCATAACAAAAACATTTACTTTTAGTCATACTGATGTGAATTCTATTTTTGAAACTCAATATATCGTCTATATGGCGCACgccagtgttacaaccaat-3’。
further, the primer sequence in step (8) is:
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’;
GS1783-MiniF-R:5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATtagggataacagggtaatcgat-3’。
further, the primer sequence in step (9) is:
CHv-UL23-F:5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’;
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’。
further, the primer sequence in step (10) is:
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’;
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’。
further, in step (10), the PCR fusion system is: ddH2O 8μl、Max DNA Polymerase 10. mu.l, template I _ SceI-Kana-MiniF fragment and UL23-MiniF fragment 1. mu.l each; the PCR fusion conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 1min for 5 cycles.
Further, the PCR amplification system in step (10) is: 20 UL of fusion template, 0.5 UL of upstream primer GS1783-MiniF-F and 0.5 UL of downstream primer CHv-UL 23-R; the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10 min.
The duck plague virus gE and gI double-gene traceless deleted strain DPV CHv-delta gE + delta gI and the construction method thereof have the following beneficial effects:
in order to obtain the duck plague virus gene deletion strain without the residual exogenous base and MiniF element, the invention firstly completes the construction of the duck plague virus double-gene traceless deletion strain without the residual exogenous base and MiniF element by utilizing a Red-based modification technology on the basis of a bacterial artificial chromosome recombinant duck plague virus rescue system platform, namely, utilizing a GS1783 escherichia coli strain containing a gene sequence capable of coding a Red operon and an I _ SceI and a plasmid pEPkan-S containing coding a Carna resistance and an I _ SceI enzyme cutting site, and carrying out homologous recombination twice on the bacterial artificial chromosome recombinant duck plague virus rescue system platform to delete the gE gene and the gI gene of the duck plague virus, and deleting the MiniF element by an intracellular spontaneous homologous recombination method. The technical scheme of the invention solves the problem of residual basic groups at the deleted sites when the duck plague virus gene is deleted, and deletes the MiniF element, thereby providing sufficient technical support for accurately researching the functions of the duck plague virus gene and constructing attenuated live vaccines.
Drawings
FIG. 1 is a map of pEPkan-S plasmid.
FIG. 2 is a flow chart of the operation of gene deletion on bacterial artificial chromosome recombinant duck plague virus rescue system platform by using Red-Based modification technology (taking gE gene deletion as an example).
FIG. 3 is a flow chart of the operation of deleting the MiniF element using the Red-Based modification technique and the intracellular spontaneous homologous recombination technique.
FIG. 4 is a picture of DPV CHv- Δ gE + Δ gI traceless deleted virus strain after virus rescue.
FIG. 5 shows PCR detection of liver DNA extracts 48h after DPV CHv- Δ gE + Δ gI inoculation of ducks.
FIG. 6 shows the missing virus content in blood, spleen and liver obtained at different time points after DPV CHv- Δ gE + Δ gI vaccination of ducks.
Detailed Description
The duck plague virus gE and gI double-gene traceless deleted strain is named as duck plague virus gE and gI double-deleted virus strain DPV CHv-delta gE + delta gI, and materials and reagents used in the construction process are as follows:
1. experimental Material
(1) Cell, strain, virus strain, plasmid
Preparing primary duck embryo fibroblasts from 10-11 day-old non-immune fertilized duck embryos by a conventional method; the GS1783 strain was stored in the Sichuan university of agriculture laboratory; the pBAC-DPV plasmid is constructed and stored in Sichuan university of agriculture laboratories; the pEPkan-S plasmid was stored by Sichuan university of agriculture laboratories.
2. Molecular biological reagent
The plasmid miniextract kit is purchased from TIANGEN company; QIAGEN Plasmid Midi Kit from QIAGEN; the common agarose gel DNA recovery kit was purchased from TIANGEN;max DNA Polymerase was purchased from Takara; TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 from TaKaRa; lipofectamine 3000 was purchased from Invitrogen; ready-to-use SABC immunohistochemical staining kit (rabbit IgG) was purchased from bosd; DAB color development kit (yellow) was purchased from Boshide.
3. Solutions for experiments and their preparation
LB liquid medium: weighing 10g of Tryptone, 5g of Yeast Extract and 10g of sodium chloride, dissolving in 800mL of deionized water, fully stirring, fixing the volume to lL, and sterilizing at high temperature and high pressure.
LB solid medium: adding 15g of agar powder into LB liquid culture medium with constant volume of 1L, sterilizing at high temperature and high pressure, cooling to about 60 ℃, adding 1.5mL of chloramphenicol (storage concentration 25mg/mL) or 1.5mL of kanamycin (storage concentration 50mg/mL), spreading, solidifying, and storing at 4 ℃.
MEM: dissolving 9.6g MEM dry powder and 2.2g sodium bicarbonate in 800mL deionized water, stirring thoroughly, adjusting pH to 7.4, diluting to a volume of lL, filtering for sterilization, and storing at 4 deg.C.
Example 1 preparation of DPV CHv-delta gE + delta gI double-gene traceless deletion strain
The construction method of the duck plague virus gE and gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI comprises the following steps:
1. preparation of GS1783 electroporation competent, electroporation pBAC-DPV plasmid
(1) Resuscitating the Escherichia coli with pBAC-DPV plasmid in LB solid medium containing chloramphenicol, culturing overnight at 37 ℃; selecting a single colony, inoculating the single colony in LB liquid culture medium containing chloramphenicol, and culturing at 37 ℃ overnight;
(2) extracting pBAC-DPV Plasmid according to the operation instruction of QIAGEN Plasmid Midi Kit;
(3) recovering GS1783 frozen bacteria, and culturing at 30 deg.C overnight in LB solid culture medium;
(4) selecting a GS1783 single colony, inoculating the single colony in 5mL LB liquid culture medium, and culturing overnight at 30 ℃ to obtain a seed solution;
(5) 5mL of the seed solution was added to 100mL of LB liquid medium and shaken at 30 ℃ to OD600The value is between 0.5 and 0.7;
(6) immediately putting the bacterial liquid obtained in the step (5) into an ice-water mixture for cooling for 20 min;
(7) centrifuging the bacterial liquid obtained in the step (6) at 4 ℃ at 4500 Xg for 10min to remove supernatant;
(8) repeatedly cleaning the bacterial body sediment in the step (7) on ice by using pre-cooled ultrapure water;
(9) adding ultrapure water into the thalli obtained in the step (8) to fix the volume of the bacterial liquid to 500 mu l, and subpackaging 100 mu l of each tube into precooled EP tubes to obtain GS1783 electrotransformation competence;
(10) adding 20ng of pBAC-DPV plasmid into 100 mul of GS1783 electroporation competence, uniformly mixing, adding competence and plasmid into the bottom of a precooled electric shock cup with the size of 2mm, and carrying out electric shock under the condition of 15 kV/cm;
(11) and (3) taking 100 mu l of LB liquid culture medium for re-suspending the electrically shocked thallus, shaking the thallus at 30 ℃ for 1h, centrifuging the thallus at 4500 Xg for 2min, removing the supernatant, adopting 200 mu l of LB liquid culture medium for suspension precipitation, coating the suspension precipitate on an LB solid culture medium containing chloramphenicol, and culturing at 30 ℃ for 24h to obtain the GS1783-pBAC-DPV strain.
2. Amplification of I _ SceI-Kana-gE targeting fragment
(1) Recovering the Escherichia coli carrying pEPkan-S plasmid in LB solid culture medium containing kanamycin, and culturing overnight at 37 ℃; selecting single colony, inoculating into LB liquid culture medium containing kanamycin, culturing at 37 deg.C overnight, and extracting pEPkan-S plasmid with plasmid miniprep kit (pEPkan-S plasmid map is shown in figure 1);
(2) amplifying an I _ SceI-Kana-gE targeting fragment by taking a pEPkan-S plasmid as a template and GS 1783-BAC-delta gE-F and GS 1783-BAC-delta gE-R as primers, and recovering the amplified fragment by adopting a common agarose gel DNA recovery kit;
GS1783-BAC-ΔgE-F:5’-ATACTGCCGGCCAGACTACGGAACCTCAACAATTGGTACGtagggataacagggtaatcgattt-3’(SEQ ID NO:1)
GS1783-BAC-ΔgE-R:5’-TAACTATTTCACTAGTGAGTCATTAGTTCAACATCCATGACGTACCAATTGTTGAGGTTCCGTAGTCTGGCCGGCAGTATgccagtgttacaaccaat-3’(SEQ ID NO:2)
the PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer GS 1783-BAC-delta gE-F1. mu.l, downstream primer GS 1783-BAC-delta gE-R1. mu.l, template pEPkan-S plasmid 1. mu.l;
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
An operation flow chart (taking gE gene deletion as an example) for carrying out gene deletion on a bacterial artificial chromosome recombinant duck plague virus rescue system platform by using a Red-Based modification technology is shown in figure 2, and the specific process comprises the following 3 and 4 processes.
3. Preparation of GS1783-pBAC-DPV electrotransferase competence for targeting fragment targeting
(1) Recovering GS1783-pBAC-DPV frozen bacteria in LB solid culture medium containing chloramphenicol, and culturing at 30 deg.C overnight;
(2) selecting a GS1783-pBAC-DPV single colony, inoculating the single colony in 5mL LB liquid culture medium containing chloramphenicol, and culturing at 30 ℃ overnight to obtain a seed solution;
(3) 5mL of the seed liquid was added to 100mL of LB liquid medium containing chloramphenicol, and the mixture was shaken at 30 ℃ to OD600The value is between 0.5 and 0.7;
(4) culturing the bacterial liquid obtained in the step (3) at 42 ℃ for 15min, immediately putting the bacterial liquid into an ice-water mixture, and cooling for 20 min;
(5) taking 50mL of the bacterial liquid obtained in the step (4), centrifuging at 4 ℃ of 4500 Xg for 10min, and removing supernatant;
(6) repeatedly cleaning the thallus precipitate in the step (5) on ice by using precooled ultrapure water;
(7) adding ultrapure water into the thalli obtained in the step (6) to fix the volume of the bacterial liquid to 500 mu l, and subpackaging 100 mu l of each tube into precooled EP tubes to obtain GS1783-pBAC-DPV electrotransformation competence;
(8) adding 200ng of I _ SceI-Kana-gE targeting segment into 100 mu l of electrotransformation competence, uniformly mixing, adding the competence and the targeting segment into the bottom of a 2mm precooled electric shock cup, and carrying out electric shock under the condition of 15 kV/cm;
(9) resuspending the shocked thallus in 100 μ l LB liquid culture medium, shaking the thallus at 30 deg.C for 1h, centrifuging the thallus at 4500 Xg for 2min, discarding the supernatant, suspending and precipitating in 200 μ l LB liquid culture medium, coating on LB solid culture medium containing kanamycin and chloramphenicol antibiotic resistance, and culturing at 30 deg.C for 48 h;
(10) identifying the single colony obtained in the step (9) by PCR to obtain a positive colony GS178-pBAC-DPV-gE-Kana, and identifying the positive colony by using an amplification I _ SceI-Kana-gE targeting fragment upstream primer GS 1783-BAC-delta gE-F and a gE gene downstream primer gE-R by using a single colony heavy suspension grown in the step (9) as a template to obtain a positive clone GS 1783-pBAC-DPV-gE-Kana;
GS1783-BAC-ΔgE-F:5’-ATACTGCCGGCCAGACTACGGAACCTCAACAATTGGTACGtagggataacagggtaatcgattt-3’(SEQ ID NO:1)
gE-R:5’-AGCGAGTACTTCTCTGCGTC-3’(SEQ ID NO:3)
the PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer GS 1783-BAC-delta gE-F1 mul, 1 mul of downstream primer gE-R, and 1 mul of template which is the single colony resuspension in the step (9);
the PCR amplification conditions were: pre-denaturing at 98 deg.C for 2min, denaturing at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extending at 72 deg.C for 5s for 30 cycles, and extending at 72 deg.C for 10min, and storing at 16 deg.C.
4. The I _ SceI-Kana fragment was deleted
(1) Selecting a GS1783-pBAC-DPV-gE-Kana single colony, inoculating the single colony in 2mL LB liquid culture medium containing chloramphenicol, and culturing overnight at 30 ℃ to obtain a seed solution;
(2) taking 10 mu l of the seed liquid obtained in the step (1) to inoculate into 2mL of LB liquid culture medium containing chloramphenicol, and culturing at 30 ℃ for 2h until the bacterial liquid presents slight haze;
(3) adding 1mL of LB liquid culture medium containing chloramphenicol and 5M of L-arabinose with the final concentration of 2% into the bacterial liquid obtained in the step (2), and culturing for 1h at 30 ℃;
(4) immediately putting the bacterial liquid obtained in the step (3) into a water bath at 42 ℃ for culturing for 30 min;
(5) culturing the bacterial liquid obtained in the step (4) at 30 ℃ for 2h, adding 1 mul of bacterial liquid into 200 mul of LB liquid culture medium, uniformly mixing, coating the mixture on an LB solid culture medium containing chloramphenicol, and culturing at 30 ℃ for 24 h-48 h;
(6) and (3) selecting the single colony obtained in the step (5) to be screened in parallel on a chloramphenicol and kanamycin double-resistant LB solid culture medium and a chloramphenicol single-resistant LB solid culture medium, not growing the chloramphenicol and kanamycin double-resistant LB solid culture medium, and identifying a colony growing in the chloramphenicol single-resistant LB solid culture medium by using a gE gene identification primer through a PCR method to obtain a positive clone GS 1783-pBAC-DPV-delta gE.
gE-F:5’-TCTCAAGACGCTCTGGAATC-3’(SEQ ID NO:4)
gE-R:5’-AGCGAGTACTTCTCTGCGTC-3’(SEQ ID NO:3)
The PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25 mul, upstream primer gE-F1 mul, downstream primer gE-R1 mul, template is step (6) single colony resuspension 1 mul;
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
5. Amplification of I _ SceI-Kana-gI targeting fragment
(1) Recovering the Escherichia coli carrying pEPkan-S plasmid in LB solid culture medium containing kanamycin, and culturing overnight at 37 ℃; selecting a single colony, inoculating the single colony in an LB liquid culture medium containing kanamycin, culturing the colony overnight at 37 ℃, and extracting a pEPkan-S plasmid by adopting a plasmid miniextraction kit;
(2) amplifying an I _ SceI-Kana-gI targeting fragment by taking a pEPkan-S plasmid as a template and GS 1783-BAC-delta gI-F and GS 1783-BAC-delta gI-R as primers, and recovering the amplified fragment by adopting a common agarose gel DNA recovery kit;
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggataacagggtaatcgattt-3’(SEQ ID NO:5)
GS1783-BAC-ΔgI-R:5’-TCATAACAAAAACATTTACTTTTAGTCATACTGATGTGAATTCTATTTTTGAAACTCAATATATCGTCTATATGGCGCACgccagtgttacaaccaat-3’(SEQ ID NO:6)
the PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer GS 1783-BAC-delta gI-F1. mu.l, downstream primer GS 1783-BAC-delta gI-R1. mu.l, template pEPkan-S plasmid 1. mu.l;
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
6. Preparation of GS 1783-pBAC-DPV-delta gE electrotransferase competent for targeting fragment targeting
(1) Recovering GS 1783-pBAC-DPV-delta gE cryopreserved bacteria in LB solid culture medium containing chloramphenicol, and culturing overnight at 30 ℃;
(2) selecting a GS 1783-pBAC-DPV-delta gE single colony, inoculating the single colony in 5mL LB liquid culture medium containing chloramphenicol, and culturing overnight at 30 ℃ to obtain a seed solution;
(3)5mL of the seed liquid was added to 100mL of LB liquid medium containing chloramphenicol, and the mixture was shaken at 30 ℃ to OD600The value is between 0.5 and 0.7;
(4) culturing the bacterial liquid obtained in the step (3) at 42 ℃ for 15min, immediately putting the bacterial liquid into an ice-water mixture, and cooling for 20 min;
(5) taking 50mL of the bacterial liquid obtained in the step (4), centrifuging at 4 ℃ of 4500 Xg for 10min, and removing supernatant;
(6) repeatedly cleaning the thallus precipitate in the step (5) on ice by using precooled ultrapure water;
(7) adding ultrapure water into the thalli obtained in the step (6) to fix the volume of the bacterial liquid to 500 mu l, and subpackaging 100 mu l of each tube into precooled EP tubes to obtain GS 1783-pBAC-DPV-delta gE electrotransformation competence;
(8) adding 200ng of I _ SceI-Kana-gI targeting segment into 100 mu l of electrotransformation competence, uniformly mixing, adding the competence and the targeting segment into the bottom of a 2mm precooled electric shock cup, and carrying out electric shock under the condition of 15 kV/cm;
(9) resuspending the shocked thallus in 100 μ l LB liquid culture medium, shaking the thallus at 30 deg.C for 1h, centrifuging the thallus at 4500 Xg for 2min, discarding the supernatant, suspending and precipitating in 200 μ l LB liquid culture medium, coating on LB solid culture medium containing kanamycin and chloramphenicol antibiotic resistance, and culturing at 30 deg.C for 48 h;
(10) identifying the single colony obtained in the step (9) by PCR to obtain a positive colony GS 178-pBAC-DPV-delta gE-gI-Kana, taking the single colony heavy suspension grown in the step (9) as a template, and identifying the positive colony by using an amplified I _ SceI-Kana-gI targeting fragment upstream primer GS 1783-BAC-delta gI-F and a downstream primer gI-R for identifying a gE gene;
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggataacagggtaatcgattt-3(SEQ ID NO:5)
gI-R:5’-GACCGGTAGTTCCAATCACT-3’(SEQ ID NO:7)
the PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25 mul, upstream primer GS 1783-BAC-delta gI-F1 mul, downstream primer gI-R1 mul, and template 1 mul;
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
7. The I _ SceI-Kana fragment was deleted
(1) Selecting a GS 1783-pBAC-DPV-delta gE-gI-Kana single colony, inoculating the single colony in 2mL LB liquid culture medium containing chloramphenicol, and culturing at 30 ℃ overnight to obtain a seed solution;
(2) inoculating 10 mu l of the seed liquid obtained in the step (1) into 2mL of LB liquid culture medium containing chloramphenicol, and culturing at 30 ℃ for 2h until the bacterial liquid is slightly cloudy;
(3) adding 1mL of LB liquid culture medium containing chloramphenicol and 5M of L-arabinose with the final concentration of 2% into the bacterial liquid obtained in the step (2), and culturing for 1h at 30 ℃;
(4) immediately putting the bacterial liquid obtained in the step (3) into a water bath at 42 ℃ for culturing for 30 min;
(5) culturing the bacterial liquid obtained in the step (4) at 30 ℃ for 2h, adding 1 mul of bacterial liquid into 200 mul of LB liquid culture medium, uniformly mixing, coating the mixture on an LB solid culture medium containing chloramphenicol, and culturing at 30 ℃ for 24 h-48 h;
(6) and (3) selecting the single colony obtained in the step (5) to be screened on a double-resistance LB solid culture medium containing chloramphenicol and kanamycin and a single-resistance LB solid culture medium containing chloramphenicol, and carrying out parallel screening, wherein the double-resistance LB solid culture medium containing chloramphenicol and kanamycin does not grow, and a colony growing on the single-resistance LB solid culture medium containing chloramphenicol is identified by a PCR method by utilizing a gI gene upstream identification primer gI-F and a gE gene downstream identification primer gE-R, so that a positive clone GS 1783-pBAC-DPV-delta gE + delta gI is obtained.
gI-F:5’-TGTGGGTGGGTCATCTACAT-3’(SEQ ID NO:14)
gE-R:5’-AGCGAGTACTTCTCTGCGTC-3’(SEQ ID NO:3)
The PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25 mul, upstream primer gE-F1 mul, downstream primer gE-R1 mul, template is step (6) single colony resuspension 1 mul;
the PCR amplification conditions were: pre-denaturing at 98 deg.C for 2min, denaturing at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extending at 72 deg.C for 5s for 30 cycles, and extending at 72 deg.C for 10min, and storing at 16 deg.C.
8. Amplification of I _ SceI-Kana-MiniF fragment
(1) Recovering the Escherichia coli carrying pEPkan-S plasmid in LB solid culture medium containing kanamycin, and culturing overnight at 37 ℃; selecting a single colony, inoculating the single colony in an LB liquid culture medium containing kanamycin, culturing the colony overnight at 37 ℃, and extracting a pEPkan-S plasmid by using a plasmid miniextraction kit;
(2) amplifying an I _ SceI-Kana-MiniF targeting fragment by taking a pEPkan-S plasmid as a template and GS1783-MiniF-F and GS1783-MiniF-R as primers, and recovering the amplified fragment by using a common agarose gel DNA recovery kit;
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’(SEQ ID NO:8)
GS1783-MiniF-R:5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATtagggataacagggtaatcgat-3’(SEQ ID NO:9)
the PCR amplification system is ddH2O 22 ul,Max DNA Polymerase 25. mu.l, upstream primer GS 1783-MiniF-F1. mu.l, downstream primer GS 1783-MiniF-R1. mu.l, template pEPkan-S plasmid 1. mu.l;
the PCR amplification conditions comprise pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
9. Amplification of the UL23-MiniF fragment
(1) Preparing Duck Embryo Fibroblast (DEF) and inoculating in a 25T cell bottle, culturing at 37 ℃ and 5% CO2 for 24h, inoculating 5MOI DPV CHv, culturing at 37 ℃ and 5% CO2 for 48h, harvesting virus, repeatedly freezing and thawing for 2 times, and extracting DPV CHv genome according to TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 instruction;
(2) DPV CHv genome is used as a template, CHv-UL23-F and CHv-UL23-R are used as primers, UL23-MiniF targeting fragments are amplified, and the amplified fragments are recovered by a common agarose gel DNA recovery kit;
CHv-UL23-F:5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’(SEQ ID NO:10)
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’(SEQ ID NO:11)
the PCR amplification system is ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer CHv-UL 23-F1. mu.l, downstream primer CHv-UL 23-R1. mu.l, template DPV CHv genome 1. mu.l;
the PCR amplification conditions comprise pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
10. Fusing the I _ SceI-Kana-MiniF fragment and the UL23-MiniF fragment to obtain the I _ SceI-Kana-MiniF-UL23 targeting fragment
(1) Fusing the I _ SceI-Kana-MiniF fragment and the UL23-MiniF fragment by taking the I _ SceI-Kana-MiniF fragment and the UL23-MiniF fragment as templates;
the fusion system is as follows: ddH2O 8μl、Max DNA Polymerase 10. mu.l, template I _ SceI-Kana-MiniF fragment and UL23-MiniF fragment 1. mu.l each;
the fusion conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 1min for 5 cycles.
(2) Adding primers GS1783-MiniF-F and CHv-UL23-R into the fusion template obtained in the step (1), amplifying an I _ SceI-Kana-MiniF-UL23 targeting fragment, and recovering the amplified fragment by using a common agarose gel DNA recovery kit;
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’(SEQ ID NO:8)
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’(SEQ ID NO:11)
the PCR amplification system is as follows: 20 UL of fusion template, 0.5 UL of upstream primer GS1783-MiniF-F and 0.5 UL of downstream primer CHv-UL 23-R;
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
The operation flow chart of the deletion of the MiniF element by using the Red-Based modification technique and the intracellular spontaneous homologous recombination technique is shown in FIG. 3, and the specific process includes the following steps 11, 12 and 13.
11. Preparation of GS 1783-pBAC-DPV-delta gE + delta gI electrotransferase competence for targeting fragment targeting
(1) Recovering GS 1783-pBAC-DPV-delta gE + delta gI cryopreserved bacteria in LB solid culture medium containing chloramphenicol, and culturing overnight at 30 ℃;
(2) selecting a single GS 1783-pBAC-DPV-delta gE + delta gI colony, inoculating the single GS 1783-pBAC-DPV-delta gE + delta gI colony in 5mL LB liquid culture medium containing chloramphenicol, and culturing overnight at 30 ℃ to obtain a seed solution;
(3) 5mL of the seed liquid was added to 100mL of LB liquid medium containing chloramphenicol, and the mixture was shaken at 30 ℃ to OD600The value is between 0.5 and 0.7;
(4) culturing the bacterial liquid obtained in the step (3) at 42 ℃ for 15min, immediately putting the bacterial liquid into an ice-water mixture, and cooling for 20 min;
(5) taking 50mL of the bacterial liquid obtained in the step (4), centrifuging at 4 ℃ of 4500 Xg for 10min, and removing supernatant;
(6) repeatedly cleaning the thallus precipitate obtained in the step (5) on ice by using precooled ultrapure water;
(7) adding ultrapure water to fix the volume of the bacterial liquid obtained in the step (6) to 500 mu l, and subpackaging 100 mu l of each tube into precooled EP tubes to obtain GS 1783-pBAC-DPV-delta gE + delta gI electrotransformation competence;
(8) adding 200ng of I _ SceI-Kana-MiniF-UL23 targeting segment into 100 mul of electrotransformation competence, uniformly mixing, adding the competence and the targeting segment into the bottom of a 2mm precooled electric shock cup, and carrying out electric shock under the condition of 15 kV/cm;
(9) adding 100 μ l LB liquid culture medium to resuspend the shocked thallus, shaking the thallus at 30 deg.C for 1h, centrifuging thallus at 4500 Xg for 2min, removing supernatant, suspending and precipitating with 200 μ l LB liquid culture medium, coating LB solid culture medium containing kanamycin and chloramphenicol antibiotic resistance, and culturing at 30 deg.C for 48 h;
(10) identifying the single colony obtained in the step (9) by PCR to obtain a positive colony GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana, and identifying the positive colony by using identification primers MiniF-F and MiniF-R by using the single colony resuspension grown in the step (9) as a template;
MiniF-F:5’-GTTATCCACTGAGAAGCGAACG-3’(SEQ ID NO:12)
MiniF-R:5’-GGCTGTAAAAGGACAGACCACA-3’(SEQ ID NO:13)
the PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer MiniF-F1. mu.l, downstream primer MiniF-R1. mu.l, template is single colony resuspension 1. mu.l of step (9);
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 15s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
12. The I _ SceI-Kana fragment was deleted
(1) Selecting a single GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana colony, inoculating the single GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana colony into 2mL of LB liquid culture medium containing chloramphenicol, and culturing overnight at 30 ℃ to obtain a seed solution;
(2) inoculating 10 mu l of the seed liquid obtained in the step (1) into 2mL of LB liquid culture medium containing chloramphenicol, and culturing at 30 ℃ for 2h until the bacterial liquid is slightly cloudy;
(3) adding 1mL of LB liquid culture medium containing chloramphenicol and 5M of L-arabinose with the final concentration of 2% into the bacterial liquid obtained in the step (2) and culturing for 1h at 30 ℃;
(4) immediately putting the bacterial liquid obtained in the step (3) into a water bath at 42 ℃ for culturing for 30 min;
(5) culturing the bacterial liquid obtained in the step (4) at 30 ℃ for 2h, adding 1 mul of bacterial liquid into 200 mul of LB liquid culture medium, uniformly mixing, coating the mixture on an LB solid culture medium containing chloramphenicol, and culturing at 30 ℃ for 24 h-48 h;
(6) and (3) selecting the single colony obtained in the step (5) to be screened on a double-resistance LB solid culture medium containing chloramphenicol and kanamycin and a single-resistance LB solid culture medium containing chloramphenicol, and performing parallel screening, wherein the double-resistance LB solid culture medium containing chloramphenicol and kanamycin does not grow, and a colony growing on the single-resistance LB solid culture medium containing chloramphenicol is identified by a PCR method by utilizing a MiniF gene identification primer, so that a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23 is obtained.
MiniF-F:5’-GTTATCCACTGAGAAGCGAACG-3’(SEQ ID NO:12)
MiniF-R:5’-GGCTGTAAAAGGACAGACCACA-3’(SEQ ID NO:13)
The PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25 mul, upstream primer MiniF-F1 mul, downstream primer MiniF-R1 mul, template is step (6) single colony resuspension 1 mul;
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
13. Rescue of DPV CHv-delta gE + delta gI virus
(1) Recovering GS 1783-pBAC-DPV-delta gE + delta gI-UL23 frozen bacteria in LB solid culture medium containing chloramphenicol, and culturing overnight at 30 ℃;
(2) extracting the pBAC-DPV-delta gE + delta gI-UL23 Plasmid according to the QIAGEN Plasmid Midi Kit operating instruction;
(3) preparing Duck Embryo Fibroblast (DEF) and inoculating in 12-well plate at 37 deg.C and 5% CO2After 24h of culture, transfecting pBAC-DPV-delta gE + delta gI-UL23 plasmid according to Lipofectamine 3000 operation instructions, observing fluorescent spots after 96h, collecting viruses, repeatedly freezing and thawing for 2 times, and then inoculating the viruses to a DEF-full 6-well plate;
(4) repeating the step (3) for three times;
(5) freezing and thawing the virus obtained in the step (4) for 2 times repeatedly, diluting by 10 times, inoculating into a 6-hole plate full of DEF, and inoculating at 37 deg.C with 5% CO2After incubation for 2h, the incubation solution was discarded, 1% methylcellulose fixed cells were added, 37 ℃ and 5% CO2Culturing for 120h, selecting non-fluorescent pathological cells, repeatedly freezing and thawing for 2 times, and re-inoculating in DEFObtaining DPV CHv-delta gE + delta gI-Q;
(6) DPV CHv-delta gE + delta gI-Q virus genomes are extracted according to TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 instructions, MiniF is used for identifying primers MiniF-F and MiniF-R and identifying a deleted MiniF element, and finally a traceless deletion virus strain DPV CHv-delta gE + delta gI positive virus strain without MiniF element residue and base residue at a deletion gene is obtained, which is shown in figure 4.
MiniF-F:5’-GTTATCCACTGAGAAGCGAACG-3’(SEQ ID NO:12)
MiniF-R:5’-GGCTGTAAAAGGACAGACCACA-3’(SEQ ID NO:13)
The PCR amplification system is as follows: ddH2O 22μl、Max DNA Polymerase 25 mul, upstream primer MiniF-F1 mul, downstream primer MiniF-R1 mul, and template DPV CHv-delta gE + delta gI-Q virus genome 1 mul extracted in the step (6);
the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10min, and storing at 16 deg.C.
Example 2 detection of the amount of virus in 7-day-old Duck inoculated with the traceless deletion strain DPV CHv-delta gE + delta gI
Inoculating the gene gE and gI and the trace-free deletion strain DPV CHv-delta gE + delta gI with 0.01MOI to DEF cells, collecting the cells 120h after inoculation, repeatedly freezing and thawing for 2 times, and measuring TCID50Then, 2MOI DPV CHv-delta gE + delta gI deletion virus is taken to inoculate 7-day-old ducks by muscles, the blood, liver and spleen of the ducks are collected at 24h, 48h, 72h and 96h respectively, and DNA of each tissue is extracted according to the instruction of TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0. And (3) detecting whether the duck liver virus is a gE gene and gI gene double-deletion virus 48h after the virus attack by using identification primers gE-F and gI-R PCR (see figure 5), identifying the primers and Taq probes by using UL30QPCR, and detecting the virus quantity in blood, liver and spleen at different time points by using QPCR (see figure 6). The result shows that the DPV CHv-delta gE + delta gI can be detected in blood, liver and spleen after being inoculated with the duck; DPV CHv-delta gE + delta gI virions in blood and liverThe sub-numbers all show the trend of increasing first and then reducing; the number of DPV CHv-delta gE + delta gI virus particles in the spleen shows a gradual reduction trend; the number of DPV CHv-delta gE + delta gI virus particles in blood is lower than that of liver and spleen tissues; liver tissue DPV CHv- Δ gE + Δ gI virion counts were highest at 48h and spleen tissue reached highest at 24 h.
In addition, the invention also performs genetic stability experiments, safety experiments and immunogenicity experiments on the duck plague virus gE and gI gene traceless deletion strain DPV CHv-delta gE + delta gI.
Genetic stability: the DPV CHv-delta gE + delta gI strain with traceless deletion of the gE and gI genes of the duck plague virus is passaged on DEF cells for 20 generations to generate lesion plaques, which shows that the obtained DPV CHv-delta gE + delta gI strain with traceless deletion of the gE and gI genes of the duck plague virus is stably inherited in DEF.
Safety is as follows: 15 4-week-old DPV antibody-negative and PCR detection-negative ducks were randomly divided into 3 groups of 5 ducks each. Group 1 was intramuscularly administered DPV CHv- Δ gE + Δ gI, group 2 was intramuscularly administered parent virus DPV CHv, the toxicity of each virus in both groups was the same, group 3 was administered with an equal amount of MEM as a control group, each group was individually kept in isolation, observed and the cases of morbidity and mortality were recorded daily. The results show that all ducks injected with the parental viruses die, but the ducks injected with the DPV CHv-delta gE + delta gI and the ducks injected with the DPV CHv-delta gE + delta gI in the control group do not die, which indicates that the DPV CHv-delta gE + delta gI has good safety.
Immunogenicity: 10 ducks that were negative for DPV antibody and negative for PCR detection at 4 weeks were randomly divided into 2 groups of 5 ducks. Group 1 was intramuscularly injected with DPV CHv- Δ gE + Δ gI, group 2 was injected with equal doses of MEM as a control group, and each group was individually kept in isolation. On day 14 post immunization, legs were intramuscularly vaccinated with DPV CHv virulent, and morbid deaths were observed and recorded. According to the results, the challenge control group is 100% dead, and the immune group is 100% protected, which indicates that the DPV CHv-delta gE + delta gI has good immunogenicity.
Sequence listing
<110> Sichuan university of agriculture
<120> duck plague virus gE and gI double-gene traceless deleted strain DPV CHv-delta gE + delta gI and construction method thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 64
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atactgccgg ccagactacg gaacctcaac aattggtacg tagggataac agggtaatcg 60
attt 64
<210> 2
<211> 98
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
taactatttc actagtgagt cattagttca acatccatga cgtaccaatt gttgaggttc 60
cgtagtctgg ccggcagtat 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
<210> 5
<211> 64
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtgcgccata tagacgatat attgagtttc aaaaatagaa tagggataac agggtaatcg 60
attt 64
<210> 6
<211> 98
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcataacaaa aacatttact tttagtcata ctgatgtgaa ttctattttt gaaactcaat 60
atatcgtcta tatggcgcac gccagtgtta caaccaat 98
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaccggtagt tccaatcact 20
<210> 8
<211> 98
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttattaatct caggagcctg tgtagcgttt ataggaagta gtgttctgtc atgatgcctg 60
caagcggtaa cgaaaacgat tgttacaacc aattaacc 98
<210> 9
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atcgttttcg ttaccgcttg caggcatcat gacagaacac tacttcctat tagggataac 60
agggtaatcg at 72
<210> 10
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gcctgcaagc ggtaacgaaa acgattcaat taattgtcat ctcgg 45
<210> 11
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ccgctccact tcaacgtaac accgcacgaa gatttctatt gttcctgaag gcatattcaa 60
cggacatatt aaaaattga 79
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gttatccact gagaagcgaa cg 22
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggctgtaaaa ggacagacca ca 22
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgtgggtggg tcatctacat 20
Claims (10)
1. The duck plague virus gE and gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI is preserved in the China Center for Type Culture Collection (CCTCC) in 2018, 7 months and 4 days, and the preservation number is CCTCC NO: and V201841.
2. The method for constructing the duck plague virus gE and gI double-gene traceless deleted strain DPV CHv-delta gE + delta gI as claimed in claim 1, which comprises the following steps:
(1) transforming the pBAC-DPV plasmid into GS1783 escherichia coli competence to obtain a GS1783-pBAC-DPV strain, and then preparing the GS1783-pBAC-DPV competence;
(2) using pEPkan-S as a template, using GS 1783-BAC-delta gE-F and GS 1783-BAC-delta gE-R as primers, amplifying a base fragment containing an I _ SceI enzyme cutting site and a Kana element and a targeting fragment I _ SceI-Kana-gE of each 40bp homologous arm at the upstream and downstream of the gE gene by a PCR method, cutting glue and recovering to obtain an I _ SceI-Kana-gE fragment;
(3) transforming the I _ SceI-Kana-gE fragment into GS1783-pBAC-DPV competence, and screening to obtain a positive clone GS 1783-pBAC-DPV-gE-Kana;
(4) removing the I _ SceI-Kana fragment in the positive clone GS1783-pBAC-DPV-gE-Kana to prepare GS 1783-pBAC-DPV-delta gE competence;
(5) using pEPkan-S as a template, using GS 1783-BAC-delta gI-F and GS 1783-BAC-delta gI-R as primers, amplifying a base fragment containing an I _ SceI enzyme cutting site and a Kana element and a targeting fragment I _ SceI-Kana-gI of each 40bp homologous arm at the upstream and downstream of the gI gene by a PCR method, cutting glue and recovering to obtain an I _ SceI-Kana-gI fragment;
(6) converting the I _ SceI-Kana-gI fragment into GS 1783-pBAC-DPV-delta gE competence, and obtaining a positive clone GS 1783-pBAC-DPV-delta gE-gI-Kana through antibiotic screening and PCR identification;
(7) removing the I _ SceI-Kana fragment in the positive clone GS 1783-pBAC-DPV-delta gE-gI-Kana to prepare GS 1783-pBAC-DPV-delta gE + delta gI competence;
(8) using pEPkan-S as a template, using GS1783-MiniF-F and GS1783-MiniF-R as primers, amplifying a base fragment containing an I _ SceI enzyme cutting site and a Kana element, a homologous arm fragment I _ SceI-Kana-MiniF located at 240bp downstream of a MiniF element ori2 gene and at 290bp downstream of a MiniF element ori2 gene by a PCR method, cutting glue and recovering to obtain an I _ SceI-Kana-MiniF fragment;
(9) using a CHv genome as a template, using CHv-UL23-F and CHv-UL23-R as primers, amplifying a homologous arm fragment which comprises a UL23 gene, an I _ SceI-Kana-MiniF downstream homologous arm overlapping 25bp and is positioned at 180bp downstream of a MiniF element ori2 gene by a PCR method, cutting glue and recovering to obtain a UL23-MiniF fragment;
(10) carrying out PCR fusion reaction by taking the I _ SceI-Kana-MiniF fragment and the UL23-MiniF fragment as templates, and then carrying out PCR amplification by taking the fusion fragment as a template and taking GS1783-MiniF-F and CHv-UL23-R as primers to obtain an I _ SceI-Kana-MiniF-UL23 targeting fragment;
(11) transforming the I _ SceI-Kana-MiniF-UL23 targeting fragment into GS 1783-pBAC-DPV-delta gE + delta gI competence, and obtaining a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana through antibiotic screening and PCR identification;
(12) removing the I _ SceI-Kana fragment in the positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23-Kana to obtain a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL 23;
(13) extracting a pBAC-DPV-delta gE + delta gI-UL23 plasmid from a positive clone GS 1783-pBAC-DPV-delta gE + delta gI-UL23, transfecting the pBAC-DPV-delta gE + delta gI-UL23 plasmid into DEF cells, and obtaining a gE and gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI by cloning and screening.
3. The method for constructing the DPV CHv-delta gE + delta gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI according to claim 2, wherein the PCR amplification system in the steps (2), (5), (8) and (9) is as follows: ddH2O 22μl、Max DNA Polymerase 25. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, template 1. mu.l; the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10 min.
4. The method for constructing the DPV CHv-delta gE + delta gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI of the duck plague virus gE and gI according to claim 2 or 3, wherein the primer sequence in the step (2) is as follows:
GS1783-BAC-ΔgE-F:5’-ATACTGCCGGCCAGACTACGGAACCTCAACAATTGGTACGtagggataacagggtaatcgattt-3’;
GS1783-BAC-ΔgE-R:5’-TAACTATTTCACTAGTGAGTCATTAGTTCAACATCCATGACGTACCAATTGTTGAGGTTCCGTAGTCTGGCCGGCAGTATgccagtgttacaaccaat-3’。
5. the method for constructing the DPV CHv-delta gE + delta gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI of the duck plague virus gE and gI according to the claim 2 or 3, characterized in that the primer sequence in the step (5) is as follows:
GS1783-BAC-ΔgI-F:5’-GTGCGCCATATAGACGATATATTGAGTTTCAAAAATAGAAtagggataacagggtaatcgattt-3’;
GS1783-BAC-ΔgI-R:5’-TCATAACAAAAACATTTACTTTTAGTCATACTGATGTGAATTCTATTTTTGAAACTCAATATATCGTCTATATGGCGCACgccagtgttacaaccaat-3’。
6. the method for constructing the DPV CHv-delta gE + delta gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI of the duck plague virus gE and gI according to the claim 2 or 3, characterized in that the primer sequence in the step (8) is as follows:
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’;
GS1783-MiniF-R:5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATtagggataacagggtaatcgat-3’。
7. the method for constructing the DPV CHv-delta gE + delta gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI of the duck plague virus gE and gI according to the claim 2 or 3, characterized in that the primer sequence in the step (9) is as follows:
CHv-UL23-F:5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’;
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’。
8. the method for constructing the DPV CHv-delta gE + delta gI double-gene traceless deletion strain DPV CHv-delta gE + delta gI of the duck plague virus gE and gI according to claim 2, wherein the primer sequence in the step (10) is as follows:
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’;
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’。
9. the method for constructing the DPV CHv-delta gE + delta gI strain with the traceless deletion of the gE and gI genes of the duck plague virus according to claim 2 or 8, wherein the PCR fusion system in the step (10) is as follows: ddH2O8μl、Max DNA Polymerase 10. mu.l, template I _ SceI-Kana-MiniF fragment and UL23-MiniF fragment 1. mu.l each; the PCR fusion conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 1min for 5 cycles.
10. The method for constructing the DPV CHv-delta gE + delta gI strain with the traceless deletion of the gE and gI genes of the duck plague virus according to claim 2 or 8, wherein the PCR amplification system in the step (10) is as follows: 20 UL of fusion template, 0.5 UL of upstream primer GS1783-MiniF-F and 0.5 UL of downstream primer CHv-UL 23-R; the PCR amplification conditions were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 5s, for 30 cycles, and final extension at 72 deg.C for 10 min.
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