WO2020134924A1 - 一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC及其构建方法 - Google Patents

一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC及其构建方法 Download PDF

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WO2020134924A1
WO2020134924A1 PCT/CN2019/122997 CN2019122997W WO2020134924A1 WO 2020134924 A1 WO2020134924 A1 WO 2020134924A1 CN 2019122997 W CN2019122997 W CN 2019122997W WO 2020134924 A1 WO2020134924 A1 WO 2020134924A1
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dpv
minif
chv
kana
gene
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程安春
周晓凤
汪铭书
刘田
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四川农业大学
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Definitions

  • the invention belongs to the technical field of genetic engineering, and particularly relates to a duck plague virus MiniF element gC gene deletion strain DPV CHv- ⁇ gC and a construction method thereof.
  • Bacterial artificial chromosome is a bacterial chromosome cloning vector based on the F plasmid. It is commonly used to clone DNA fragments of about 150kd in size, and can store up to 300kd base pairs.
  • the plasmid mainly includes oris, RepE (controlling F plasmid replication) and parA, parB (controlling copy number), etc.
  • RepE controlling F plasmid replication
  • parA, parB controlling copy number
  • the vector cloned on the basis of BAC has a low efficiency of chimerism and high transformation efficiency, and it exists in the bacteria in a circular structure, which is easy to distinguish Separation and purification are currently mainly used for the construction of large fragment genomic libraries and related research on large gene clusters.
  • E. coli minimal fertility factor replicon
  • Mini-F minimal fertility factor replicon
  • the most commonly used E. coli gene localization and modification technologies include Red/ET-mediated homologous recombination technology, RecA protein-mediated homologous recombination technology, Cre/loxP-mediated homologous recombination technology, and Tn transposon-mediated Random insertion and mutation technology.
  • the molecular cloning technology has now obtained a mature bacterial artificial chromosome duck plague virus rescue system platform, and the use of E. coli gene positioning modification Red/ET-mediated homologous recombination technology can be used on the bacterial artificial chromosome duck plague virus rescue system platform
  • E. coli gene positioning modification Red/ET-mediated homologous recombination technology can be used on the bacterial artificial chromosome duck plague virus rescue system platform.
  • the MiniF element as the minimum reproductive factor replicon that maintains the replication of the BAC vector, is mainly composed of the repE and repF genes that regulate the origin of BAC replication (oriS), the sopA and sopB genes that regulate the distribution of replicons, and the sopC genes that encode centromere regions.
  • MiniF element added resistance screening gene and fluorescent marker gene to complete bacterial resistance screening and BAC marker screening.
  • MiniF elements are inserted into the viral genome and increase the length of the genome, which has an uncertain effect on viral replication. At the same time, MiniF elements remain as bacterial sequences on the viral genome, which is not conducive to the development and licensing of live attenuated vaccines. Therefore, obtaining the MiniF element deletion strain has become the focus of exploring the method of duck plague virus gene deletion.
  • Duck plague is an acute contact highly lethal infectious disease caused by ducks, geese and other waterfowl caused by Duck Plague virus (DPV) in the subfamily of ⁇ -herpes virus.
  • DDV Duck Plague virus
  • the disease was first reported by the Netherlands, and then spread in more developed areas of duck farming in South China, Central China, and East China, causing serious economic losses to the duck farming industry in my country. Therefore, in-depth understanding of duck plague virus gene function and strengthening research on duck plague are particularly important to ensure the healthy and sustainable development of duck farming in my country.
  • the genomic DNA of duck plague virus DPV-CHv strain is 162175 bp in length and contains 78 open reading frames. It can encode structural proteins and functional proteins that participate in the life cycle of duck plague virus.
  • the structural proteins mainly include the envelope, interlayer and coat that constitute the virus Shell and DNA binding protein.
  • Envelope proteins are glycosylated proteins, including twelve kinds of gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM, and gN.
  • the herpes virus envelope glycoprotein plays an important role in the structure, function and virulence of the virus. Therefore, exploring the role of capsular glycoproteins in the life cycle of duck plague virus is essential for further exploration of the gene function of duck plague virus and the prevention and control of duck plague.
  • coli DH10B extract the plasmid and transfect duck embryo fibroblasts (DEF) to rescue the artificial bacteria that can produce green fluorescence and plaque on the host cell DEF Chromosome recombinant duck plague virus DEVCHv-BAC-G
  • the recombinant virus DEVCHv-BAC-G can survive in the form of a virus, and can also replicate in E. coli in the form of a plasmid, allowing us to mature using the E. coli system
  • the genetic manipulation method of the DPVCHv genome is modified, modified and studied at any position. However, after using the Red/ET modification technology to delete the duck plague virus gene on the platform of the bacterial artificial chromosome recombinant duck plague virus rescue system, MiniF elements will remain. The residue of MiniF element has an impact on the study of gene function, the development and licensing of live attenuated vaccines.
  • the present invention provides a duck plague virus MiniF element gC gene deletion strain DPV CHV- ⁇ gC and a construction method thereof, which can effectively solve the problem of residual MiniF elements.
  • a method for constructing DPV plague virus-free MiniC element gC gene deletion strain DPV CHv- ⁇ gC includes the following steps:
  • GS1783-MiniF-F and GS1783-MiniF-R as primers, amplify the base fragment containing the I_SceI cleavage site and Kana element by PCR, and 240bp downstream of the ori2 gene of MiniF element
  • the homologous arm fragment I_SceI-Kana-MiniF located at 290 bp downstream of the ori2 gene of MiniF element was obtained by excising the I_SceI-Kana-MiniF fragment;
  • the PCR amplification method includes the UL23 gene, I_SceI-Kana-MiniF downstream homology arm overlap 25bp and is located downstream of the MiniF element ori2 gene
  • the homology arm fragment at 180bp was recovered by excising the gel to obtain the UL23-MiniF fragment;
  • the I_SceI-Kana-MiniF-UL23 target fragment was transformed into GS1783-DPV CHv-BAC-G ⁇ gC competent state, and after antibiotic screening and PCR identification, a positive clone GS1783-DPV CHv-BAC-G ⁇ gC was obtained -UL23-Kana;
  • the PCR amplification system in step (1) is: ddH 2 O 14 ⁇ L, Max DNA Polymerase 20 ⁇ L, upstream primer 2 ⁇ L, downstream primer 2 ⁇ L, template 2 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 1min, denaturation at 94°C for 15s, annealing at 55°C for 15s, extension at 72°C for 1min, a total of 30 cycles, and finally extension at 72°C for 10min.
  • primer sequence in step (1) is:
  • the PCR amplification system in steps (2) and (3) is: ddH 2 O 6 ⁇ L, 2 ⁇ Taq PCR MasterMin 10 ⁇ L, upstream primer 1 ⁇ L, downstream primer 1 ⁇ L, template 2 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 95°C for 5min, denaturation at 94°C for 4min, annealing at 55°C for 30s, extension at 72°C for 2min, a total of 30 cycles, and finally extension at 72°C for 10min.
  • primer sequences in steps (2) and (3) are:
  • ⁇ gC identification upstream primer 5’-ATGGTAAGCACATAAAAGTGTCGT-3’;
  • ⁇ gC identifies the downstream primer: 5'-ATTGCTATCCTATCAGTCCGTA-3'.
  • primer sequence in step (5) is:
  • GS1783-MiniF-F 5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’;
  • GS1783-MiniF-R 5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATtagggataacagggtaatcgat-3’.
  • primer sequence in step (6) is:
  • CHv-UL23-F 5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’;
  • CHv-UL23-R 5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCC TGAAGGCATATTCAACGGACATATTAAAAATTGA-3’.
  • the PCR fusion system in step (7) is: ddH 2 O 8 ⁇ L, Max DNA Polymerase 10 ⁇ L, template I_SceI-Kana-MiniF fragment and UL23-MiniF fragment 1 ⁇ L each;
  • the PCR fusion conditions were: pre-denaturation at 95°C for 5min, denaturation at 95°C for 15s, annealing at 55°C for 5s, and extension at 72°C for 1min for 5 cycles.
  • the PCR amplification system in step (7) is: fusion template 20 ⁇ L, upstream primer GS1783-MiniF-F 0.5 ⁇ L, downstream primer CHv-UL23-R 0.5 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5s, a total of 30 cycles, and finally extension at 72°C for 10min.
  • the duck plague virus constructed by the above method removes the MiniF element gC gene deletion strain DPV CHv- ⁇ gC.
  • the present invention utilizes Red-based modification technology on the basis of the bacterial artificial chromosome recombinant duck plague virus rescue system platform, that is, the GS1783 E. coli strain containing the gene sequence encoding the Red operon and the I_SceI enzyme and the encoding kana resistance and The plasmid pEPKan-S at the I_SceI cleavage site deleted the MiniF element by the spontaneous homologous recombination method in the cell, and for the first time completed the construction of the duck plague virus deletion strain without the MiniF element residue; solved the MiniF element residue when the duck plague virus gene was deleted
  • the problem provides sufficient technical support for accurately exploring the gene function of duck plague virus and the construction of live attenuated vaccine.
  • Figure 1 is the pEPKan-S plasmid map
  • Figure 2 is a flowchart of the operation of deleting MiniF elements using Red-Based modification technology and intracellular spontaneous homologous recombination technology;
  • Figure 3 is a picture of the virus rescued from the MiniF element DPV CHv- ⁇ gC-deleted virus strain
  • Figure 4 shows the PCR detection of liver DNA extracts 48h and 72h after DPV CHv- ⁇ gC inoculation of ducks
  • Fig. 5 is a graph comparing the plaque size of the DPV-less CHV- ⁇ gC-deleted strain and the DPV-CHv-deleted strain;
  • Fig. 6 is a schematic diagram of plaque detection of DPV-CHv- ⁇ gC-deleted strain and DPV-CHv-deleted strain with MiniF element removed; where, Fig. 6a is plaque of DPV-CHv- ⁇ gC-deficient strain, and Fig. 6b is plaque of DPV-CHv strain.
  • a duck plague virus gC gene trace-free deletion strain DPV CHv- ⁇ gC the materials and reagents used in the construction process are as follows:
  • Plasmid small extraction kit was purchased from TIANGEN; QIAGEN Plasmid Midi Kit was purchased from QIAGEN; Ordinary 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 was purchased from TaKaRa; Lipofectamine 3000 was purchased from Invitrogen;
  • LB liquid culture medium Weigh Tryptone 10g, Yeast Extract 5g, sodium chloride 10g and dissolve in 800mL deionized water, stir thoroughly, bring the volume to 1L, and sterilize at high temperature and high pressure.
  • LB solid medium add 15g agar powder to LB liquid medium with a fixed volume to 1L, after high temperature and high pressure sterilization, cool to about 60°C, add 1.5mL chloramphenicol (storage concentration 25mg/ml) or 1.5mL card Namycin (storage concentration 50mg/mL), spread the plate, after coagulation, store at 4 °C.
  • MEM Dissolve 9.6g of MEM dry powder and 2.2g of sodium bicarbonate in 800mL of deionized water, stir thoroughly, adjust the pH to 7.4, bring the volume to 1L, filter and sterilize, and store at 4°C.
  • the construction of DPV plague virus-free MiniF element gC gene deletion strain DPV CHv- ⁇ gC includes the following steps:
  • the PCR amplification system is: ddH 2 O 14 ⁇ L, Max DNA Polymerase 20 ⁇ L, construction of ⁇ gC expansion kana fragment upstream primer 2 ⁇ L, construction of ⁇ gC expansion kana fragment downstream primer 2 ⁇ L, template Pkd4 plasmid 2 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 1 min, denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min for a total of 30 cycles, and finally extension at 72°C for 10 min and storage at 16°C.
  • the preparation method of LB liquid culture medium is as follows: Weigh Tryptone 10g, Yeast Extract 5g, sodium chloride 10g, dissolve in 800mL deionized water, stir well, bring the volume to 1L, sterilize at high temperature and high pressure
  • ⁇ gC-R 5, -ATTGCTGTCCTATCAGTCCGTA-3; (SEQ ID NO: 4)
  • the PCR amplification system is: ddH 2 O 6 ⁇ L, 2 ⁇ Taq PCR MasterMix 10 ⁇ L, DPV CHv- ⁇ gc identification primer F 1 ⁇ L, CHv- ⁇ gc identification primer R 1 ⁇ L, and the template is a single colony resuspended solution 2 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 4 min, annealing at 55°C for 30 s, extension at 72°C for 2 min for a total of 30 cycles, and finally extension at 72°C for 10 min and storage at 16°C.
  • the PCR amplification system is: ddH 2 O 6 ⁇ L, 2 ⁇ Taq PCR MasterMix 10 ⁇ L, DPV CHv- ⁇ gC identification primer F 1 ⁇ L, CHv- ⁇ gC identification primer R 1 ⁇ L, and the template is a single colony resuspended solution 2 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 4 min, annealing at 55°C for 30 s, extension at 72°C for 2 min for a total of 30 cycles, and finally extension at 72°C for 10 min and storage at 16°C.
  • the PCR amplification system is ddH 2 O 22 ⁇ L, Max DNA Polymerase (purchased from Takara) 25 ⁇ L, GS1783-MiniF-F 1 ⁇ L, GS1783-MiniF-R 1 ⁇ L, template pEPKan-S plasmid 1 ⁇ L;
  • the PCR amplification conditions were pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5s for a total of 30 cycles, and finally extension at 72°C for 10min and storage at 16°C.
  • the PCR amplification system is: ddH 2 O 22 ⁇ L, Max DNA Polymerase 25 ⁇ L, CHv-UL23-F1 ⁇ L, CHv-UL23-R 1 ⁇ L, template DPV CHv genome 1 ⁇ L;
  • the PCR amplification conditions were pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5s for a total of 30 cycles, and finally extension at 72°C for 10min and storage at 16°C.
  • the PCR fusion system is: ddH 2 O 8 ⁇ L, Max DNA Polymerase 10 ⁇ L, template I_SceI-Kana-MiniF fragment and UL23-MiniF fragment 1 ⁇ L each;
  • the PCR fusion conditions were: pre-denaturation at 95°C for 5min, denaturation at 95°C for 15s, annealing at 55°C for 5s, and extension at 72°C for 1min, a total of 5 cycles;
  • the PCR amplification system is: fusion template 20 ⁇ L, upstream primer GS1783-MiniF-F 0.5 ⁇ L, downstream primer CHv-UL23-R 0.5 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5s, a total of 30 cycles, and finally extension at 72°C for 10min.
  • step (3) Incubate the bacterial solution obtained in step (3) at 42°C for 15 min and immediately put it in an ice-water mixture to cool for 20 min;
  • step (4) Take 50 mL of the bacterial solution obtained in step (4), centrifuge at 4500 ⁇ g for 10 min at 4°C, and remove the supernatant;
  • the PCR amplification system is: ddH 2 O 22 ⁇ L, Max DNA Polymerase 25 ⁇ L, MiniF-F 1 ⁇ L, MiniF-R 1 ⁇ L, template as step (9) Single colony resuspended solution 1 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 15s for a total of 30 cycles, and finally extension at 72°C for 10min and storage at 16°C.
  • step (2) Take 10 ⁇ L of the seed solution obtained in step (1) and inoculate it in 2 mL of LB liquid medium containing chloramphenicol, and incubate at 30°C for 2 hours until the bacterial solution appears slightly cloudy;
  • step (3) Add 1mL of LB liquid medium containing chloramphenicol and 5M L-arabinose with a final concentration of 2% to the bacterial liquid obtained in step (2) at 30°C, and incubate for 1 hour;
  • step (3) Immediately put the bacterial solution obtained in step (3) into a 42°C water bath and incubate for 30 min;
  • step (4) After the bacterial solution obtained in step (4) is incubated at 30°C for 2h, take 1 ⁇ l of the bacterial solution into 200 ⁇ l of LB liquid medium and mix well, then apply to LB solid medium containing chloramphenicol and incubate at 30°C for 24h ⁇ 48h;
  • step (6) Pick the single colony obtained in step (5) on the LB solid medium containing chloramphenicol and kanamycin double resistance and LB solid medium containing chloramphenicol to conduct parallel screening, and then select chloramphenicol It does not grow with kanamycin double-resistant LB solid medium, and colonies grown with chloramphenicol-resistant LB solid medium are identified by PCR amplification using primers SEQ ID NO: 9 and SEQ ID NO: 10, The positive clone GS1783-DPV CHv-BAC-G ⁇ gC-UL23 was obtained.
  • the PCR amplification system is: ddH 2 O 22 ⁇ L, Max DNA Polymerase 25 ⁇ L, upstream primer MiniF-F 1 ⁇ L, downstream primer MiniF-R 1 ⁇ L, template as step (6) single colony resuspension 1 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5s for a total of 30 cycles, and finally extension at 72°C for 10min and storage at 16°C.
  • step (4) The virus obtained in step (4) was repeatedly frozen and thawed twice, diluted 10 times, and inoculated in a 6-well plate overgrown with DEF. After incubation at 37°C and 5% CO 2 for 2 hours, the incubation solution was discarded and added Cells were fixed with 1% methylcellulose, cultured at 37°C and 5% CO 2 for 120 hours, and then selected non-fluorescent diseased cells. After repeated freezing and thawing twice, they were re-seeded in DEF to obtain DPV CHv- ⁇ gC-Q;
  • the PCR amplification system is: ddH 2 O 22 ⁇ L, Max DNA Polymerase 25 ⁇ L, upstream primer MiniF-F 1 ⁇ L, downstream primer MiniF-R 1 ⁇ L, template as step (6) extracted DPV CHv- ⁇ gC-Q virus genome 1 ⁇ L;
  • the PCR amplification conditions were: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 5s for a total of 30 cycles, and finally extension at 72°C for 10min and storage at 16°C.
  • the parental virus DPV CHV and the MiniF element DPV CHV- ⁇ gC-deleted strain were inoculated with DEF cells at 2 MOI, and the supernatant and cells were collected 6h, 12h, 18h, and 24h after inoculation, and repeated three times at each time point. After the collection was completed, freeze-thaw was repeated twice, and the virus titer was detected in a 96-well plate. The results showed that the deletion of gC gene partly affected the replication of DPV CHv virus.
  • the parental virus DPV CHV and the MiniF element DPV CHV- ⁇ gC-deficient strain were inoculated with DEF cells at 0.01 MOI, and the supernatant and cells were collected 12h, 24h, 48h, 72h after inoculation, and repeated three times at each time point. After the collection was completed, freeze-thaw was repeated twice, and the virus titer was detected in 96-well plates. The results showed that the deletion of gC gene significantly affected the proliferation of DPV CHv virus.
  • Example 3 The plaque test of the MiniV element DPV CHV- ⁇ gC-deleted strain and DPV CHv strain
  • the element to MiniF DPV CHV- ⁇ gC deletion strain was inoculated with DPV CHv DEF cells, 72h after poisoning were measured for yield TCID 50, wherein, DPV CHV- ⁇ gC deletion mutant TCID 50 of 10-3.75, DPV CHv strain TCID 50 is 10 -6.75 , and then the DPV CHV- ⁇ gC-deleted strain with MiniF element and DPV CHv strain were diluted by 10 times, 10 -2 -10 -7 were inoculated into six wells with DEF cells overgrown with slides In the plate, incubate in a 37°C incubator containing 5% CO 2 for 2h. During this period, shake it back and forth, left and right every 15 minutes to evenly distribute the virus.
  • Discard the virus solution wash the cells twice with PBS, add 4mL of 1% methylcellulose to each cell well, incubate in a 37°C incubator containing 5% CO 2 for 30h, observe the cells under a microscope, and remove the MiniF element DPV CHV - ⁇ gC-deficient strain selected 10 -3 dilution well, DPV CHv strain selected 10 -5 dilution well for follow-up test, aspirate methylcellulose, wash twice with PBS and add 1 mL of pre-cooled 4% paraformaldehyde, 4°C Fix for 30 minutes, add 1 mL of 30% H 2 O 2 after sucking out the liquid, soak for 30 Min at room temperature, suck out the liquid, and wash twice with distilled water.
  • Boshide ready-to-use SABC immunohistochemical staining kit (rabbit IgG), add 5% BSA blocking solution, incubate at room temperature for 30min, and aspirate the liquid; add DPV rabbit antibody diluted 1:100, the volume is 1mL/ Wells, incubate at 37°C for 3h; aspirate liquid, wash 3 times with PBS, add biotinylated goat anti-rabbit IgG dropwise, incubate at 37°C for 30min, wash 3 times with PBS, 5min/time; add SBAC reagent, incubate at 37°C for 30min, PBS Wash 3 times, 5min/time; aspirate the PBS, follow the steps of the Dr.
  • DAB color development kit add 800 ⁇ L of DAB mixed solution to each well, avoid light reaction for 30min, observe the background dyed light yellow under the microscope, discard the color Solution, washed with distilled water 3 times, 5 min each time, take the slide from the cell well and place it on the glass slide, observe under the microscope and make sure the size of the two plaques (see Figure 5, Figure 6, where Figure 6a is DPV plaque detection map of CHV- ⁇ gC-deficient strain; Figure 6b is the plaque detection map of DPV CHv strain); The plaque was 18.65% smaller than that of DPV CHv strain.
  • Pathogenicity Fifteen 4-week-old DPV antibody-negative and PCR-negative ducks were randomly divided into 3 groups of 5 ducks in each group. Group 1 was intramuscularly injected with DPV CHV- ⁇ gC, group 2 was intramuscularly injected with parental virus DPV CHV, each group was injected with the same virus, and group 3 was injected with an equal dose of MEM as a control group. , Observe and record the morbidity and mortality every day. It can be seen from the results that all the ducks injected with the parental virus died, while the ducks injected with DPV CHv- ⁇ gC and the control group did not die, indicating that the pathogenicity of DPV CHv- ⁇ gC decreased.

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Abstract

一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC及其构建方法。通过利用GS1783大肠杆菌菌株及pEPKan-S质粒，在细菌人工染色体重组鸭瘟病毒拯救系统平台上经过细胞内自发同源重组方法缺失MiniF元件，首次完成了无MiniF元件残留的鸭瘟病毒无痕缺失株的构建，解决了缺失鸭瘟病毒基因时在鸭瘟病毒基因组上残留MiniF元件的问题，为准确探究鸭瘟病毒基因功能及减毒活疫苗的构建提供了充分的技术支持。

Description

一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC及其构建方法 技术领域
本发明属于基因工程技术领域，具体涉及一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC及其构建方法。
背景技术
细菌人工染色体(bacterial artificial chromosome，BAC)是一种以F质粒为基础构成的细菌染色体克隆载体，常用来克隆150kd左右大小的DNA片段，最多可保存300kd个碱基对，该质粒主要包括oris，repE(控制F质粒复制)和parA，parB(控制拷贝数)等构成，以BAC为基础克隆的载体成嵌合体的效率较低，转化效率高，而且以环状结构存在于细菌体内，易于分辨和分离纯化，目前主要用于大片段基因组文库的构建和大的基因簇的相关研究。将完整病毒基因组DNA分子插入BAC载体，利用该载体编码的最小生育力因子复制子(Minimal fertility factor replicon，Mini-F)获得分子克隆化病毒，并结合大肠杆菌中成熟的基因定位修饰技术，从而实现在原核系统中病毒基因的缺失及外源基因的插入。目前较为常用的大肠杆菌基因定位修饰技术主要包括Red/ET介导的同源重组技术、RecA蛋白介导的同源重组技术、Cre/loxP介导的同源重组技术以及Tn转座子介导的随机插入和突变技术。利用分子克隆化技术手段现已获得成熟的细菌人工染色体鸭瘟病毒拯救系统平台，同时利用大肠杆菌基因定位修饰Red/ET介导的同源重组技术可在细菌人工染色体鸭瘟病毒拯救系统平台上进行鸭瘟病毒基因的缺失和外源基因的插入，该成果极大的推动了鸭瘟病毒基因功能的研究进程。
MiniF元件作为维持BAC载体复制的最小生育力因子复制子，主要由调控BAC复制起点(oriS)的repE、repF基因、调控复制子分配的sopA、sopB基因以及编码着丝粒区域的sopC基因构成。MiniF元件加入了抗性筛选基因和荧光标记基因，以完成细菌抗性筛选及BAC标记筛选，。MiniF元件插入病毒基因组，增加基因组长度，对病毒复制产生不确定影响。同时MiniF元件作为细菌序列在病毒基因组上残留，不利于减毒活疫苗的开发和许可。因此获得去除MiniF元件缺失株，已成为探究鸭瘟病毒基因缺失方法的重点。
鸭瘟(Duck Plague，DP)是由α-疱疹病毒亚科中鸭瘟病毒(Duck Plague virus，DPV)引起的鸭、鹅等水禽的急性接触性高度致死性传染病。该病首先由荷兰报道，随即在我国华南、华中和华东等养鸭业较为发达的地区流行，给我国的养鸭业造成了严重的经济损失。因此深入了解鸭瘟病毒基因功能、加强对鸭瘟疫病的研究对确保我国养鸭业健康、可持续发展尤为重要。
鸭瘟病毒DPV-CHv株基因组DNA全长162175bp，包含78个开放阅读框，可编码参与鸭瘟病毒生命周期的结构蛋白和功能蛋白，其中结构蛋白主要包括构成病毒的囊膜、间层、衣壳及DNA结合蛋白。囊膜蛋白为糖基化蛋白，包括gB、gC、gD、gE、gG、gH、gI、gJ、gK、gL、gM、gN十二种。疱疹病毒囊膜糖蛋白在病毒的结构、功能和毒力等方面发挥着重要作用。因此探究囊膜糖蛋白在鸭瘟病毒生命周期中的作用对深入探究鸭瘟病毒基因功能及开展鸭瘟疫病防治工作至关重要。
现有技术中利用以BAC为平台分子克隆化病毒的技术，将鸭瘟病毒基因组重组到含有BAC的病毒转移载体中，通过将长达162kb左右的鸭瘟病毒中国强毒株(DPVCHv)基因组克隆到细菌人工染色体(BAC)中，电转化大肠杆菌DH10B后获得的阳性克隆提取质粒后转染鸭胚成纤维细胞(DEF)，拯救出能在宿主细胞DEF上产生绿色荧光和空斑的细菌人工染色体重组鸭瘟病毒DEV CHv-BAC-G所获重组病毒DEV CHv-BAC-G，能够以病毒形式在细胞内生存，亦可以质粒的形式在大肠杆菌内复制，使得我们可以运用大肠杆菌系统成熟的遗传操作手段对DPVCHv基因组的任意位置进行修饰、改造、研究。但利用Red/ET修饰技术在细菌人工染色体重组鸭瘟病毒拯救系统平台上对鸭瘟病毒基因缺失后，会残留MiniF元件。MiniF元件的残留对基因功能的探究、减毒活疫苗的开发和许可存在影响。
发明内容
针对现有技术中的上述不足，本发明提供一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC及其构建方法，可有效解决残留MiniF元件的问题。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，包括以下步骤：
(1)以pKD4质粒为扩增模板，构建△gC扩kana上游引物和构建△gC扩kana下游引物，通过PCR方法扩增包含两侧带有gC基因同源臂及FRT序列的kana抗性基因片段，切胶回收，获得DPV gC左臂-FRT-kana-FRT-gC右臂片段；
(2)诱导含有pKD46和DPV CHv-BAC-G克隆菌DH10B RED重组系统表达；并制备含有pKD46和DPV CHv-BAC-G的DH10B阳性克隆菌感受态；电转化切胶回收DPV gC左臂-FRT-kana-FRT-gC右臂打靶片段进入含有pKD46和感染性克隆质粒的感受态中；筛选阳性克隆子，利用gC基因鉴定引物gC-R；gC-F进行PCR鉴定，获得阳性克隆子DPV CHv-BAC-G△gC-kana；
(3)去除阳性克隆子DPV CHv-BAC-G△gC-kana中的kana抗性基因，利用gC基因鉴定引物gC-R；gC-F进行PCR鉴定，获得阳性克隆子DPV CHv-BAC-G△gC；
(4)提取重组DPV CHv-BAC-G△gC质粒，将其电转化进入GS1783感受态中，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC，并制备GS1783-DPV CHv-BAC-G△gC感受态；
(5)以pEPKan-S为模板，GS1783-MiniF-F和GS1783-MiniF-R为引物，通过PCR方法扩增包含I_SceI酶切位点和Kana元件的碱基片段、位于MiniF元件ori2基因下游240bp处和位于MiniF元件ori2基因下游290bp处的同源臂片段I_SceI-Kana-MiniF，切胶回收获得I_SceI-Kana-MiniF片段；
(6)以CHv基因组为模板，CHv-UL23-F和CHv-UL23-R为引物，通过PCR方法扩增包含UL23基因、I_SceI-Kana-MiniF下游同源臂重叠25bp和位于MiniF元件ori2基因下游180bp处的同源臂片段，切胶回收，获得UL23-MiniF片段；
(7)以I_SceI-Kana-MiniF片段和UL23-MiniF片段为模板，进行PCR融合反应，然后以融合片段为模板，以GS1783-MiniF-F和CHv-UL23-R作为引物进行PCR扩增，获得I_SceI-Kana-MiniF-UL23打靶片段；
(8)将I_SceI-Kana-MiniF-UL23打靶片段转化到GS1783-DPV CHv-BAC-G△gC感受态中，经抗生素筛选和PCR鉴定，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23-Kana；
(9)将阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23-Kana中的I_SceI-Kana片段去掉，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23；
(10)从阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23中提取DPV  CHv-BAC-G△gC-UL23质粒，用DPV CHv-BAC-G△gC-UL23质粒转染DEF细胞，通过克隆筛选，获得去除MiniF元件的DPV CHv-△gC。
进一步地，步骤(1)中PCR扩增体系为：ddH 2O 14μL、
Figure PCTCN2019122997-appb-000001
Max DNA Polymerase 20μL、上游引物2μL、下游引物2μL、模板2μL；
PCR扩增条件为：98℃预变性1min、94℃变性15s、55℃退火15s、72℃延伸1min，共30个循环，最后72℃延伸10min。
进一步地，步骤(1)中引物序列为：
△gC扩kana片段上游引物:
5’-GCCGGTGTATCCTTCGTACATCATCAAGTTGTAACAATACTTAACGATTCTATATCGTGTAGGCTGGAGCTGCTTC-3’；
△gC扩kana片段下游引物:
5’-TAAAACGTCGTTTATTTATCAAAAGCTTTATTAAACATTTTATATTAAACCAGTATCATATGAATATCCTAATTAG-3’。
进一步地，步骤(2)和(3)中PCR扩增体系为：ddH 2O 6μL、2×Taq PCR MasterMin10μL、上游引物1μL、下游引物1μL、模板2μL；
PCR扩增条件为：95℃预变性5min、94℃变性4min、55℃退火30s、72℃延伸2min，共30个循环，最后72℃延伸10min。
进一步地，步骤(2)和(3)中引物序列为：
△gC鉴定上游引物:5’-ATGGTAAGCACATAAAAGTGTCGT-3’；
△gC鉴定下游引物:5’-ATTGCTATCCTATCAGTCCGTA-3’。
进一步地，步骤(5)中所述引物序列为：
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’；
GS1783-MiniF-R:5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATtagggataacagggtaatcgat-3’。
进一步地，步骤(6)中引物序列为：
CHv-UL23-F:5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’；
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCC TGAAGGCATATTCAACGGACATATTAAAAATTGA-3’。
进一步地，步骤(7)中PCR融合体系为：ddH 2O 8μL、
Figure PCTCN2019122997-appb-000002
Max DNA Polymerase 10μL、模板I_SceI-Kana-MiniF片段和UL23-MiniF片段各1μL；
PCR融合条件为：95℃预变性5min、95℃变性15s、55℃退火5s、72℃延伸1min，共5个循环。
进一步地，步骤(7)中PCR扩增体系为：融合模板20μL、上游引物GS1783-MiniF-F0.5μL、下游引物CHv-UL23-R 0.5μL；
PCR扩增条件为：98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min。
上述方法构建的鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC。
本发明的有益效果为：
本发明在细菌人工染色体重组鸭瘟病毒拯救系统平台的基础上，利用Red-based修饰技术，即利用含有可编码Red操纵子和I_SceI酶基因序列的GS1783大肠杆菌菌株及含有编码卡纳抗性及I_SceI酶切位点的质粒pEPKan-S，经细胞内自发同源重组方法缺失MiniF元件，首次完成了无MiniF元件残留的鸭瘟病毒缺失株的构建；解决了缺失鸭瘟病毒基因时MiniF元件残留的问题，为准确探究鸭瘟病毒基因功能及减毒活疫苗的构建提供了充分的技术支持。
附图说明
图1为pEPKan-S质粒图谱；
图2为利用Red-Based修饰技术和细胞内自发同源重组技术对MiniF元件进行缺失的操作流程图；
图3为去MiniF元件DPV CHv-ΔgC缺失病毒株病毒拯救后图片；
图4为DPV CHv-ΔgC接种鸭子48h和72h后肝脏DNA提取物的PCR检测；
图5为去MiniF元件DPV CHv-ΔgC缺失株与DPV-CHv株空斑大小对比结果图；
图6为去MiniF元件DPV CHv-ΔgC缺失株与DPV-CHv株空斑检测示意图；其中，图6a为DPV CHv-ΔgC缺失株空斑，图6b为DPV-CHv株空斑。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见 的，一切利用本发明构思的发明创造均在保护之列。
一种鸭瘟病毒gC基因无痕缺失株DPV CHv-ΔgC，构建过程所用材料及试剂如下：
1、实验材料
(1)细胞、菌株、病毒毒株、质粒
原代鸭胚成纤维细胞由10-11日龄非免疫受精鸭胚按常规方法制备；GS1783菌株由四川农业大学实验室保存；细菌人工染色体重组鸭瘟病毒DPV CHv-BAC-G及其感染性克隆pBAC-DPV由四川农业大学预防兽医研究所禽病中心制备并保存；pKD4和pEPKan-S质粒由四川农业大学实验室保存。
2、分子生物学试剂
质粒小提试剂盒购自TIANGEN公司；QIAGEN Plasmid Midi Kit购自QIAGEN公司；普通琼脂糖凝胶DNA回收试剂盒购自TIANGEN公司；
Figure PCTCN2019122997-appb-000003
Max DNA Polymerase购自Takara公司；TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0购自TaKaRa公司；Lipofectamine 3000购自Invitrogen公司；
3、实验所用溶液及其配制
LB液体培养基：称取Tryptone 10g、Yeast Extract 5g、氯化钠10g溶于800mL去离子水中，充分搅拌，定容至lL，高温高压灭菌。
LB固体培养基：在定容至1L的LB液体培养基中加入15g琼脂粉，高温高压灭菌后，冷却至60℃左右，加入1.5mL氯霉素(储存浓度25mg/ml)或1.5mL卡那霉素(储存浓度50mg/mL)，铺制平板，待凝固后，4℃保存。
MEM：将9.6g MEM干粉和2.2g碳酸氢钠溶于800mL去离子水，充分搅拌，调节pH值至7.4，定容至lL，过滤除菌，4℃保存。
实施例1 制备去MiniF元件的鸭瘟病毒gC基因缺失株DPV CHv-ΔgC
构建鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC，其构建方法包括以下步骤：
1、按照质粒抽提试剂盒(购自TIANGEN公司)提取pKD4质粒，并设计引物△gC扩kana-F和△gC扩kana-R，引物具体序列如下：
△gC扩kana-F:5’-GCCGGTGTATCCTTCGTACATCATCAAGTTGTAACAATACTTAACGATTCTATATCGTGTAGGCTGGAGCTGCTTC-3’；(SEQ ID NO:1)
△gC扩kana-R:5’-TAAAACGTCGTTTATTTATCAAAAGCTTTATTAAACATTTTATATTAAACCAGTATCATATGAATATCCTAATTAG-3’；(SEQ ID NO:2)
(1)以pKD4质粒作为扩增模板，以△gC扩kana-F和△gC扩kana-R为引物，通过PCR扩增包含两侧带有gC基因同源臂及FRT序列的kana抗性基因片段，切胶回收，获得DPV gC左臂-FRT-kana-FRT-gC右臂片段；
PCR扩增体系为：ddH 2O 14μL、
Figure PCTCN2019122997-appb-000004
Max DNA Polymerase 20μL、构建△gC扩kana片段上游引物2μL、构建△gC扩kana片段下游引物2μL、模板Pkd4质粒2μL；
PCR扩增条件为：98℃预变性1min、98℃变性15s、55℃退火15s、72℃延伸1min，共30个循环，最后72℃延伸10min，于16℃保存。
2、含pKD46和DPV CHv-BAC-G感染性克隆(pBAC-DPV)DH10B中RED重组系统的诱导表达及感受态的制备及电转gC左臂-FRT-kana-FRT-△gC右臂打靶片段
(1)从保存菌种的冻存管中挑取含pKD46和pBAC-DPV的DH10B阳性克隆菌，氨苄抗性和氯霉素抗性的LB平板30℃过夜，扩大培养。
(2)挑取鉴定正确的单菌落，接种到5mL含氨苄的LB液体培养基中，30℃，180rpm 培养过夜。
LB液体培养基的制备方法为：称取Tryptone 10g、Yeast Extract 5g、氯化钠10g溶于800mL去离子水中，充分搅拌，定容至lL，高温高压灭菌
(3)将过夜菌体按2％接种量接种到100mL含氯霉素和氨苄抗性LB液体培养基中，30℃，180r摇菌。
(4)当OD 600＝0.2时，1：10加入1mol/L的L-阿拉伯糖，使其终浓度为100mmol/L 30℃诱导90min(OD 600≈0.6)，使pKD46上的3个蛋白充分表达。冰上预冷15-30min。
(5)预先4℃降温的冷冻离心机离心收集菌体，5000rpm离心5min，倒尽上清。
(6)用50mL的冰预冷灭菌超纯水洗菌体2次，用枪吹打时一定要轻柔，并时刻保持在冰上，6000rpm离心5min，离心后要立即倒掉上清，以免菌体重新悬浮造成损失。
(7)用200μL的冰预冷灭菌超纯水轻轻地溶解菌体，置于冰上，备用。
(8)转化PCR产物到含有pKD46和感染性克隆pBAC-DPV质粒的感受态中，在λ-Red重组酶介导下，通过同源重组，实现卡那霉素抗性基因对gc基因的替换。操作步骤如下：
(9)将约200ng PCR产物片段gC左臂-FRT-kana-FRT-gC右臂加入制备的200μl感受态，混匀，转入0.2cm电击杯中，电击仪作电转化，电击条件：200Ω，25μF，电击电压2.3kV。
(10)电击后迅速加入1mL的LB重悬细菌，150r/min，37℃培养，复苏2h。
(11)取200μL菌液涂布于一个氯酶和氨苄抗性LB平板，剩下的离心后涂布于另一个氯酶和氨苄LB平板，37℃培养箱倒置培养16h-24h。PCR鉴定所得单菌落，获得阳性菌落DPV CHv-BAC-G△gC-kana，以该步骤中生长的单菌落重悬液为模板，利用鉴定引物gC-F和gC-R进行PCR鉴定，获得阳性克隆子CHv-BAC-G△gC-kana
△gC-F:5，-ATGGATTGCACATAAAAGTGTCGT-3；(SEQ ID NO:3)
△gC-R:5，-ATTGCTGTCCTATCAGTCCGTA-3；(SEQ ID NO:4)
PCR扩增体系为：ddH 2O 6μL、2×Taq PCR MasterMix 10μL、DPV CHv-△gc鉴定引物F 1μL、CHv-△gc鉴定引物R 1μL、模板为单菌落重悬液2μL；
PCR扩增条件为：95℃预变性5min、95℃变性4min、55℃退火30s、72℃延伸2min，共30个循环，最后72℃延伸10min，于16℃保存。
3、去掉kana抗性基因片段
(1)按照质粒抽提试剂盒提取pCP20质粒DNA，以1：50的比例将CHv-BAC-G△gC-kana接种至100mL含卡那和氯酶抗生素LB液体培养基中，于37℃剧烈培养2～3h至OD 600为0.4左右；然后迅速将CHv-BAC-G△gC-kana重组菌液冰浴30min以上。
(2)取pCP20质粒1～2ΜL加入100上述
Figure PCTCN2019122997-appb-000005
-BAC-G△gC-kana感受态中，轻轻混匀，冰上放置30min；42℃加热90S后，再在冰上放置1～2min；加入500μL LB，30℃水浴中以150r/min震荡培养1～2h；取200ΜL培养物LB平板上，30℃培养18h；挑取菌落进行菌液PCR鉴定，同时将阳性菌液划线氯酶LB平板42℃过夜诱导FLP重组酶表达，挑出单菌落到卡那抗性平板上未生长，则卡那抗性基因己经被FLP重组酶删除，利用引物SEQ ID NO:3和SEQ ID NO:4进行PCR扩增，鉴定阳性菌落，命名为DPV CHv-BAC-G△gC；
PCR扩增体系为：ddH 2O 6μL、2×Taq PCR MasterMix 10μL、DPV CHv-△gC鉴定引物F 1μL、CHv-△gC鉴定引物R 1μL、模板为单菌落重悬液2μL；
PCR扩增条件为：95℃预变性5min、95℃变性4min、55℃退火30s、72℃延伸2min， 共30个循环，最后72℃延伸10min，于16℃保存。
4、将鉴定正确的DPV CHv-BAC-G△gC克隆扩大培养至200ml氯酶抗性LB中培养过夜，提取重组质粒。
5、将提取的重组质粒，电转化至GS1783菌株中，获得GS1783-DPV CHv-BAC-G△gC。
6、扩增I_SceI-Kana-MiniF片段
(1)将带有pEPKan-S质粒的大肠杆菌置于含有卡那霉素的LB固体培养基中复苏，37℃培养过夜；挑取单菌落接种于含有卡那霉素的LB液体培养基中，37℃培养过夜，质粒小提试剂盒提取pEPKan-S质粒(pEPKan-S质粒图谱见图1)；
(2)以pEPKan-S质粒为模板，并设计引物GS1783-MiniF-F和GS1783-MiniF-R，包含I_SceI酶切位点和Kana元件的碱基片段，以及位于MiniF元件ori2基因下游240bp处和位于MiniF元件ori2基因下游290bp处的同源臂片段I_SceI-Kana-MiniF，普通琼脂糖凝胶DNA回收试剂盒(购自TIANGEN公司)回收扩增片段，获得I_SceI-Kana-MiniF片段；引物具体序列如下：
GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’(SEQ ID NO:5)
GS1783-MiniF-R:5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATtagggataacagggtaatcgat-3’(SEQ ID NO:6)
PCR扩增体系为ddH 2O 22μL、
Figure PCTCN2019122997-appb-000006
Max DNA Polymerase(购自Takara公司)25μL、GS1783-MiniF-F 1μL、GS1783-MiniF-R 1μL、模板pEPKan-S质粒1μL；
PCR扩增条件为98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min，于16℃保存。
8、扩增UL23-MiniF片段
(1)制备鸭胚成纤维细胞(DEF)并接种于25T细胞瓶，37℃，5％CO 2培养24h后，接种5MOI DPV CHv，37℃，5％CO 2培养48h后收获病毒，反复冻融2次后，按照TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0(购自TaKaRa公司)说明书提取DPV CHv基因组；
(2)以DPV CHv基因组为模板，设计引物CHv-UL23-F和CHv-UL23-R，通过PCR扩增包含UL23基因、I_SceI-Kana-MiniF下游同源臂重叠25bp和位于MiniF元件ori2基因下游180bp处的同源臂片段，普通琼脂糖凝胶DNA回收试剂盒回收扩增片段，获得UL23-MiniF片段；具体序列如下：
CHv-UL23-F:5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’(SEQ ID NO:7)
CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCC TGAAGGCATATTCAACGGACATATTAAAAATTGA-3’(SEQ ID NO:8)
PCR扩增体系为：ddH 2O 22μL、
Figure PCTCN2019122997-appb-000007
Max DNA Polymerase 25μL、CHv-UL23-F1μL、CHv-UL23-R 1μL、模板DPV CHv基因组1μL；
PCR扩增条件为98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min，于16℃保存。
9、融合I_SceI-Kana-MiniF片段和UL23-MiniF片段获得I_SceI-Kana-MiniF-UL23打靶片段
(1)以I_SceI-Kana-MiniF片段和UL23-MiniF片段为模板，进行PCR融合，然后以 融合后的片段为模板，SEQ ID NO:6和SEQ ID NO:8为引物，PCR扩增获得I_SceI-Kana-MiniF-UL23片段；
PCR融合体系为：ddH 2O 8μL、
Figure PCTCN2019122997-appb-000008
Max DNA Polymerase 10μL、模板I_SceI-Kana-MiniF片段和UL23-MiniF片段各1μL；
PCR融合条件为：95℃预变性5min、95℃变性15s、55℃退火5s、72℃延伸1min，共5个循环；
PCR扩增体系为：融合模板20μL、上游引物GS1783-MiniF-F 0.5μL、下游引物CHv-UL23-R 0.5μL；
PCR扩增条件为：98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min。
再利用Red-Based修饰技术和细胞内自发同源重组技术对MiniF元件进行缺失的操作流程图(见图2)，具体过程包括步骤10～12。
10、制备GS1783-DPV CHv-BAC-G△gC电转感受态进行打靶片段打靶
(1)将GS1783-DPV CHv-BAC-G△gC冻存菌置于含有氯霉素的LB固体培养基中复苏，30℃培养过夜；
(2)挑取GS1783-DPV CHv-BAC-G△gC单菌落接种于5mL含氯霉素的LB液体培养基中，30℃培养过夜，获得种子液；
(3)将5mL种子液加入100mL含氯霉素的LB液体培养基中，于30℃摇至OD 600值在0.5～0.7之间；
(4)将步骤(3)所得菌液于42℃培养15min后立即放入冰水混合物中冷却20min；
(5)取50mL步骤(4)所得菌液，于4℃4500×g离心10min，去上清；
(6)用预冷超纯水在冰上反复清洗步骤(5)所得菌体沉淀；
(7)加入超纯水将步骤(6)所得菌液定容至500μL，每管100μL分装至预冷EP管，获得GS1783-DPV CHv-BAC-G△gC电转感受态；
(8)在100μL电转感受态中加入200ng I SceI-Kana-MiniF-UL23打靶片段，混匀后将感受态和打靶片段一起加入2mm预冷的电击杯底部，15kV/cm条件下进行电击；
(9)加入100μL LB液体培养基重悬电击后菌体，30℃摇菌1h后，4500×g离心菌体2min，弃上清，200μL LB液体培养基悬浮沉淀，涂布含有卡那霉素和氯霉素双抗生素抗性的LB固体培养基，于30℃培养48h；
(10)PCR鉴定步骤(9)所得单菌落，获得阳性菌落GS1783-DPV CHv-BAC-G△gC-UL23-Kana，以步骤(9)中生长的单菌落重悬液为模板，利用鉴定引物MiniF-F和MiniF-R鉴定阳性菌落；
MiniF-F:5’-GTTATCCACTGAGAAGCGAACG-3’；(SEQ ID NO:9)
MiniF-R:5’-GGCTGTAAAAGGACAGACCACA-3’；(SEQ ID NO:10)
PCR扩增体系为：ddH 2O 22μL、
Figure PCTCN2019122997-appb-000009
Max DNA Polymerase 25μL、MiniF-F 1μL、MiniF-R 1μL、模板为步骤(9)单菌落重悬液1μL；
PCR扩增条件为：98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸15s，共30个循环，最后72℃延伸10min，于16℃保存。
11、去掉I_SceI-Kana片段
(1)挑取GS1783-DPV CHv-BAC-G△gC-UL23-Kana单菌落接种于2mL含氯霉素的LB液体培养基中，30℃培养过夜，获得种子液；
(2)取10μL步骤(1)所得种子液接种于2mL含氯霉素的LB液体培养基中，30℃培养2h至菌液呈现轻微云雾状；
(3)向步骤(2)所得菌液中加入1mL含氯霉素的LB液体培养基和5M终浓度为2％的L-阿拉伯糖30℃，培养1h；
(4)将步骤(3)所得菌液立即放入42℃水浴中培养30min；
(5)将步骤(4)所得菌液置于30℃培养2h后，取1μl菌液加入200μl LB液体培养基中混匀后涂布到含有氯霉素的LB固体培养基，30℃培养24h～48h；
(6)挑取步骤(5)中所得单菌落在含氯霉素和卡纳霉素双抗性LB固体培养基和氯霉素单抗性LB固体培养基上进行平行筛选，将氯霉素和卡纳霉素双抗性LB固体培养基不生长，氯霉素单抗性LB固体培养基生长的菌落通过PCR方法利用引物SEQ ID NO:9和SEQ ID NO:10进行PCR扩增鉴定，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23。
PCR扩增体系为：ddH 2O 22μL、
Figure PCTCN2019122997-appb-000010
Max DNA Polymerase 25μL、上游引物MiniF-F 1μL、下游引物MiniF-R 1μL、模板为步骤(6)单菌落重悬液1μL；
PCR扩增条件为：98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min，于16℃保存。
12、拯救DPV CHv-ΔgC病毒
(1)将GS1783-DPV CHv-BAC-G△gC-UL23冻存菌置于含有氯霉素的LB固体培养基中复苏，30℃培养过夜；
(2)按照QIAGEN Plasmid Midi Kit(购自QIAGEN公司)操作说明提取DPV CHv-BAC-G△gC-UL23质粒；
(3)制备鸭胚成纤维细胞(DEF)并接种于12孔板，37℃，5％CO2培养24h后，按照Lipofectamine 3000(购自Invitrogen公司)操作说明转染DPV CHv-BAC-G△gC-UL23质粒，96h后观察荧光斑，收集病毒，反复冻融2次后接种于长满DEF的6孔板；
(4)重复步骤(3)三次；
(5)步骤(4)获得的病毒反复冻融2次后，10倍倍比稀释，接种于长满DEF的6孔板中，37℃，5％CO 2孵育2h后，弃孵育液，加入1％甲基纤维素固定细胞，37℃，5％CO 2培养120h后，挑取挑取无荧光病变细胞，反复冻融2次后重新接种于DEF中，获得DPV CHv-ΔgC-Q；
(6)按照TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0说明书提取DPV CHv-ΔgC病毒基因组，利用引物SEQ ID NO:9和SEQ ID NO:10进行PCR扩增，鉴定缺失的MiniF元件，最终获得无MiniF元件残留缺失病毒株DPV CHv-ΔgC阳性病毒株，见图3。
PCR扩增体系为：ddH 2O 22μL、
Figure PCTCN2019122997-appb-000011
Max DNA Polymerase 25μL、上游引物MiniF-F 1μL、下游引物MiniF-R 1μL、模板为步骤(6)提取的DPV CHv-ΔgC-Q病毒基因组1μL；
PCR扩增条件为：98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min，于16℃保存。
实施例2 去MiniF元件DPV CHV-△gC缺失株生长曲线的测定
1、一步生长曲线的测定
将亲本病毒DPV CHv和去MiniF元件DPV CHV-△gC缺失株分别以2MOI接种DEF细胞，接毒后6h、12h、18h、24h收集上清和细胞，每个时间点做三次重复。待收集完全后，反复冻融2次，于96孔板检测病毒滴度，结果表明gC基因的缺失部分影响DPV CHv 病毒的复制。
2、多步生长曲线的测定
将亲本病毒DPV CHv和去MiniF元件DPV CHV-△gC缺失株分别以0.01MOI接种DEF细胞，接毒后12h、24h、48h、72h收集上清和细胞，每个时间点做三次重复。待收集完全后，反复冻融2次，于96孔板检测病毒滴度，结果表明gC基因的缺失显著影响DPV CHv病毒的增殖情况。
实施例3 去MiniF元件DPV CHV-△gC缺失株与DPV CHv株空斑试验
将去MiniF元件DPV CHV-△gC缺失株与DPV CHv株接种于DEF细胞中，72h收毒后分别测其TCID 50，其中，DPV CHV-△gC缺失株TCID 50为10-3.75，DPV CHv株TCID 50为10 -6.75，再分别将去MiniF元件DPV CHV-△gC缺失株与DPV CHv株进行10倍倍比稀释，10 -2-10 -7接种于含爬片长满DEF细胞的六孔板中，于含5％CO 2的37℃培养箱中中培养2h，期间每隔15min进行前后左右摇晃，使病毒分散均匀。
弃病毒液，用PBS洗涤细胞2次，每个细胞孔加入4mL 1％甲基纤维素，于含5％CO 2的37℃培养箱中中培养30h，显微镜下观察细胞，去MiniF元件DPV CHV-△gC缺失株选取10 -3稀释孔，DPV CHv株选取10 -5稀释孔进行后续试验，吸出甲基纤维素，用PBS洗涤2次后加入1mL预冷的4％多聚甲醛，4℃固定30min，吸出液体后加入1mL 30％H 2O 2，室温浸泡30Min，吸出液体，蒸馏水洗涤2次。
按照博士德即用型SABC免疫组化染色试剂盒(兔IgG)操作步骤，滴加5％BSA封闭液，室温孵育30min，吸出液体；加入1:100稀释的DPV兔抗，加入体积为1mL/孔，37℃孵育3h；吸出液体，PBS洗涤3次，滴加生物素化山羊抗兔IgG，37℃孵育30min，PBS洗涤3次，5min/次；滴加SBAC试剂，37℃孵育30min，PBS洗涤3次，5min/次；吸出PBS，按照博士德DAB显色试剂盒操作步骤，每孔滴加800μL的DAB混合液，避光反应30min，于显微镜下观察背景染为淡黄色，弃显色液，用蒸馏水洗涤3次，每次5min，将细胞孔中的爬片取出置于载玻片上，显微镜下观察并必将两者空斑大小(见图5，图6，其中，图6a为DPV CHV-△gC缺失株的空斑检测图；图6b为DPV CHv株的空斑检测图)；如图5和图6的检测结果所示，去MiniF元件DPV CHV-△gC缺失株的空斑较DPV CHv株的空斑小18.65％。
本发明还对鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC做了遗传稳定性实验、致病性实验和免疫原性实验。
遗传稳定性：鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC在DEF细胞上传代20代，均出现蚀斑，表明所获得的鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC在DEF中稳定遗传。
致病性：将15只4周龄DPV抗体阴性和PCR检测阴性鸭随机分成3组，每组5只。第1组肌肉注射DPV CHv-ΔgC，第2组肌肉注射亲本病毒DPV CHv，两组中每只注射病毒的毒价相同，第3组注射等剂量的MEM，作为对照组，每组单独隔离饲养，观察，每天记录发病死亡情况。由结果可知，注射有亲本病毒的鸭全部死亡，而注射有DPV CHv-ΔgC以及对照组中的鸭无死亡，说明DPV CHv-ΔgC致病性降低。
免疫原性：将10只4周龄DPV抗体阴性和PCR检测阴性鸭随机分成2组，每组5只。第1组肌肉注射DPV CHv-ΔgC，第2组注射等剂量的MEM，作为对照组，每组单独隔离饲养。在免疫后14天采血分离血清，利用纯化的DPV作为包被抗原进行间接ELISA检测，结果可检测到注射DPV CHv-ΔgC鸭血清中的抗体，说明DPV CHv-ΔgC具有良好的免疫原性。

Claims (10)

  1. 一种鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，包括以下步骤：
    (1)以pKD4质粒为扩增模板，构建△gC扩kana上游引物和构建△gC扩kana下游引物，通过PCR方法扩增包含两侧带有gC基因同源臂及FRT序列的kana抗性基因片段，切胶回收，获得DPV gC左臂-FRT-kana-FRT-gC右臂片段；
    (2)诱导含有pKD46和DPV CHv-BAC-G克隆菌DH10B RED重组系统表达；并制备含有pKD46和DPV CHv-BAC-G的DH10B阳性克隆菌感受态；电转化切胶回收DPV gC左臂-FRTkana-FRT-gC右臂打靶片段进入含有pKD46和感染性克隆质粒的感受态中；筛选阳性克隆子，利用gC基因鉴定引物gC-R；gC-F进行PCR鉴定，获得阳性克隆子DPV CHv-BAC-G△gCkana；
    (3)去除阳性克隆子DPV CHv-BAC-G△gC-kana中的kana抗性基因，利用gC基因鉴定引物gC-R；gC-F进行PCR鉴定，获得阳性克隆子DPV CHv-BAC-G△gC；
    (4)提取重组DPV CHv-BAC-G△gC质粒，将其电转化进入GS1783感受态中，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC，并制备GS1783-DPV CHv-BAC-G△gC感受态；
    (5)以pEPKan-S为模板，GS1783-MiniF-F和GS1783-MiniF-R为引物，通过PCR方法扩增包含I_SceI酶切位点和Kana元件的碱基片段、位于MiniF元件ori2基因下游240bp处和位于MiniF元件ori2基因下游290bp处的同源臂片段I_SceI-Kana-MiniF，切胶回收获得I_SceI-Kana-MiniF片段；
    (6)以CHv基因组为模板，CHv-UL23-F和CHv-UL23-R为引物，通过 PCR方法扩增包含UL23基因、I_SceI-Kana-MiniF下游同源臂重叠25bp和位于MiniF元件ori2基因下游180bp处的同源臂片段，切胶回收，获得UL23-MiniF片段；
    (7)以I_SceI-Kana-MiniF片段和UL23-MiniF片段为模板，进行PCR融合反应，然后以融合片段为模板，以GS1783-MiniF-F和CHv-UL23-R作为引物进行PCR扩增获得I_SceI-KanaMiniF-UL23打靶片段；
    (8)将I_SceI-Kana-MiniF-UL23打靶片段转化到GS1783-DPV CHv-BAC-G△gC感受态中，经抗生素筛选和PCR鉴定，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23-Kana；
    (9)将阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23-Kana中的I_SceI-Kana片段去掉，获得阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23；
    (10)从阳性克隆子GS1783-DPV CHv-BAC-G△gC-UL23中提取DPV CHv-BAC-G△gC-UL23质粒，用DPV CHv-BAC-G△gC-UL23质粒转染DEF细胞，通过克隆筛选，获得去除MiniF元件的DPV CHv-△gC。
  2. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(1)中所述PCR扩增体系为：ddH 2O14μL、
    Figure PCTCN2019122997-appb-100001
    Max DNA Polymerase 20μL、上游引物2μL、下游引物2μL、模板2μL；
    PCR扩增条件为：98℃预变性1min、94℃变性15s、55℃退火15s、72℃延伸1min，共30个循环，最后72℃延伸10min。
  3. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(1)中所述引物序列为：
    △gC扩kana片段上游引物:
    5’-GCCGGTGTATCCTTCGTACATCATCAAGTTGTAACAATACTTAACGATTCTATATCGTGTAGGCTGGAGCTGCTTC-3’；
    △gC扩kana片段下游引物:
    5’-TAAAACGTCGTTTATTTATCAAAAGCTTTATTAAACATTTTATATTAAACCAGTATCATATGAATATCCTAATTAG-3’。
  4. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(2)和(3)中所述PCR扩增体系为：ddH 2O 6μL、2×Taq PCR MasterMin10μL、上游引物1μL、下游引物1μL、模板2μL；
    PCR扩增条件为：95℃预变性5min、94℃变性4min、55℃退火30s、72℃延伸2min，共30个循环，最后72℃延伸10min。
  5. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(2)和(3)中所述引物序列为：
    △gC鉴定上游引物:5’-ATGGTAAGCACATAAAAGTGTCGT-3’；
    △gC鉴定下游引物:5’-ATTGCTATCCTATCAGTCCGTA-3’。
  6. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(5)中所述引物序列为：
    GS1783-MiniF-F:5’-TTATTAATCTCAGGAGCCTGTGTAGCGTTTATAGGAAGTAGTGTTCTGTCATGATGCCTGCAAGCGGTAACGAAAACGATtgttacaaccaattaacc-3’；
    GS1783-MiniF-R:5’-ATCGTTTTCGTTACCGCTTGCAGGCATCATGACA GAACACTACTTCCTATtagggataacagggtaatcgat-3’。
  7. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(6)中所述引物序列为：
    CHv-UL23-F:5’-GCCTGCAAGCGGTAACGAAAACGATtcaattaattgtcatctcgg-3’；
    CHv-UL23-R:5’-CCGCTCCACTTCAACGTAACACCGCACGAAGATTTCTATTGTTCCTGAAGGCATATTCAACGGACATATTAAAAATTGA-3’。
  8. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(7)中所述PCR融合体系为：ddH 2O8μL、
    Figure PCTCN2019122997-appb-100002
    Max DNA Polymerase 10μL、模板I_SceI-Kana-MiniF片段和UL23-MiniF片段各1μL；
    PCR融合条件为：95℃预变性5min、95℃变性15s、55℃退火5s、72℃延伸1min，共5个循环。
  9. 根据权利要求1中所述鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC的构建方法，其特征在于，步骤(7)中所述PCR扩增体系为：融合模板20μL、上游引物GS1783-MiniF-F 0.5μL、下游引物CHv-UL23-R 0.5μL；
    PCR扩增条件为：98℃预变性2min、98℃变性10s、55℃退火15s、72℃延伸5s，共30个循环，最后72℃延伸10min。
  10. 权利要求1～9任一项所述方法构建的鸭瘟病毒去MiniF元件gC基因缺失株DPV CHv-ΔgC。
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