搜索

Construction of FWPV Chimaeric MVA
FWPV 嵌合 MVA的建立   

引用 收藏 1 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Journal of Virology
May 2013

Abstract

Construction of chimaeric MVA is a useful tool with which to study gene function of related viruses. The protocol given here describes MVA chimaeras containing genes from Fowlpox virus (FWPV), although this can be applied to DNA derived from other organisms. There are a number of steps required to make the chimaeric MVA: 1) Purification of viral particles; 2) Extraction of DNA from purified viral particles; 3) Assembly of linear recombination templates; 4) Transfection of linear recombination templates; 5) Selection of chimaeric MVA.
Note: This procedure uses live virus, and should be conducted using Good Microbiological Practice, in accordance with international and national biocontainment requirements. This procedure also involves Genetic Modification of microorganisms, and appropriate safety approval should be obtained before commencing.

Materials and Reagents

  1. Purification of viral particles
    1. Fowlpox virus (FWPV) (e.g. ATCC, catalog number: VR-251 )
    2. Modified Vaccinia virus Ankara (MVA) (ATCC, catalog number: VR-1508 )
    3. CEF cells (Institute for Animal Health)
    4. Tris-EDTA buffer (Sigma-Aldrich, catalog number: 93283 )
    5. DMEM (e.g. Sigma-Aldrich, catalog number: D6429 )
    6. Foetal bovine serum (FBS) (e.g. Sigma-Aldrich, catalog number: F9665 )
    7. 10x medium 199 with Earle’s salts (Sigma-Aldrich, catalog number: M0650 )
    8. Heat Inactivated newborn bovine serum (Life Technologies, catalog number: 26010074 )
    9. Tryptose phosphate broth (Sigma-Aldrich, catalog number: T8159 )
    10. Nystatin (Sigma-Aldrich, catalog number: N1638 )
    11. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333 )
    12. L-glutamine solution (Sigma-Aldrich, catalog number: G7513 )
    13. Sodium bicarbonate (Sigma-Aldrich, catalog number: S8761 )
    14. Amicon Ultra-15 Centrifugal filter unit (100 kDa membrane NMWL) (Merck Millipore, catalog number: UFC910024 )
    15. FWPV virus growth medium (see Recipes)
    16. MVA virus growth medium (see Recipes)
    17. TE (see Recipes)

  2. Extraction of DNA from purified viral particles
    1. Phenol saturated with 10 mM Tris-HCl (Sigma-Aldrich, catalog number: P4557 )
    2. Phenol-chloroform-isoamyl alcohol (25:24:1, saturated with 10 mM Tris, pH 8.0) (Sigma-Aldrich, catalog number: P2069 )
    3. Chloroform-isoamyl alcohol (24:1) (Sigma-Aldrich, catalog number: 25666 )
    4. 3 M Sodium acetate (Sigma-Aldrich, catalog number: 71196 )
    5. Ethanol (Sigma-Aldrich, catalog number: E7023 )
    6. 10 mM Tris-Cl (pH 8.5) (QIAGEN, catalog number: 19086 )
    7. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
    8. EDTA (Sigma-Aldrich, catalog number: EDS )
    9. NaCl (Sigma-Aldrich, catalog number: S3014 )
    10. SDS solution (Sigma-Aldrich, catalog number: 71736 )
    11. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
    12. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
    13. 2x extraction buffer (see Recipes)

  3. Assembly of linear recombination templates
    1. PCR oligonucleotides (Sigma-Aldrich)
    2. Accuprime Taq high fidelity polymerase (Life Technologies, catalog number: 12346086 )
    3. pNEB193 (New England Biolabs)
    4. Plasmid encoding Escherichia coli (E.coli) gpt under p7.5 promoter (e.g. pGNR, available on request)
    5. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
    6. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )

  4. Transfection of linear recombination templates
    1. DF-1 cells (ATCC, catalog number: CRL-12203 )
    2. DMEM (e.g. Sigma-Aldrich, catalog number: D6429)
    3. Foetal bovine serum (FBS) (e.g. Sigma-Aldrich, catalog number: F9665)
    4. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333)
    5. Salmon sperm DNA solution (Life Technologies, catalog number: 15632011 )
    6. Opti-MEM (Life Technologies, catalog number: 31985062 )
    7. Polyfect (QIAGEN, catalog number: 301105 )
    8. DF-1 culture medium (see Recipes)

  5. Selection of chimaeric MVA
    1. DMEM (e.g. Sigma-Aldrich, catalog number: D6429)
    2. Foetal bovine serum (FBS) (e.g. Sigma-Aldrich, catalog number: F9665)
    3. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333)
    4. Mycophenolic acid (Sigma-Aldrich, catalog number: M5255 )
    5. Xanthine (Sigma-Aldrich, catalog number: X3627 )
    6. Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
    7. Hydrochloric acid (Sigma-Aldrich, catalog number: H9892 )
    8. Sodium hydroxide (Sigma-Aldrich, catalog number: S2770 )
    9. 2% DMEM + MXH (see Recipes)

Equipment

  1. Benchtop centrifuge (e.g. Hettich®, model: Rotanta 460 )
  2. Thermal cycler (Techne Progene®)
  3. Laminar flow microbiological safety cabinet (e.g. HeraSafe®, model: 51022738 )
  4. Humidified CO2 incubator (e.g. Thermo Fisher Scientific, model: BB15 )
  5. Cuphorn sonicator (e.g. UltrasonicsTM, model: W-375 )

Procedure

  1. Purification of viral particles
    Use aseptic technique, in a laminar flow microbiological safety cabinet.
    1. Infect confluent CEF cells with FWPV or MVA at 0.01 plaque-forming units (pfu) per cell.
      Note: The number of cells to infect will depend on the number of chimaeric MVA viruses to be made. As a guide, 30 infected 175 cm2 flasks of FWPV yields ~150 μg DNA; 2 infected175 cm2 flasks of MVA yields ~800 ng DNA.
    2. Culture in virus growth medium at 37 °C 5% CO2 for 5-7 days, until extensive cytopathic effects are visible.
    3. Freeze-thaw the culture (still in the flask) 3 times.
      Note: Freeze at -80 °C. The flask may be thawed at room temperature or 37 °C, but do not leave at elevated temperature longer than absolutely necessary.
    4. Transfer the infected cell lysate to a centrifuge tube.
    5. Centrifuge at 1,500 x g for 15 min.
    6. Transfer the supernatant to an Amicon Ultra-15 Centrifugal filter unit (100 kDa NMWL).
    7. Centrifuge at 1,500 x g for 60 min at 4 °C.
    8. Resuspend the lysate retained on the membrane (approx. 200 μl) containing virus particles in 2 ml TE.
    9. Store at 4 °C overnight, or proceed directly to Section B.

  2. Extraction of DNA from purified viral particles
    1. Combine the purified viral particles with an equal volume of 2x extraction buffer.
    2. Incubate at 55 °C for 2 h.
    3. Add an equal volume of phenol saturated with 10 mM Tris-Cl at pH 8.0.
    4. Agitate at room temperature for 195 min.
    5. Centrifuge at 1,500 x g for 5 min.
    6. Transfer the aqueous layer to an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, saturated with 10 mM Tris, pH 8.0).
    7. Agitate at room temperature for 1 h.
    8. Centrifuge at 1,500 x g for 5 min.
    9. Transfer the aqueous layer to an equal volume of 24:1 chloroform-isoamyl alcohol.
    10. Agitate at room temperature for 1 h.
    11. Centrifuge at 1,500 x g for 5 min.
    12. Transfer the aqueous layer to a clean tube.
    13. Add 0.1x volume 3 M sodium acetate and 2.5x volume ethanol and mix.
    14. Centrifuge at 1,500 x g for 15 min.
    15. Aspirate the supernatant.
    16. Wash the DNA pellet in 70% ethanol.
    17. Air-dry the DNA pellet.
    18. Dissolve the DNA pellet in 200 μl 10 mM Tris-Cl (pH 8.5).
    19. Store DNA at -80 or -20 °C.

  3. Assembly of linear recombination templates. See Figure 1 for a schematic showing the final assembled recombination template.
    Note: For steps C1-3, the following conditions are recommended: initial denaturation at 94 °C, followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing for 30 sec, and extension at 68 °C for 1 min per kilobase. Add a final extension step at 68 °C for 10 min. Reactions should contain 300 nM of each primer. See “Standard PCR Protocol” (He, 2011).
    1. Using PCR and 0.02 IU/μl of Accuprime Taq High Fidelity polymerase with the supplied polymerase Buffer II, amplify a 489 bp product from 20 ng of purified MVA DNA template with primers KB1F (TGGTCAATCGCCATTTGTTCG) and KB1R (GGACATAGTGCCTTGTATTATG). Set the annealing temperature to 43 °C, and cycling extension to 30 sec. This product will be referred to as FLL.
    2. Using PCR and a high fidelity polymerase, amplify a 557 bp PCR product from 20 ng of purified MVA DNA template with primers KB4F (GCTATTCGACATCGAGAACAATCTTTGC-TAGAGAGAAAGC) and KB4R (GAGGGATATTAAGTCGATAG). Set the annealing temperature to 40 °C, and cycling extension to 30 sec. This product will be referred to as FLR.
    3. Using PCR and a high fidelity polymerase, amplify a 853 bp PCR product from a plasmid containing the E. coli GPT gene downstream of the Vaccinia virus p7.5 promoter [(e.g. plasmid pGNR, as described in Boulanger et al. (1998)]. In this publication, a cassette containing the E. coli gpt gene under the control of the vaccinia virus p7.5 early/late promoter was cloned into the SmaI site of pNEB193. Primers are KB3F (GGATGCAGCCTCCAG-AATAC) and KB3R (GCTTTCTCTCTAGCAAAGATTGTTCTCGATGTCGAATAGC). Set the annealing temperature to 46 °C, and cycling extension to 60 sec.
    4. After confirming that all PCRs contain a single product of the expected size, purify each product using a QIAquick PCR Purification kit and elute into 10 mM Tris-Cl (pH 8.5).
    5. Use splice-overlap-extension PCR to anneal the GPT and FLR products together: The first 5 cycles are annealed at 57 °C in the absence of primers. Primers KB3F and KB4R are then added prior to an additional 30 cycles with annealing at 43 °C. This 1,390 bp product, referred to as RHS, should be purified by agarose gel electrophoresis, but avoid exposure to ultraviolet light.
    6.  Design primers to amplify the FWPV gene of interest. The forward primer should have a 5’ tail complementary to primer KB1R. The reverse primer should have a 5’ tail complementary to primer KB3F. The FWPV product can be up to 8 kbp long.
    7. Using PCR and a high fidelity polymerase, amplify the FWPV product designed in step C6 from 100 ng of purified FWPV DNA (from step B). Purify this product by agarose gel electrophoresis and a QIAquick Gel Extraction kit. Elute into 10 mM Tris-Cl (pH 8.5). Products over 2 kbp should not be exposed to ultraviolet light, to avoid photo-nicking.
    8. Use splice-overlap-extension PCR to assemble the FWPV product with FLL and RHS, using equimolar amounts of each component. For a 8 kbp FWPV product, initially combine 10 ng of FLL with 160 ng of the FWPV product, in the absence of primers, for 16 cycles, with annealing at 43 °C and 30 sec extension time. Then add 30 ng RHS for a further 6 cycles, with annealing at 45 °C and 90 sec extension time. Then add primers KB1F and KB4R for a final 20 cycles with annealing at 39 °C and 9 min extension time.
      Note: Extension times are given assuming use of a high fidelity polymerase with an extension time of 1 min per kbp.
    9. Purify the assembled FLL-FWPV-RHS cassette using gel electrophoresis and the QIAquick gel extraction kit. Elute into 10 mM Tris-Cl, 1 mM EDTA (pH 8.0). Products over 2 kbp should not be exposed to ultraviolet light.

  4. Transfection of linear recombination templates
    1. Culture DF-1 cells in DF-1 culture medium.
    2. In a 12.5 cm2 tissue culture flask, infect DF-1 cells at 70% confluency with MVA at 3 pfu/cell:
      1. De-aggregate virus in a cuphorn sonicator for 30 sec. Set sonicator to 100% output power, with pulse on/off at 1 sec intervals.
        Note: During sonication, vial should be floating approximately 2-4 cm above the horn. Ice may be added to the water in the cup, to prevent overheating.
      2. Dilute virus in serum-free DMEM.
      3. Aspirate medium from DF-1 cells and replace with diluted virus in a volume just sufficient to cover the cell monolayer (e.g. 1 ml for 12.5 cm2).
      4. Incubate at 37 °C, 5% CO2 for 90 min.
    3. Meanwhile, mix the linear recombination template from step C (at least 35 ng) with 2.5 μg of salmon sperm carrier DNA in Opti-MEM to a final volume of 100 microlitres. Add 12 μl of Polyfect. Allow to complex at room temperature for 5-15 min. Add 5 ml DMEM supplemented with 2% FBS (v/v) and mix by pipetting.
    4. Aspirate the virus from the DF-1 cells infected in step D2. Add the transfection mix prepared in step D3.
    5. Incubate the infected/transfected cells at 37 °C, 5% CO2 for 3 days.
    6. Harvest the cells with a cell scraper to scrape the cells off the plastic and into the culture medium. Transfer to a centrifuge tube and centrifuge at 1,500 x g for 5 min.
    7. Resuspend the pellet in 1 ml of supernatant, discarding the rest of the supernatant. Typically, this yields approximately 1.4 ml cell lysate. Transfer to a screw-cap microtube and freeze-thaw 3 times.

  5. Selection of chimaeric MVA
    Note: Three serial passages of chimaeric MVA in selection media are usually sufficient to remove parental MVA, but this should be confirmed by PCR analysis.
    1. Defrost the transfection-infection harvest from step D7 and de-aggregate the virus particles in a cuphorn sonicator for 30 sec, as in step D2a.
    2. Dilute 0.1% of the harvested volume (e.g. 1.4 μl of a 1.4 ml harvest) from step D6 into 2 ml serum-free DMEM.
    3. Aspirate the culture media from 80-100% confluent CEF cells in a 25 cm2 tissue culture flask and replace with the diluted virus.
    4. Incubate at 37 °C, 5% CO2 for 90 min.
    5. Aspirate virus from cells and replace with 2% DMEM + MXH.
    6. Culture at 37 °C, 5% CO2 for 6 days.
    7. Harvest this passaged virus (P1) by using a cell scraper to scrape the cells off the plastic and into the culture medium. Transfer to a centrifuge tube and centrifuge at 1,500 x g for 5 min.
    8. Resuspend the pellet in 1 ml of supernatant, discarding the rest of the supernatant. Transfer to a screw-cap microtube and freeze-thaw 3 times.
    9. Defrost and de-aggregate the harvested material in a cuphorn sonicator for 30 sec.
    10. Repeat steps E2-7 using the P1 material, to generate P2 material.
    11. Defrost and de-aggregate the harvested material in a cuphorn sonicator for 30 sec.
    12. Repeat steps E2-7 using the P2 material, to generate P3 material.
    13. Store virus at -80 °C.

Recipes

  1. TE
    10 mM Tris-Cl
    1 mM EDTA
    pH 8.0
  2. FWPV virus growth medium
    E199
    2% (v/v) newborn bovine serum
    10% tryptose phosphate broth
    250 Units/ml nystatin
    100 U/ml penicillin
    100 μg/ml streptomycin
    2 mM L-Glutamine
    0.225% sodium bicarbonate
  3. MVA virus growth medium
    DMEM
    2% (v/v) foetal bovine serum
    100 U/ml penicillin
    100 μg/ml streptomycin
  4. 2x extraction buffer
    20 mM Tris-Cl
    20 mM EDTA
    200 mM NaCl
    1% SDS
    4% β-mercaptoethanol
    2.5 mg/ml proteinase K
  5. DF-1 culture medium
    DMEM
    8% (v/v) foetal bovine serum
    100 U/ml penicillin
    100 μg/ml streptomycin
  6. 2% DMEM + MXH
    DMEM
    2% (v/v) foetal bovine serum
    100 U/ml penicillin
    100 μg/ml streptomycin
    25 μg/ml mycophenolic acid (stock solution of 10 mg/ml in 0.1 N NaOH)
    250 μg/ml xanthine (stock solution of 10 mg/ml in 0.1 N NaOH)
    15 μg/ml hypoxanthine (stock solution of 10 mg/ml in sterile water)
    0.1 N hydrochloric acid (add an equivalent volume to xanthine and mycophenolic acid)

Acknowledgments

This protocol was adapted from Buttigieg et al. (2013). This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), studentship BBS/S/K/2005/12558, grants BBS/B/00115/2, BB/E009956/1, and BB/G018545/1.

References

  1. Boulanger, D., Green, P., Smith, T., Czerny, C. P. and Skinner, M. A. (1998). The 131-amino-acid repeat region of the essential 39-kilodalton core protein of fowlpox virus FP9, equivalent to vaccinia virus A4L protein, is nonessential and highly immunogenic. J Virol 72(1): 170-179.
  2. Buttigieg, K., Laidlaw, S. M., Ross, C., Davies, M., Goodbourn, S. and Skinner, M. A. (2013). Genetic screen of a library of chimeric poxviruses identifies an ankyrin repeat protein involved in resistance to the avian type I interferon response. J Virol 87(9): 5028-5040.
  3. He, F. (2011). Standard PCR protocol. Bio-protocol Bio101: e53.

简介

嵌合MVA的构建是研究相关病毒的基因功能的有用工具。 本文给出的方案描述了含有来自鸡痘病毒(FWPV)的基因的MVA嵌合体,尽管这可以应用于来自其他生物体的DNA。 制备嵌合MVA需要许多步骤:1)病毒颗粒的纯化; 2)从纯化的病毒颗粒中提取DNA; 3)装配线性重组模板; 4)线性重组模板的转染; 5)嵌合MVA的选择。
注意:此程序使用活病毒,并应根据国际和国家生物承诺要求使用良好微生物实践进行。 该程序还涉及微生物的遗传修饰,并且在开始前应获得适当的安全批准。

材料和试剂

  1. 病毒颗粒的纯化
    1. 禽痘病毒(FWPV)(例如ATCC,目录号:VR-251)
    2. 修饰的痘苗病毒安卡拉(MVA)(ATCC,目录号:VR-1508)
    3. CEF细胞(动物健康研究所)
    4. Tris-EDTA缓冲液(Sigma-Aldrich,目录号:93283)
    5. DMEM(例如Sigma-Aldrich,目录号:D6429)
    6. 胎牛血清(FBS)(例如Sigma-Aldrich,目录号:F9665)
    7. 10x培养基199与Earle's盐(Sigma-Aldrich,目录号:M0650)
    8. 热灭活的新生牛血清(Life Technologies,目录号:26010074)
    9. 磷酸胰蛋白酶肉汤(Sigma-Aldrich,目录号:T8159)
    10. 制霉菌素(Sigma-Aldrich,目录号:N1638)
    11. 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:P4333)
    12. L-谷氨酰胺溶液(Sigma-Aldrich,目录号:G7513)
    13. 碳酸氢钠(Sigma-Aldrich,目录号:S8761)
    14. Amicon Ultra-15离心过滤器单元(100kDa膜NMWL)(Merck Millipore,目录号:UFC910024)
    15. FWPV病毒生长培养基(参见Recipes)
    16. MVA病毒生长培养基(参见配方)
    17. TE(参见食谱)

  2. 从纯化的病毒颗粒中提取DNA
    1. 用10mM Tris-HCl(Sigma-Aldrich,目录号:P4557)饱和的苯酚
    2. 苯酚 - 氯仿 - 异戊醇(25:24:1,用10mM Tris饱和,pH8.0)(Sigma-Aldrich,目录号:P2069)
    3. 氯仿 - 异戊醇(24:1)(Sigma-Aldrich,目录号:25666)
    4. 3M乙酸钠(Sigma-Aldrich,目录号:71196)
    5. 乙醇(Sigma-Aldrich,目录号:E7023)
    6. 10mM Tris-Cl(pH 8.5)(QIAGEN,目录号:19086)
    7. Tris-HCl(Sigma-Aldrich,目录号:T5941)
    8. EDTA(Sigma-Aldrich,目录号:EDS)
    9. NaCl(Sigma-Aldrich,目录号:S3014)
    10. SDS溶液(Sigma-Aldrich,目录号:71736)
    11. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
    12. 蛋白酶K(Sigma-Aldrich,目录号:P2308)
    13. 2x提取缓冲液(参见配方)

  3. 装配线性重组模板
    1. PCR寡核苷酸(Sigma-Aldrich)
    2. Accuprime Taq高保真聚合酶(Life Technologies,目录号:12346086)
    3. pNEB193(New England Biolabs)
    4. 在p7.5启动子(例如, pGNR,可根据要求提供)上编码大肠杆菌(<大肠杆菌)gpt的质粒
    5. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
    6. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)

  4. 线性重组模板的转染
    1. DF-1细胞(ATCC,目录号:CRL-12203)
    2. DMEM(例如Sigma-Aldrich,目录号:D6429)
    3. 胎牛血清(FBS)(例如Sigma-Aldrich,目录号:F9665)
    4. 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:P4333)
    5. 鲑精DNA溶液(Life Technologies,目录号:15632011)
    6. Opti-MEM(Life Technologies,目录号:31985062)
    7. Polyfect(QIAGEN,目录号:301105)
    8. DF-1培养基(见Recipes)

  5. 选择嵌合MVA
    1. DMEM(例如Sigma-Aldrich,目录号:D6429)
    2. 胎牛血清(FBS)(例如Sigma-Aldrich,目录号:F9665)
    3. 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:P4333)
    4. 霉酚酸(Sigma-Aldrich,目录号:M5255)
    5. 黄嘌呤(Sigma-Aldrich,目录号:X3627)
    6. 次黄嘌呤(Sigma-Aldrich,目录号:H9636)
    7. 盐酸(Sigma-Aldrich,目录号:H9892)
    8. 氢氧化钠(Sigma-Aldrich,目录号:S2770)
    9. 2%DMEM + MXH(参见配方)

设备

  1. 台式离心机(例如 Hettich ®,型号:Rotanta 460)
  2. 热循环仪(Techne Progene ®
  3. 层流微生物安全柜(例如 HeraSafe ®,型号:51022738)
  4. 加湿CO 2培养箱(例如Thermo Fisher Scientific,型号:BB15)
  5. Cuphorn超声仪(例如 Ultrasonics TM ,型号:W-375)

程序

  1. 病毒颗粒的纯化
    使用无菌技术,在层流微生物安全柜
    1. 使用FWPV或MVA以0.01个噬斑形成单位(pfu)/细胞感染融合的CEF细胞。
      注意:感染的细胞数量取决于数量 嵌合MVA病毒。 作为指导,30个感染的175cm 2烧瓶   的FWPV产生〜150μgDNA; 2感染的175cm 2 MVA的烧瓶中产生〜800   ng DNA。
    2. 在37℃,5%CO 2下在病毒生长培养基中培养5-7天,直到可见广泛的细胞病变效应。
    3. 将培养物(仍然在烧瓶中)冻融3次 注意:在-80°C下冷冻。 所述烧瓶可以在室温或0℃下解冻 37℃,但不要在高于绝对温度的温度下离开 必要。
    4. 将感染的细胞裂解液转移到离心管中
    5. 以1,500 x g离心15分钟。
    6. 将上清液转移至Amicon Ultra-15离心过滤装置(100kDa NMWL)
    7. 在4℃下以1500×g离心60分钟。
    8. 将保留在含有病毒颗粒的膜(约200μl)上的裂解物重悬在2ml TE中
    9. 储存于4°C过夜,或直接进入B部分。

  2. 从纯化的病毒颗粒中提取DNA
    1. 将纯化的病毒颗粒与等体积的2x提取缓冲液混合
    2. 在55℃孵育2小时
    3. 加入等体积的用10mM Tris-Cl在pH 8.0饱和的苯酚
    4. 在室温下搅拌195分钟
    5. 以1,500 x g离心5分钟。
    6. 将水层转移至等体积 苯酚 - 氯仿 - 异戊醇(25:24:1,用10mM Tris饱和, pH 8.0)。
    7. 在室温下搅拌1小时
    8. 以1,500 x g离心5分钟。
    9. 将水层转移至等体积的24:1氯仿 - 异戊醇
    10. 在室温下搅拌1小时
    11. 以1,500 x g离心5分钟。
    12. 将水层转移到干净的管中。
    13. 加入0.1倍体积的3M乙酸钠和2.5倍体积乙醇并混合
    14. 以1,500 x g离心15分钟。
    15. 吸出上清液。
    16. 在70%乙醇中洗涤DNA沉淀
    17. 风干DNA颗粒。
    18. 将DNA沉淀溶解在200μl10mM Tris-Cl(pH8.5)中
    19. 将DNA储存在-80或-20°C。

  3. 线性重组模板的装配。参见图1,显示最终组装的重组模板的示意图 注意:对于步骤C1-3,推荐以下条件:在94℃初始变性,接着是30个循环的94℃变性30秒,退火30秒,和延伸在68℃,每千碱基1分钟。在68℃下添加最终延伸步骤10分钟。反应应含有300nM的每种引物。请参阅"标准PCR方案"(他,2011年)。
    1. 使用Accuprime Taq High Fidelity聚合酶的PCR和0.02IU/
    2. 在DF-1培养基中培养DF-1细胞。
    3. 在12.5cm 2组织培养瓶中,以3pfu /细胞用MVA以70%融合感染DF-1细胞:
      1. 在杯形超声仪中解聚集病毒30秒。 设置超声波仪 到100%输出功率,脉冲以1秒间隔打开/关闭。
        注意: 在超声处理期间,小瓶应该漂浮约2-4厘米以上 喇叭。 冰可以添加到杯中的水中,以防止 过热。
      2. 在无血清DMEM中稀释病毒
      3. 吸出 来自DF-1细胞的培养基,并用体积中的稀释的病毒替换 足以覆盖细胞单层(例如,对于12.5cm 2 ,1ml)。
      4. 在37℃,5%CO 2孵育90分钟。
    4. 同时,混合来自步骤C的线性重组模板(at 至少35ng)与2.5μg鲑鱼精子载体DNA在Opti-MEM中混合 最终体积为100微升。 加入12μl的Polyfect。 允许复杂   在室温下5-15分钟。 加入5 ml DMEM补充2% FBS(v/v)并通过吸移混合。
    5. 从步骤D2中感染的DF-1细胞吸出病毒。 加入步骤D3中制备的转染混合物
    6. 将感染/转染的细胞在37℃,5%CO 2下孵育3天。
    7. 用细胞刮刀收获细胞以刮除细胞 塑料并进入培养基。 转移到离心管和 在1,500×g离心5分钟。
    8. 将沉淀重悬在1ml中 的上清液,丢弃剩余的上清液。 通常,这 产生约1.4ml细胞裂解物。 转移到螺旋盖 微管和冻融3次。

  4. 选择嵌合MVA
    注意:选择培养基中嵌合MVA的三次连续传代通常足以除去亲本MVA,但这应通过PCR分析确认。
    1. 解冻步骤D7的转染 - 感染收获物并解聚   病毒颗粒在杯形超声仪中30秒,如步骤D2a
    2. 稀释收获体积的0.1%( 1.4 μ 的1.4ml收获物)装入2ml无血清DMEM中
    3. 在25℃下从80-100%汇合的CEF细胞吸出培养基 cm 2组织培养瓶中并用稀释的病毒替换。
    4. 在37℃,5%CO 2孵育90分钟。
    5. 从细胞吸出病毒,并更换2%的DMEM + MXH
    6. 在37℃,5%CO 2下培养6天。
    7. 收获此传代病毒(P1)通过使用细胞刮刀刮 细胞脱离塑料并进入培养基。 转移到 离心管并在1,500×g离心5分钟。
    8. 重悬   沉淀在1ml上清液中,弃去剩余部分 上清液。 转移到螺旋盖微量管中,冻融3次
    9. 在铜杯超声仪中除霜和解聚收获的物料30秒
    10. 使用P1材料重复步骤E2-7,生成P2材料。
    11. 在铜杯超声仪中除霜和解聚收获的物料30秒
    12. 使用P2材料重复步骤E2-7,生成P3材料。
    13. 将病毒储存于-80℃。

食谱

  1. TE
    10mM Tris-Cl 1mM EDTA
    pH 8.0
  2. FWPV病毒生长培养基
    E199
    2%(v/v)新生牛血清 10%胰蛋白酶磷酸盐培养基
    250单位/ml制霉菌素 100 U/ml青霉素
    100μg/ml链霉素 2mM L-谷氨酰胺 0.225%碳酸氢钠
  3. MVA病毒生长培养基
    DMEM
    2%(v/v)胎牛血清 100 U/ml青霉素
    100μg/ml链霉素
  4. 2x提取缓冲液
    20mM Tris-Cl 20 mM EDTA
    200 mM NaCl
    1%SDS
    4%β-巯基乙醇 2.5mg/ml蛋白酶K
  5. DF-1培养基
    DMEM
    8%(v/v)胎牛血清 100 U/ml青霉素
    100μg/ml链霉素
  6. 2%DMEM + MXH
    DMEM
    2%(v/v)胎牛血清 100 U/ml青霉素
    100μg/ml链霉素 25μg/ml霉酚酸(在0.1N NaOH中的10mg/ml的储备溶液) 250μg/ml黄嘌呤(10mg/ml的储备溶液,在0.1N NaOH中) 15μg/ml次黄嘌呤(10mg/ml的储备溶液,在无菌水中) 0.1 N盐酸(添加等量的黄嘌呤和霉酚酸)

致谢

该协议改编自Buttigieg等人(2013)。这项工作由生物技术和生物科学研究委员会(BBSRC),学生资格BBS/S/K/2005/12558,赠款BBS/B/00115/2,BB/E009956/1和BB/G018545/1资助。

参考文献

  1. Boulanger,D.,Green,P.,Smith,T.,Czerny,C.P.and Skinner,M.A。(1998)。 鸡痘病毒FP9的基本39千道尔顿核心蛋白的131个氨基酸重复区,相当于痘苗病毒A4L蛋白,是非必需的和高度免疫原性的。

    72(1):170-179。
  2. Buttigieg,K.,Laidlaw,S.M.,Ross,C.,Davies,M.,Goodbourn,S.and Skinner,M.A。(2013)。 嵌合痘病毒文库的遗传筛选识别涉及对禽I型抗性的锚蛋白重复蛋白干扰素反应。病毒学87(9):5028-5040。
  3. 他,F.(2011)。 标准PCR协议 生物协议 Bio101:e53。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Buttigieg, K. R. and Skinner, M. A. (2015). Construction of FWPV Chimaeric MVA. Bio-protocol 5(1): e1376. DOI: 10.21769/BioProtoc.1376.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。

Farhad Gilavand
shahid Behshti university/sbu
Excellent work
1/15/2015 9:41:48 AM Reply