Characterization of Recombinant Vaccinia Viruses and Their Products

Patricia L. Earl1, Bernard Moss1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.18
DOI:  10.1002/0471142727.mb1618s43
Online Posting Date:  May, 2001
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Abstract

After a recombinant vaccinia virus is made, its DNA and protein products can be analyzed in several ways. Protocols are provided in this unit for identification of the recombinant virus by PCR (with verification of correct insertion of the DNA by Southern blotting) and by dotā€blot hybridization. Also, when antibodies are available, protein expression can be analyzed by immunological methods detailed here such as dot blotting with an antibody, immunoblotting and/or immunoprecipitation. In addition, immunostaining can be used for identification of recombinant plaques as well as for determination of the purity of a recombinant virus stock. All of the protocols in this unit can be used for characterization of modified vaccinia virus Ankara (MVA) recombinant viruses.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Vaccinia DNA Using PCR
  • Basic Protocol 2: Detection of Vaccinia DNA Using Southern Blot Hybridization
  • Basic Protocol 3: Detection of Vaccinia DNA Using Dot‐Blot Hybridization
  • Alternate Protocol 1: Detection of Expressed Protein by a Dot‐Blot Procedure
  • Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting
  • Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Detection of Vaccinia DNA Using PCR

  Materials
  • Confluent BS‐C‐1 or HuTK 143B cell monolayer (unit 16.16)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Trypsin/EDTA (0.25%:0.02%), 37°C
  • Complete MEM‐10, ‐2.5, and ‐5 media (unit 16.16)
  • Complete MEM‐10/BrdU medium (unit 16.16)
  • Recombinant virus plaques, picked and resuspended in tubes (unit 16.17)
  • Selective agents (filter sterilize and store at −20°C; also see unit 16.17):
  •   10 mg/ml (400×) mycophenolic acid (MPA; Calbiochem) in 0.1 N NaOH (for XGPRT selection)
  •   10 mg/ml (40×) xanthine in 0.1 N NaOH (for XGPRT selection)
  •   10 mg/ml (670×) hypoxanthine in 0.1 N NaOH (for XGPRT selection)
  •   5 mg/ml (200×) 5‐bromodeoxyuridine (BrdU) in water (for TK selection)
  • Dry ice/ethanol bath
  • recipeDNA extraction buffer (see recipe)
  • 3 M sodium acetate, pH 6.0 ( appendix 22)
  • 95% and 70% ethanol, ice‐cold
  • 10× MgCl 2‐free PCR amplification buffer (unit 15.1)
  • 10 mM 4dNTP mix (unit 15.1)
  • 100 µM primer (sequence depends on where foreign gene is inserted; see unit 2.11 for oligonucleotide synthesis strategies)
  • 15 mM MgCl 2
  • 24‐well tissue culture plates
  • Cup sonicator
  • Additional reagents and equipment for tissue culture and counting cells ( appendix 3F), phenol/chloroform extraction of DNA, PCR amplification (unit 15.1), and agarose gel electrophoresis (unit 2.52.5)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Detection of Vaccinia DNA Using Southern Blot Hybridization

  Materials
  • Confluent BS‐C‐1 cell monolayer (unit 16.16)
  • Complete complete MEM‐10 and ‐5 media (unit 16.16)
  • Recombinant vaccinia virus stock (unit 16.16)
  • 0.25 mg/ml trypsin (2× crystallized and salt‐free;Worthington; filter sterilize and store at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • recipeLow‐salt buffer (see recipe)
  • 20 mg/ml proteinase K
  • Buffered phenol (unit 2.12.1)
  • 1:1 phenol/chloroform
  • 3 M sodium acetate, pH 6.0 ( appendix 22)
  • 95% and 70% ethanol, ice‐cold
  • TE buffer, pH 7.8 ( appendix 22)
  • HindIII restriction endonuclease (unit 3.1) and appropriate buffer
  • 12‐well tissue culture plates
  • Additional reagents and equipment for preparing BS‐C‐1 cells for vaccinia infection (see protocol 1), tissue culture and counting cells ( appendix 3F), phenol/chloroform extraction of DNA (unit 2.12.1), restriction endonuclease digestion of DNA (unit 3.1), Southern blotting (unit 2.9), and hybridization (unit 2.10)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Detection of Vaccinia DNA Using Dot‐Blot Hybridization

  Materials
  • 0.5 N NaOH
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 22)
  • 2× SSC ( appendix 22)
  • Nitrocellulose membrane (see Table 97.80.47112.9.1)
  • Whatman 3MM filter paper
  • 80°C vacuum oven
  • Additional reagents and equipment for preparing cells, infecting with vaccinia virus, and performing selection (see protocol 1), dot blotting (unit 2.9), and hybridization (unit 2.10)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 1: Detection of Expressed Protein by a Dot‐Blot Procedure

  • recipePBS/Tween (see recipe) with and without 4% (w/v) BSA
  • Antibody to foreign protein
  • 100 µCi/ml [125I]‐labeled protein A (30 mCi/mg; Amersham)
  • Additional reagents and equipment for autoradiography ( appendix 3A)

Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting

  Materials
  • Confluent BS‐C‐1 cell monolayer (unit 16.16)
  • Complete MEM‐5 medium (unit 16.16)
  • Recombinant vaccinia virus stock (unit 16.16)
  • 0.25 mg/ml trypsin (2× crystallized and salt‐free;Worthington; filter sterilize and store at −20°C)
  • recipeCell lysis buffer (see recipe)
  • 5× SDS sample buffer (unit 10.210.2)
  • 6‐well tissue culture plates
  • Sorvall centrifuge with H‐6000A rotor (or equivalent)
  • 95°C water bath
  • Additional reagents and equipment for preparing cell cultures for infection (see protocol 1) and immunoblotting (unit 10.8)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation

  Materials
  • Complete MEM‐5 medium (unit 16.16)
  • Complete methionine‐ or cysteine‐free MEM‐5 medium (see recipe in unit 16.16 but use methionine‐ or cysteine‐free MEM, e.g., from Life Technologies) prepared with dialyzed FBS (e.g., HyClone)
  • [35S]methionine or [35S]cysteine (depending on which amino acid was omitted from the medium; see )
  • Phosphate‐buffered saline (PBS; appendix 22)
  • recipeCell lysis buffer (see recipe)
  • Additional reagents and equipment for preparing cells and infecting with vaccinia virus (see protocol 5) and immunoprecipitation (unit 10.16)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
   Barrett, N., Mitterer, A., Mundt, W., Eibl, J., Eibl, M., Gallo, R.C., Moss, B., and Dorner, F. 1989. Large‐scale production and purification of a vaccinia recombinant‐derived HIV‐1 gp 160 and analysis of its immunogenicity. AIDS Res. Hum. Retroviruses 5:159‐171.
   Zhang, Y. and Moss, B. 1991. Inducer‐dependent conditional‐lethal mutant animal viruses. Proc. Natl. Acad. Sci. U.S.A. 88:1511‐1515.
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