Characterization of Recombinant Vaccinia Viruses and Their Products

Patricia L. Earl1, Bernard Moss1

1 National Institute of Allergy and Infectious Diseases, Bethesda
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.14
DOI:  10.1002/0471140864.ps0514s13
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Detection of Vaccinia DNA Using PCR

  Materials
  • Confluent BS‐C‐1 or HuTK 143B cell monolayer (unit 5.12)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Trypsin/EDTA (0.25%:0.02%), 37°C
  • Complete MEM‐10, ‐2.5, and ‐5 media (unit 5.12)
  • Complete MEM‐10/BrdU medium (unit 5.12)
  • Recombinant virus plaques, picked and resuspended in tubes (unit 5.13)
  • Selective agents (filter sterilize and store at −20°C; also see unit 5.13):
    •   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 2E)
  • 95% and 70% ethanol, ice cold
  • 10× MgCl 2‐free PCR amplification buffer ( appendix 4J)
  • 10 mM 4dNTP mix ( appendix 4J)
  • 100 µM primer (sequence depends on where foreign gene is inserted)
  • 15 mM MgCl 2
  • 24‐well tissue culture plates
  • Cup sonicator
  • Additional reagents and equipment for tissue culture and counting cells ( appendix 3C), phenol/chloroform extraction of DNA ( appendix 4E), PCR amplification ( appendix 4J), and agarose gel electrophoresis ( appendix 4F)
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 5.12)
  • Complete complete MEM‐10 and ‐5 media (unit 5.12)
  • Recombinant vaccinia virus stock (unit 5.12)
  • 0.25 mg/ml trypsin (2× crystallized and salt‐free;Worthington; filter sterilize and store at −20°C)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • recipeLow‐salt buffer (see recipe)
  • 20 mg/ml proteinase K
  • Buffered phenol ( appendix 2E)
  • 1:1 phenol/chloroform
  • 3 M sodium acetate, pH 6.0 ( appendix 2E)
  • 95% and 70% ethanol, ice‐cold
  • TE buffer, pH 7.8 ( appendix 2E)
  • HindIII restriction endonuclease ( appendix 4I) 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 3C), phenol/chloroform extraction of DNA ( appendix 4E), restriction endonuclease digestion of DNA ( appendix 4I), Southern blotting ( appendix 4G), and hybridization ( appendix 4H)
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 2E)
  • 2× SSC ( appendix 2E)
  • Nitrocellulose membrane
  • 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 ( appendix 4G), and hybridization ( appendix 4H)
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 (unit 10.11)

Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting

  Materials
  • Confluent BS‐C‐1 cell monolayer (unit 5.12)
  • Complete MEM‐5 medium (unit 5.12)
  • Recombinant vaccinia virus stock (unit 5.12)
  • 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 10.1)
  • 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 10.10)
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 5: Detection of Expressed Protein Using Immunoprecipitation

  Materials
  • Complete MEM‐5 medium (unit 5.12)
  • Complete methionine‐ or cysteine‐free recipeMEM‐5 medium (see recipe in UNIT 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 2E)
  • recipeCell lysis buffer (see recipe)
  • Additional reagents and equipment for preparing cells and infecting with vaccinia virus (see protocol 5) and immunoprecipitation (unit 10.10)
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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library