Rescue, Purification, and Characterization of a Recombinant HSV Expressing a Transgenic Protein

Methods Mol Biol. 2020:2060:153-168. doi: 10.1007/978-1-4939-9814-2_8.

Abstract

In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1-US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.

Keywords: HSV rescue; Plaque assay; Plaque purification; Virion purification; mIL12 transgene expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Chromosomes, Artificial, Bacterial / genetics*
  • Gene Expression*
  • Herpesvirus 1, Human* / genetics
  • Herpesvirus 1, Human* / metabolism
  • Interleukin-12* / biosynthesis
  • Interleukin-12* / genetics
  • Mice
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombination, Genetic*

Substances

  • Recombinant Proteins
  • Interleukin-12