Optimization of a GFP-PAC co-expression cassette-based purification system for efficient construction and rapid...

Area of Science:

• Virology
• Oncolytic Virus Therapy
• Molecular Biology

Background:

• Oncolytic viruses (OVs) are engineered to target and destroy cancer cells.
• Recombinant Herpes Simplex Virus type 1 (HSV-1) is a leading OV candidate, but isolating modified viruses is challenging.
• Current methods for purifying recombinant HSV-1 are inefficient and labor-intensive due to low recombination rates and parental virus persistence.

Purpose of the Study:

• To develop and optimize a novel protocol for efficient isolation of recombinant HSV-1.
• To simplify and accelerate the purification process of engineered oncolytic viruses.
• To enhance the purity and yield of recombinant HSV-1 constructs for therapeutic applications.

Main Methods:

• Developed a novel purification protocol using a GFP-PAC co-expression cassette.
• Implemented simultaneous puromycin selection and fluorescence-based identification.
• Optimized the dual-selection strategy for recombinant HSV-1 isolation.

Main Results:

• The new protocol significantly accelerates and simplifies the isolation of recombinant HSV-1.
• Achieved enhanced efficiency and purity in isolating engineered viral constructs.
• Demonstrated a robust and scalable strategy for generating recombinant HSV-1.

Conclusions:

• The developed dual-selection protocol offers a significant improvement over traditional plaque purification methods.
• This strategy facilitates the generation of high-purity recombinant HSV-1.
• The protocol has broad applicability in vaccine development, gene therapy, and virology research.