[Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System]

Area of Science:

• Virology
• Molecular Biology
• Biotechnology

Background:

• Recombinant modified vaccinia virus Ankara (rMVA) requires stringent quality control for clinical applications.
• Efficient screening methods are crucial for developing clinical-grade rMVA vaccines.

Purpose of the Study:

• To develop a novel shuttle vector for rapid and accurate screening of rMVA.
• To construct a vector enabling automatic marker gene deletion during rMVA propagation.

Main Methods:

• Modification of the pSC11 shuttle vector to create pZL-EGFP, incorporating enhanced green fluorescent protein (EGFP) and TKL homologous sequences.
• Cotransfection of Baby Hamster Kidney (BHK)-21 cells with pZL-EGFP and MVA, followed by serial passages and plaque screening.
• Verification of pZL-EGFP function and EGFP-free rMVA using polymerase chain reaction (PCR) and Western blotting.

Main Results:

• Successful construction of the pZL-EGFP shuttle vector with an auto-deleting EGFP marker gene.
• Generation of EGFP-free rMVA carrying exogenous genes through homologous recombination and intramolecular recombination.
• Demonstration that marker gene deletion does not impact rMVA activity or exogenous gene expression.

Conclusions:

• The pZL-EGFP vector facilitates efficient packaging and screening of rMVA.
• Automatic marker gene removal ensures the quality of rMVA for clinical trials.
• This approach provides a robust foundation for future rMVA research and development.