Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System.
- Author:
Lili LI
;
Zhan WANG
;
Yubai ZHOU
;
Fang ZHANG
;
Sisi SHEN
;
Zelin LI
;
Yi ZENG
- Publication Type:Journal Article
- MeSH:
Animals;
Biomarkers;
Cricetinae;
Epithelial Cells;
virology;
Gene Deletion;
Genetic Engineering;
methods;
Genetic Vectors;
genetics;
metabolism;
Green Fluorescent Proteins;
genetics;
metabolism;
Humans;
Recombination, Genetic;
Vaccinia;
virology;
Vaccinia virus;
genetics;
physiology;
Virus Replication
- From:
Chinese Journal of Virology
2015;31(5):507-514
- CountryChina
- Language:Chinese
-
Abstract:
For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.
