Deletion of IV a2 gene from adenoviral genome by lambda-Red recombinase system and packaging of the recombinant adenovirus.
- Author:
Yun-Fan LIU
1
;
Chi-Jie YU
;
Gang WANG
;
Wen-Hong TIAN
;
Yue LU
;
Xue-Rong LIU
;
Xiao-Yan DONG
;
Gang ZHENG
;
Wei SHEN
;
Xiao-Bing WU
;
Li RUAN
Author Information
1. Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
Genome, Viral;
HEK293 Cells;
Humans;
Polymerase Chain Reaction;
Recombinases;
metabolism;
Sequence Deletion;
Viral Proteins;
genetics;
Virus Assembly
- From:
Chinese Journal of Virology
2011;27(3):257-264
- CountryChina
- Language:Chinese
-
Abstract:
This investigation is to delete the most of the coding sequence (1104 bp) of the IV a2 gene in an adenovirus genome by a lambda-Red recombinase system-mediated PCR-targeting approach and rescue a recombinant adenovirus with IV a2 deletion. First, the template pAK of PCR targeting, containing kanamycin cassette, was constructed. Then, a linear fragment for PCR targeting, which had 39 bp homologous arms at both of its terminus, was amplified by PCR from the pAK. The pFG140 and the linear fragment were electroporated into E. coli BW25113/pIJ790 sequentially and the recombinant pFG140-deltaIV a2 (1104) was established by homologous recombination between the linear fragment and the pFG140 with aid of X-Red recombinase. The precise deletion of 1 104 bp fragment from IV a2 was confirmed by restriction endonucleases digestion and DNA sequencing. ORF of IV a2 was amplified by PCR from pFG140 and then cloned into the pAAV2neo vector. The recombinant adenovirus Ad5delta IV a2 (1104) was rescued by co-transfection of pFG140-deltaIV a2 (1104) and pAAV2neo-IV a2 into HEK293 cells. It was shown by Western Blot that IV a2 could not be detected in the Ad5deltaIV a2 (1104)- infected HEK293 cells. This study established a PCR-targeting strategy for manipulating adenovirus genome directly by a lambda-Red recombinase system, and a recombinant adenovirus with IV a2 deletion was obtained.
