[Reconstituting turkey herpesvirus with bacterial artificial chromosome clones].
Herpesvirus of turkey (HVT) is an alpherpesvirus and widely used as a live vaccine against Marek's disease (MD) because of its antigenic relationship with Marek's disease virus (MDV).


OBJECTIVE
The aim of this study was to construct Herpesvirus of turkey Fc126 strain as an infectious bacterial artificial chromosome (BAC).


METHODS
Using the selection marker Eco-gpt (Xanthine-guanine phosphoribosyl transferase)(1.3 kb) and BAC vector pBeloBAC11(7.4 kb), we constructed the transfer plasmid pGAB-gpt-BAC11. Then, the transfer plasmid and HVT-infected cells' total DNA were cotransfected into primary chicken embryo fibroblasts (CEFs). After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, we obtained purified recombinant viruses. Genomic DNA was extracted and electroporated into Escherichia coli DH10B competent cells. BAC clones were identified by restriction enzyme digestion and PCR analysis, and then tested for infectivity after transfection into CEFs using calcium phosphate.


RESULTS
We obtained 25 BAC clones, and reconstituted recombinant viruses by transfection of HVT-BAC6 DNA, HVT-BAC8 DNA and HVT-BAC10 DNA into CEFs respectively.


CONCLUSIONS
In this study, we cloned the complete genome of HVT Fc126 strain as an infectious bacterial artificial chromosome.
Lan
Desong
D
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute the Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Shi
Xingming
X
Wang
Yunfeng
Y
Cui
Hongyu
H
Liu
Changjun
C
Wang
Mei
M
Hu
Wenwei
W
Tong
Guangzhi
G
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D026002:Herpesvirus 1, Meleagrid / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D014162:Transfection; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral