| Herpesvirus of turkey (HVT) is an alpherpesvirus that is widely used as a live vaccine against Marek's disease (MD) because of its antigenic relationship with Marek's disease virus (MDV).The aim of this study was to construct Herpesvirus of turkey Fc126 strain as an infectious bacterial artificial chromosome( BAC).Using the selection marker Eco-gpt(Xanthine-guanine phosphoribosyl transferase) (1.3 kb) which was designed by ourselves 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.Chloramphenicol-resistant colonies containing high molecμLar mass DNA (BAC clones )were identified by restriction enzyme digestion and PCR analysis,and then tested for infectivity after transfection into CEFs using calcium phosphate.We obtained twenty-five BAC clones,and three recombinant viruses were reconstituted by transfection of HVT-BAC6 DNA, HVT-BAC8 DNA and HVT-BAC10 DNA into CEFs respectively.In this study, we cloned the complete genome of HVT Fc126 strain as an infectious bacterial artificial chromosome,with these cloned genomes in hand,we will utilize the Red/ET or Cre/loxP recombination system to modify the HVT BACs,which acclerate the understanding of HVT gene functions and promote the using of HVT as a vector for expressing foreign genes.In addition,it may be possible to generate novel HVT live vector vaccines based on the BACs.Utilizing the Red/ET recombination system which is a new selection and counter-selection system based on the rpsL gene (rpsL-neo) and Streptomycin selection,we are trying to develop a biologically safe live vaccine based on the HVT BACs.Using this two step red-mediated mutagenesis system in E.coli,firstly The DH10B competent cells carrying the HVT BACs were transformed with the expression plasmid pRedET,then induced by the addition of L-arabinose and a temperature shift from 30℃to 37℃,after induction the cells were prepared for electroporation and the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms was electroporated into the cells,RecE and RecT catalyze the homologous recombination reaction,the functional cassette was inserted into the US2 locus to be modified of HVT.Only colonies carrying the modified BAC wound suivive Kanamycin selection on the agar plates.The successful integration of the rpsL-neo cassette was moniotored by PCR and Streptomycin selection,for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive.Secondly,in the same way,the rpsL-neo cassette was replaced by the non-selectable target DNA—hemagglutinin (HA) gene of the highly pathogenic avian influenza virus(HPAIV)A/Goose/Guangdong/1/96(H5N1)flanked by the same homology arms.Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates.Finally,we obtained many BAC colonies of which the HA gene of the AIV was inserted into the US2 locus to be modified of HVT, and one recombinant virus was reconstituted by transfection of one of these BAC clones DNA into CEFs,the rescued virus was designated as rHVT-HA.The H5 HA gene expression in this recombinant virus rHVT-HA was confirmed by immunofluorescence assay. |
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