Generation of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 by Rde/ET

Abstract:

[Objective] In recent years, manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). [Methods] With these BAC clones in hand, we manipulated the genome of HVT by utilizing Red/ET recombination system, and we were trying to develop a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs, and then added inducer L-arabinose into the cells and shifted the temperature from 30℃ to 37℃, after 45 minutes induction, we prepared the cells into competent cells,then we electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms 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 would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin(HA )gene of (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. [Results]Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the US2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts(CEFs). [Conclusion]We achieved one rescued recombinant virus which designated as rHVT-HA3.The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.