Restriction digests of the clone give the following sizes (kb): BamHI--4.2; EcoRI--4.2; SalI/HindIII--3.2, 1.0. Recombinants can be identified by inactivation of the hemagglutinin gene (HA-). Shuttle vector component of a virulence tester system to be used with the avirulent rabbit poxvirus deletion mutant RPmuhr23HA+ (ATCC VR-2379). DNA cloned into the MCS is integrated into VR-2379 by homologous recombination, possibly altering the host range and virulence of the virus. The hemagglutinin insert is a 1.8 kb 5'SalI/3'HindIII fragment containing approximately 443 bp 5' flanking sequence, 988 bp coding sequence interrupted by a polylinker, and 408 bp 3' flanking sequence. The gene is interrupted at the AccI site (nt 43 of the coding region) by a 47 bp MCS from pUC9. Constructed by deleting lacZ and the polylinker from pUC9 by a HaeII digest. The order of the major features in the plasmid is: pUC9 delta lacZ - KpnI - 5' end of insert - PstI/MCS/EcoRI - 3' end of insert - KpnI. |
