[Construction and characterization of a pseudorabies virus TK-/gG- mutant].
To construct a TK-/gG- mutant of pseudorabies virus, the gG-detected transfer vector pUSKKBB and genomic DNA of pseudorabies virus TK-/gG-/LacZ+ were co-transfected into IBRS-2 cells. Transfection progeny were plated onto PK-15 cells and incubated for 2 days under methylcellulose. Then the overlay was removed and replaced by 1% low melting point agarose in DMEM supplemented with 150 microg/mL X-gal. After 2 days, white plaques were screened for and purified 4 times. By PCR amplification of gG-deleted gene and LacZ gene, a recombinant virus with TK-/gG- phenotype was confirmed. Sequence of the PCR product revealed that there were 1,176 bp detection in gG gene of the PRV TK-/gG- mutant. Amplifying the gG-deleted gene of different generations of the TK-/gG- mutant showed that the mutant was stable within PK-15 cells. TCID50 assay indicated that the recombinant virus grows well on PK-15 cells. The mice immunized with the TK-/gG- virus showed no sign of abnormality. As a control, all mice inoculated with PRV strain died from the infection. All mice that received TK-/gG- survived after a lethal PRV challenge. However none of the mice injected with phosphate-buffer saline (PBS) survived from the challenge. The above results demonstrated that the recombinant virus could be a candidate marker vaccine strain for eradicating pseudorabies in pig herds.
Xu
Xiao-Juan
XJ
Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Xu
Gao-Yuan
GY
Chen
Huan-Chun
HC
Liu
Zheng-Fei
ZF
He
Qi-Gai
QG
D000818:Animals; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000472:pathogenicity; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D022421:Pseudorabies Vaccines / Q000276:immunology; D013552:Swine; D013937:Thymidine Kinase / Q000235:genetics*; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology
D022421:Pseudorabies Vaccines; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins; D013937:Thymidine Kinase