Construction and characterization of a pseudorabies virus TK-/gG- mutant.
- Author:
Xiao-Juan XU
1
;
Gao-Yuan XU
;
Huan-Chun CHEN
;
Zheng-Fei LIU
;
Qi-Gai HE
Author Information
1. Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Herpesvirus 1, Suid;
genetics;
pathogenicity;
Mice;
Mice, Inbred BALB C;
Mutation;
Pseudorabies Vaccines;
immunology;
Swine;
Thymidine Kinase;
genetics;
Vaccines, Synthetic;
Viral Envelope Proteins;
genetics;
immunology
- From:
Chinese Journal of Biotechnology
2004;20(4):532-535
- CountryChina
- Language:Chinese
-
Abstract:
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.
