伪狂犬病病毒TK^-/gG^-缺失株的构建及其生物学特性研究  

Construction and Characterization of a Pseudorabies Virus TK^-/gG^- Mutant

作　　者：徐晓娟[1] 徐高原[1] 陈焕春[1] 刘正飞[1] 何启盖[1] 

机构地区：[1]华中农业大学动物医学院病毒室,武汉430070

出　　处：《生物工程学报》2004年第4期532-535,共4页Chinese Journal of Biotechnology

基　　金：国家 8 63项目基金资助 (No.2 0 0 2AA2 45 0 71)~~

摘　　要：将伪狂犬病病毒TK- gG- LacZ+ 突变株的基因组DNA与含有缺失的gG基因的转移质粒pUSKBB共转染猪肾传代细胞PK 1 5 ,待完全病变后收获病毒进行空斑试验 ,用PCR筛选gG缺失的重组病毒。空斑纯化 3次后 ,随机挑取空斑进行PCR扩增 ,证实所获得的病毒为均一的TK- gG- 缺失株。遗传稳定性试验表明该重组病毒能在PK 1 5细胞上稳定遗传 ,动物试验表明该缺失株对Balb c小鼠极为安全且能保护Balb c小鼠抵抗致死量PRV强毒的攻击。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 150μg/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. TCID 50 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.

关 键 词：伪狂犬病病毒 TK^-/gG^-/LacZ^+突变株 TK^-/gG^-缺失株 

分 类 号：S852.65[农业科学—基础兽医学]