【作者】 徐晓娟； 徐高原； 陈焕春； 刘正飞； 何启盖；

【Author】 Xu Xiao-Juan, Xu Gao-Yuan, Chen HuanChun, Liu Zhen-Fei, He Qi-Gai(Laboratory of Animal Virology, College of Veterinary Medicine,Huazhong Agricultural University, Wuhan, Hubei Province 430070, China)

【机构】 华中农业大学动物医学院病毒室；

【摘要】 将伪狂犬病病毒FK-/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-15,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。空斑纯化三次后,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-15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。更多还原

【Abstract】 To develop pseudorabies Virus TK-/gG- Mutant, the gG-detected transfer vector pUSKKBB and the genome of pseudorabies virus TK-/gG-/LacZ+ mutant were cotransfected into IBRS-2 celis. Transfection progeny were plated onto PK-15 celis and incubated for 2 days under methlcellulose. Then the overlay was renioved and replaced by 1% low melting point agarose in DMEM supplemented with 150ug/ml X-gal. After 2 days, white plaques were screened and purified 4 times. By PCR amplification of gG-deleted gene and LacZ gene, a recombinant virus with TK-/gG- phenotype was confiemed. PCR product sequence indicates there were 1176 bp detetion in gG gene of PRV TK-/gG- mutant. Amplifying gG-deleted gene of different generations of the TK-/gG- mutant show that the mutant is stable within PK-15 celis. TCID50 assay revealed that the recombinant grows well on PK-15 celis. The mice immunized with the TK-/gG- virus showed no signs of illness. As a control, all mice inoculated with PRV strain died from 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.更多还原