摘要
将SV40启动子控制下的LacZ基因表达盒和CMV启动子控制下的H3N2亚型猪流感病毒(SIV H3N2)的HA基因插入到伪狂犬病病毒(PRV)通用转移载体pBdTK-Uni中,获得转移载体pLTK-HA。将该载体与PRV Bartha-K61株基因组DNA通过脂质体法共转染Vero细胞,经过10代蓝斑筛选、纯化和PCR鉴定获得了一株插入SIV HA基因的重组伪狂犬病病毒,命名为rPRV-HA。Western blotting和间接免疫荧光试验证实HA基因在重组病毒感染的细胞中获得了表达。用不同的细胞(PK-15I、BRS-2、Vero和鸡胚成纤维细胞)对该重组病毒与亲本病毒的增殖滴度和致细胞病变进行比较,未见显著差异,对第30代重组病毒的HA基因进行序列分析,表明该重组病毒遗传性状稳定。
A pseudorabies virus （PRV） transfer vector pLTK-HA containing a LacZ gene expression cassette and the HA gene of swine influenza virus （SIV） H3N2 subtype under the control of human cytomegalovirus （CMV） promoter was constructed and used to cotransfect with genomic DNA of strain Bartha-K61 into Vero cells using lipofectin transfection procedure. A recombinant PRV haboring SIV HA gene, designated as rPRV-HA, was generated after ten cycles of blue plague purification and PCR identification. The expression of the HA by rPRV-HA infected cells were characterized by Western blotting and indirect immunefluorescenee assay （IFA）. Compared to its parental virus Bartha-K61 strain, rPRV-HA showed no obvious difference in virus multiplication and cytopathogenic effects in Vero, PK-15, IBRS-2, chicken embryo fibroblast cells. The results of analyzing 30th passage rPRV-HA indicated that HA gene can be stably expressed by rPRV-HA.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2005年第10期1043-1048,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家863计划项目(2001AA213051)
国家科技攻关项目(2004BA519A21)
