摘要
在伪狂犬病病毒转移载体pBdTK＿Uni的多克隆位点中插入由SV40启动子控制下的LacZ基因表达盒,同时在右侧同源臂下游插入一个1 7kb的KpnI片段,构建成一个新的转移载体pUni＿LacZ。用该载体与Bartha＿K61株基因组通过脂质体法共转染Vero细胞,经过10代蓝斑筛选纯化和PCR鉴定获得了一株稳定表达LacZ基因的伪狂犬病病毒gE/TK基因缺失突变株,命名为rPrV＿LacZ。在不同的细胞(PK＿15、IBRS＿2、Vero和CEF)上,对该重组病毒与亲本病毒的增殖滴度和细胞病变进行比较,未见显著差异。结果表明转移载体pBdTK＿Uni具有实用性,可用于构建伪狂犬病病毒基因工程活载体疫苗。
A 1.7kb KpnI fragment and a LacZ gene expression cassette were inserted into the transfer vector pBdTK-Uni of pseudorabies virus (PrV), produced a transfer vector pUni-LacZ. The purified genomic DNA of Bartha-K61 strain and transfer vector pUni-LacZ were used to cotransfect Vero cells using lipofection transfection procedure. A recombinant virus stably expressing LacZ gene was generated after ten cycles of blue plague purification and PCR identification, and designated as rPrV-LacZ. Compared to its parental virus Bartha-K61 strain, rPrV-LacZ showed no obvious defferences in virus multiplication and cytopathogenic effect in defferent cell cultures (PK-15, IBRS-2,Vero and CEF). The results showed that the transfer vector pBdTK-Uni can be used for creation of PrV recombinants expressing foreign gene(s).
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2004年第3期181-185,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家863计划资助项目(2001AA213051)
