摘要
本研究以地高辛标记的含TK基因的BamHI/KpnI片段为探针 ,通过Southern杂交确定伪狂犬病病毒鄂A株基因组中一大小约 5 9kb的KpnI片段中含有TK基因 ,回收该片段并克隆于pUC18的KpnI位点。然后进一步克隆其中含TK基因的PstI/KpnI片段 ,并将LacZ表达盒插入到该片段中的BamHI位点 ,构建转移质粒 pUEKPZ。将该质粒与伪狂犬病病毒鄂A株基因组共转染PK 15细胞 ,待完全病变后在X gal存在下筛选蓝斑 ,蓝斑纯化 3次后 ,经PCR扩增、Southern杂交证实获得的病毒为伪狂犬病病毒鄂A株TK- /LacZ+ 突变株。
A plasmid pUEKPZ was constructed by insertation of LacZ expression cassette into TK gene region of pUEKP,then co transfected into PK 15 cell with PRV strain Ea DNA.After CPE was observed,virus contained supernatants were harvested.Transfection progeny were plated onto PK 15 cells.Then blue plaques were screened in PK 15 cells in the presence of X gal and plaque purified three times.As a result,TK -/LacZ + recombinant virus was obtained.The TK -/LacZ + mutant was further confirmed by Southern blotting hybridization using digoxin labelled TK gene and LacZ gene respectively.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2001年第3期244-248,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
"九五"国家科技攻关计划生物技术项目资助!(项目编号 96 -C0 1- 0 4- 0 3)
