摘要
将含有牛传染性鼻气管炎病毒（ Ｉ Ｂ Ｒ Ｖ） Ｔ Ｋ 基因的片段克隆于ｐ Ｂｌｕｅｓｃｒｉｐｔ Ｓ Ｋ 中， 再用 Ｂｇ１ Ⅱ和 Ｓａｃ Ｉ切去 Ｔ Ｋ 基因中３４７ｂｐ 的片段， 获得了含缺失 Ｔ Ｋ 基因的重组质粒， 然后插入 Ｌａｃ Ｚ 报告基因， 构建成转移载体。以脂质体转染法将此转移载体与 Ｉ Ｂ Ｒ Ｖ Ｂａｒｔｈａ 株在牛肾细胞（ Ｍ Ｄ Ｂ Ｋ） 上共转染， 经过蓝色蚀斑筛选和蚀斑克隆纯化， 获得了能稳定遗传的重组 Ｉ Ｂ Ｒ Ｖ。经 Ｐ Ｃ Ｒ 和 Ｓｏｕｔｈｅｒｎ 印迹杂交鉴定， 证实该重组病毒为 Ｔ Ｋ 基因缺失突变株。
A fragment containing TK gene of infectious bovine rhinotracheitis virus(IBRV)was cloned into pBluescript SK,and the recombinant plasmid was designated as pTK.A 347bp fragment within TK gene was cut off by overdigestion with BglII and partial digestion with SacI and then self ligated.The LacZ gene from E.coli was then inserted into the deleted TK region.The recombinant plasmid with deleted TK and LacZ genes was used to co transfect MDBK cells with IBRV Bartha strain.Through five times blue plaque purifications,a genetically stable recombinant IBRV was obtained.The recombinant virus was identified as TK gene deleted mutant by PCR and Southern hybridization.
出处
《中国预防兽医学报》
CAS
CSCD
1999年第4期275-277,共3页
Chinese Journal of Preventive Veterinary Medicine
