牛Ⅰ型疱疹病毒感染性细菌人工染色体的构建和gN基因缺失病毒的细胞繁殖特性

Bovine herpesvirus type 1 genome cloned as infectious bacterial artificial chromosome and replication of gN gene deletion mutant in vitro

通过同源重组将pHA2质粒插入到牛Ⅰ型疱疹病毒ZJ分离株基因组的UL15和UL18基因之间,构建了重组病毒rBHV1-HA;提取重组病毒的环状基因组DNA,转化大肠杆菌DH10B,获得了含有BHV1全基因组的感染性细菌人工染色体克隆pBHV1.pBHV1转染MDBK细胞可以拯救出病毒,该病毒与野生毒株在细胞上的繁殖特性未见差异.通过两步Red E/T重组,构建了gN基因跨膜区缺失的pBHV1突变体,并转染获得了重组病毒rBHV1-△gN.病毒繁殖动态曲线显示,rBHV1-△gN的滴度比野生毒株低9%～20%.BHV1感染性克隆的成功构建,将为研究新型BHV-1基因缺失疫苗和牛的病毒载体疫苗提供技术平台.

The recombinant bovine herpesvirus type 1 （rBHV1-HA） was constructed by inserting pHA2 plasmid into the viral genome between UL15 and UL18 cassettes. BHV1 infectious bacterial artificial chromosome had been confirmed after rBHV1-HA circular genome was extracted and transformed into E. coil strain DH10B. The resulting Bac clone, pBHV-1, was transfected into bovine kidney cells （MDBK） and the virus was rescued. The rescued virus, BHVl-res, have almost the same titres as the wildtype in replication in vitro. The transmembrane domain in gN open reading fram was deleted from pBHV-1 by Red E/T mutagenesis in E.coli. BHV-1 with partial deletion of gN, rBHV1-AgN, was reconstituted from the mutated Bac. rBHV1-AgN had lower titres by from 9% to 20% than rBHV-res in growth property in MDBK. This construction of infectious BHV1 clone should contribute greatly to the recombinant BHV1 vaccine with genes deletion and bovine universal viral vector.