缺失gE基因的牛Ⅰ型疱疹病毒构建及其体外增殖特性比较  

Construction of gE gene deleted bovine herpesvirus type 1

作　　者：尹龙勃[1,2] 尹文玲[1,2] 叶伟成[2] 孙学强[3] 杨鸣琦[1] 王一成[2] 袁秀芳[2] 张存[2] 

机构地区：[1]西北农林科技大学动物医学学院,陕西杨凌712100 [2]浙江省农科院畜牧兽医研究所,浙江杭州310021 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《中国预防兽医学报》2010年第11期839-843,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省农业科学院国际合作专项资助项目

摘　　要：为构建gE基因缺失的牛Ⅰ型疱疹病毒(BHV1),本研究在BHV1全基因组感染性细菌人工染色体克隆pBHV1-ZJ的基础上,利用Red E/T重组技术,分别构建了gE基因缺失、gE和gN双基因缺失的pBHV1突变体,通过转染获得了重组病毒rBHV1-△gE,而双基因缺失突变体pBHV1-△gN-△gE则未能观察到细胞病变。病毒噬斑大小和生长曲线试验表明,在感染细胞内和培养基中,rBHV1-△gE滴度与亲本毒株BHV1无明显差异,而rBHV1-△gN显著降低;重组病毒rBHV1-△gE和前期构建的rBHV1-△gN形成的噬斑均显著小于亲本病毒,其大小分别为亲本病毒的18%和64%。rBHV1-△gE和rBHV1-△gN的构建将有助于阐明gE与gN协同作用机制和为研制BHV1新型基因标记疫苗奠定基础。Using two-step Red-mediated recombination technology in Escherichia coli,the glycoprotein E（gE） gene or gE and glycoprotein N（gN） gene of Bovine herpesvirus type 1（BHV1） were deleted respectively from the bacterial artificial chromosome（BAC） clone pBHV1-ZJ that carried whole BHV1 genome.The resulting BAC clones,pBHV1-ΔgE and pBHV1-ΔgE-ΔgN were transfected into bovine kidney cells（MDBK） respectively.Recombinant virus rBHV1-ΔgE was successfully obtained,however no virus with gE and gN double deletions was rescued after transfection.The growth kinetics of rBHV1-ΔgE was similar to the parental virus rBHV1 in MDBK cells,whereas the rBHV1-ΔgN（previously constructed） exhibited markedly lowered virus titres.The plaque sizes of rBHV1-ΔgE and rBHV1-ΔgN were reduced to 18% and 64%,respectively,compared to the parental strain.These mutant BHV1 provided useful resources for further studies on the interaction between gE and gN in the viral replication.

关 键 词：牛Ⅰ型疱疹病毒 感染性克隆 RedE/T重组 囊膜蛋白gN 囊膜蛋白gE 

分 类 号：S852.65[农业科学—基础兽医学]