利用BAC/gal K系统构建缺失潜伏相关转录物启动子LAP1的伪狂犬病突变病毒

CONSTRUCTION OF PSEUDORABIES VIRUS MUTANT WITH DELETION OF LATENCY-ASSOCIATED TRANSCRIPTS PROMOTER LAP1 USING BAC/GALK SYSTEM

伪狂犬病病毒感染猪后,在耐过猪中均能形成潜伏感染。在潜伏感染期间,病毒基因组仅大量转录病毒潜伏相关转录物(latency-associated transcripts,LATs)。为了研究潜伏相关转录物启动子对LAT表达特征的影响,通过构建缺失LAT启动子LAP1、并添加绝缘子cHS4的供体质粒,利用感染性细菌人工染色体克隆(bacterial artificial chromosome,BAC)操作平台和galK(半乳糖激酶)正负筛选系统,获得BAC载体pBAC-JS-cHS4-△LAP1b,BAC载体与质粒pcDNA3.1-cre共转染Vero细胞后获得突变病毒vJS-cHS4-△LAP1b。空斑和生长曲线结果显示,vJS-cHS4-△LAP1b与亲本病毒具有相似的生物学特性。vJS-cHS4-△LAP1b感染Vero细胞后,RT-PCR检测显示表达大的潜伏相关转录本(large latency transcripts,LLT)。vJS-cHS4-△LAP1b的构建为深入研究LAT表达特征及功能提供了依据和基础。

Pseudorabies virus(PRV)establishes latent infection in the survived pigs.During the latent infection,the viral genome only transcribes a large number of latency-associated transcripts(LATs).In the present study,a donor vector with LAT promoter LAP1 deletion and cHS4 addition was constructed to study its effect on LAT expression.Using an infective bacterial artificial chromosome(BAC)clone of PRV and galK-based positive-negative screening method,we obtained a mutant BAC clone pBAC-JS-cHS4-△LAP1b.The BAC clone was co-transfected with pcDNA3.1-cre into Vero cells,generating the mutant virus vJS-cHS4-△LAP1b.The mutant virus vJScHS4-△LAP1b showed similar properties to that of the parental virus in various virological assays.LAT expression was demonstrated in the Vero cells infected by vJS-cHS4-△LAP1b.In conclusion,construction of mutant virus vJS-cHS4-△LAP1b would provide a solid foundation for further investigation on the role of LAT of PRV.