用于山羊痘病毒载体的强痘病毒启动子筛选  

Screening for strong poxvirus promoter for goat poxvirus vector

作　　者：谢梅梅 李翠翠[1] 张家林[1] 王子龙[1] 陈伟业[1] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第7期502-505,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高科技研究发展计划(863计划)(2011AA10A208);国家科技重大专项(2012ZX1000214);国家科技支撑计划课题(2013BAD12B05)

摘　　要：为提高重组山羊痘病毒（GPV）中外源基因的表达水平及筛选强转录效力的启动子,本研究选择桑灯蛾昆虫痘病毒42K启动子（42K）、痘病毒合成晚期441启动子（SL441）、痘病毒合成晚期454启动子（SL454）、牛痘病毒（VV）早晚期P7.5启动子（P7.5）、VV晚期P11启动子（P11）、VV H6启动子（H6）、VV合成早晚期启动子（E/L）及人工优化早晚期启动子（LEO）8种启动子,并在其下游插入萤火虫荧光素酶（Fluc）报告基因,分别构建了启动子效力检测重组质粒。并分别将其转染于预感染GPV的DF-1（鸡）、LT（绵羊）、BHK-21（仓鼠）和Vero（猴）4种动物源细胞中,以表达海肾荧光素酶（Rluc）的p RL-TK为内参质粒,24 h后检测荧光素酶活性。结果表明,在LT细胞中,SL441转录效力最强;在DF-1细胞中,LEO转录效力最强,为其他启动子的1.19-14.46倍;在Vero细胞中,H6和SL441的转录效力较强,达到其他启动子的1.21-11.24倍;在BHK-21中,H6启动子的转录效力也显著强于其它启动子。本实验最终确定了GPV在4种动物源细胞中转录效力强的启动子,该研究结果为进一步提高以GPV为载体疫苗的免疫效力奠定了基础。To improve the expression of the foreign genes in recombinant goat poxvirus（GPV） and screen for strong promoter, the recombinant plasmids were constructed with the firefly luciferase（Fluc） gene as report gene under control of 8poxvirus promoters, respectively, which included the promoters of Amsacta moorei entomopoxvirus 42K（42K）, poxvirus synthetic late 441（SL441）, poxvirus synthetic late 454（SL454）, vaccine virus（VV） early/late P7.5（P7.5）, VV late P11（P11）, VV H6（H6）, VV synthetic early/late（E/L） and synthetic late-early optimized（LEO）. The recombinant plasmids were transfected into four animal cells [DF-1（chicken）, LT（sheep）, BHK-21（hamster） and Vero（monkey）] pre-infected with GPV for 1 hour, respectively,and plasmid p RL-TK expressing Renilla luciferase（Rluc） was used as internal control. Luciferase activities were then detected at24 hours. The results showed that in LT cells, SL441 promoter was the strongest; in DF-1 cells, LEO promoter was the strongest,and was 1.19 to 14.46 times to others; in Vero cells, the transcription efficiency of H6 and SL441 promoters were similar, and were 1.21 to 11.24 times to others; in BHK-21 cells, H6 promoter was significantly stronger than the others. Taken together, we identified different promoters＇ activities in four cells infected by GPV, which established the foundation for improving immune efficacy of GPV recombinant vaccine.

关 键 词：启动子 山羊痘病毒 荧光素酶 转录效力 

分 类 号：S852.65[农业科学—基础兽医学]