鸡氨肽酶N基因转染BHK-21细胞后IBV感染情况的改变  

IBV intection difference before and after the chicken aminopeptidase N gene transfected the BHK-21 cell

作　　者：明晓波[1] 张颖[1] 刘澜澜[1] 魏萍 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030

出　　处：《中国兽医学报》2010年第6期793-797,861,共6页Chinese Journal of Veterinary Science

摘　　要：构建了带绿色荧光蛋白基因及鸡氨肽酶N全长基因的真核表达重组载体,重组载体转染BHK-21细胞后12h开始可见绿色荧光,36h荧光最强,试验中转染24h后感染IBV鸡胚肾细胞适应毒,72h收获病毒接下一次转染的BHK-21细胞,如此在转染细胞上连续传5代,用间接免疫荧光检测各代次病毒感染转染BHK-21细胞,可见随着代次增加,感染程度增加;病毒毒力EID50逐渐增加,第5代时原病毒毒力基本恢复,半定量RT-PCR检测各代次病毒也可见病毒含量逐渐增加。结果表明,鸡氨肽酶N转染至IBV非易感的BHK-21细胞系后,BHK-21细胞变得对IBV敏感,鸡氨肽酶N可能用作IBV感染的细胞受体。An eukaryotic expression vector containing the green fluorescent protein gene and the 12 h full length chicken amninopeptidase N gene was successfully constructed,the BHK-21 showed the green fluorescent 12 h after the cells were transfected with the recombinant vector,then the fluorescence intensity increased gradually,the fluorescence intensity reached its peak at 48 h,then the fluorescence intensity decreased gradually.IBV which was chicken embryo kidney（CEK）cell-adapted was inoculated the transfected BHK-21 cell 24 h after tansfection.72 h After transfected the virus was harvested,and then the virus were inoculated into the next transfected BHK-21 cells,and the next harvested virus was denominated the next generation.Using the same method,we propagated the virus for five passages serially on the transfected cells.Each generation virus was called as F1,F2,F3,F4,and F5.The harvested virus IBV of each generation was determined by indirect fluorescent assay （IFA）,the results indicated that the infected level increased with the passages;the virulence EID50 strengthened gradually,and recovered to the primary virulence in the fifth passage.The quantities of IBV increased with the passages using semiquantitative RT-PCR assay;the results indicated that the unsusceptible BHK-21 cells became susceptible to IBV after transfected with cAPN,suggesting the cAPN probably serve as a receptor for IBV.

关 键 词：氨肽酶N 基因克隆 真核转染 RT-PCR 间接免疫荧光 绿色荧光蛋白 红色荧光标记二抗 BHK-21 

分 类 号：S852.23[农业科学—基础兽医学] S852.65[农业科学—兽医学]