基于甲病毒复制子的猪繁殖与呼吸综合征DNA疫苗的构建及在BHK-21细胞上的表达特性  

Construction and characterization of alphavirus replicon based DNA vaccine against porcine reproductive and respiratory syndrome in vitro

作　　者：梁欠欠[1] 侯绍华[1] 李秀岭 贾红[1] 袁维峰[1] 鑫婷[1] 孙建涛[3] 朱鸿飞[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物医学研究室,北京100193 [2]北京市大兴区动物疾病控制中心,北京102600 [3]天津农学院,天津300384

出　　处：《中国兽医科学》2013年第4期364-371,共8页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2012AA101302);农业部动物疫情监测与防治项目;国家科技支撑计划重点项目(2010BAD04B02);公益性行业(农业)科研专项经费项目(200903027)

摘　　要：以猪繁殖与呼吸综合征病毒(PRRSV)FZ06A株全长cDNA为模板,扩增并修饰GP3、GP5、M蛋白的基因,同时融合牛疱疹病毒VP22基因,构建了基于甲病毒复制子的PRRSV DNA疫苗pSCA-VPm3、pSCA-VPm5、pSCA-VP6和pSCA-V56。将其分别转染BHK-21细胞,通过检测转染细胞中目的基因的mRNA水平、目的蛋白的表达水平和转染细胞的凋亡情况,对该疫苗的体外表达特性进行初步研究。结果显示,PCR扩增得到长度分别为765、603、525bp的GP3、GP5、M蛋白的基因。经置换、敲除、融合VP22基因等修饰后获得长度分别为1 545、1 386、1 320bp的修饰后基因。疫苗转染细胞后,用RT-PCR能检测到目的蛋白的mRNA。IFA和Western-blot结果显示,转染细胞中均有目的蛋白表达,转染48h后细胞发生核固缩,甚至核裂解现象。证实,成功构建了基于甲病毒复制子的疫苗pSCA-VPm3、pSCA-VPm5、pSCA-VP6和pSCA-V56,在转染的BHK-21细胞中能实现目的蛋白的表达,且能诱导转染细胞发生凋亡。The structure protein genes, GP3, GP5 and M of PRRSV were amplified by RT-PCR, and then cloned into pSCA1. The recombinant plasmids were verified by restriction enzyme analysis and nucleic acid sequencing. The resultant plasmids were named pSCA-VPm3, pSCA-VPm5, pSCA-VP6 and pSCA- V56,respectively. These plasmids were transfected into BHK-21 cells. Expression of he recombinant proteins in BHK-21 cells were confirmed by indirect immunofluorescence test and Western-blot. Forty eight hours after transfection of EGFP-pSCA into BHK-21 cell 48 hours,we detected the apoptosis cells by fluorescence microscopy. The results suggested that the target proteins were expressed in the BHK 21 cells.

关 键 词：猪繁殖与呼吸综合征病毒 甲病毒复制子 构建 表达特性 

分 类 号：S852.659.6[农业科学—基础兽医学]