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

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.