抗猪繁殖与呼吸综合征病毒转基因细胞系的建立

Establishment of a transgenic cell line with resistance to porcine reproductive and respiratory syndrome virus

采用脂质体法将具有抑制猪繁殖与呼吸综合征病毒(PRRSV)复制的shRNA质粒pEGFP-N1-shRNA导入PK-15细胞中,经G418药物筛选后,分离扩增绿色荧光蛋白阳性细胞,获得抗PRRSV转基因PK细胞系。通过对转染方法和条件的优化,确立最佳的转染及筛选步骤;对得到的阳性转基因细胞进行冷冻-解冻,并作PCR检测。结果表明,G418最佳筛选浓度为600μg/mL,脂质体与质粒的最佳转染比例为7∶2,最佳转染时间为24 h。本研究成功建立抗PRRSV转基因细胞系,为进一步的功能验证及体细胞核移植奠定了基础。

The objective of this study is to construct a transgenic cell line resistant to porcine reproductive and respiratory syndrome virus （PRRSV） ihfection. The lipid method was used to introduce the anti-PRRSV shRNA plasmid, pEGFP-NI-shRNA, into PK-15 cells. After screened with G418, the fluorecent cells were separated and cultured repeatedly, and therefore an anti-PRRSV transgenic PK-15 cell line was established. The transfection methods and conditions were further optimized, and the constructed cell line was detected by PCR after frozen-thawed. The results showed that the optimal concentration of G418 was 600μg/mL, the transfection ratio of liposome to plasmid was 7 ： 2, and the transfection time was 24 h. This study established successfully an anti-PRRSV transgenic PK-15 cell line, and thus applies a basis for function validation of anti-disease gene and somatic cell nuclear transplantation.