| 抗猪繁殖与呼吸综合征病毒转基因细胞系的建立 |
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| 引用本文： | 马汝钧,罗碧平,剧世强,芮荣.抗猪繁殖与呼吸综合征病毒转基因细胞系的建立[J].畜牧与兽医,2012,44(1):8-11. |
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| 作者姓名： | 马汝钧 罗碧平 剧世强 芮荣 |
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| 作者单位： | 南京农业大学动物医学院,江苏南京,210095 |
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| 基金项目： | 博士学科点专项科研基金，国家转基因生物新品种培育重大专项 |
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| 摘 要： | 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。
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| 关 键 词： | 猪繁殖与呼吸综合征病毒 PK-15细胞 转基因细胞系 |
Establishment of a transgenic cell line with resistance to porcine reproductive and respiratory syndrome virus |
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| MA Ru-jun
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LUO Bi-ping
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JU Shi-qiang
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RUI Rong.Establishment of a transgenic cell line with resistance to porcine reproductive and respiratory syndrome virus[J].Animal Husbandry & Veterinary Medicine,2012,44(1):8-11. |
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| Authors: | MA Ru-jun LUO Bi-ping JU Shi-qiang RUI Rong |
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| Institution: | (College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China) |
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| Abstract: | The objective of this study is to construct a transgenic cell line resistant to porcine reproductive and respiratory syndrome virus(PRRSV) infection.The lipid method was used to introduce the anti-PRRSV shRNA plasmid,pEGFP-N1-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 600 μg/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. |
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| Keywords: | porcine reproductive and respiratory syndrome virus(PRRSV) PK-15 cells transgenic cell line |
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