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2025年8月4日 星期一
  2018, Vol. 26 Issue (10): 1814-1820    
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
稳定表达小反刍兽疫病毒受体Nectin-4的MDBK细胞系的建立
杨侃侃1,张成1,王元红1,殷冬冬1,俞赵荣1,刘光清2,朱杰2,李刚3,赵长城4,李永东5,王勇1
1. 安徽农业大学 动物科技学院
2. 中国农业科学院 上海畜牧兽医研究所
3. 中国农业科学院北京畜牧兽医研究所
4. 安徽省立医院感染病院
5. 宁波市疾病预防控制中心
Establishment of MDBK Cell Lines Stably Expressing Peste des petits ruminants virus (PPRV) Receptor Nectin-4
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摘要 小反刍兽疫病毒(Peste des petits ruminants virus, PPRV) 是小反刍兽疫(Peste des petits ruminants, PPR)的病原。Nectin-4为PPRV感染宿主上皮细胞的受体。本研究采用合成的Nectin-4基因,构建重组质粒pLOV-eGFP-Nectin-4。将该重组质粒与包装质粒pSPAX2和外膜质粒pMD2.G质粒共转染293T细胞,获得逆转录病毒样颗粒。为了构建稳定表达PPRV受体Nectin-4的MDBK (Madin-Darby bovine kidney)细胞系,将含有逆转录病毒样颗粒的细胞培养上清液感染MDBK细胞,利用嘌呤霉素筛选、纯化,获得了稳定表达Nectin-4蛋白的MDBK细胞,将其命名为MDBK-Nectin-4。pLOV-eGFP-Nectin-4、pSPAX2和pMD2.G三质粒共转染293T细胞后荧光结果表明,慢病毒成功包装。嘌呤霉素对MDBK细胞最小致死浓度检测结果表明,1 μg/mL的嘌呤霉素为最佳的筛选工作浓度。RT-PCR检测结果表明,Nectin-4基因能够在MDBK-Nectin-4细胞中转录成mRNA。Western blot检测结果表明,Nectin-4在MDBK-Nectin-4细胞中能够稳定表达。间接免疫荧光(indirect immunofluorescence assay, IFA)结果表明,表达的Nectin-4蛋白主要分布在细胞膜上。构建的MDBK-Nectin-4细胞系在连续传代至20代,仍然能够稳定表达Nectin-4蛋白,结果表明具有良好的遗传稳定性。PPRV分别感染 MDBK细胞与MDBK-Nectin-4细胞系,后者较于前者能够更快地出现细胞病变。该细胞系的建立为深入研究PPRV与受体Nectin-4的相互作用机制,以及临床快速地分离PPRV提供了实验材料。
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杨侃侃
张成
王元红
殷冬冬
俞赵荣
刘光清
朱杰
李刚
赵长城
李永东
王勇
关键词 小反刍兽疫病毒受体Nectin-4MDBK细胞系稳定表达    
Abstract:Peste des petits ruminants virus (PPRV) is the causative agent of Peste des petits ruminants (PPR). Nectin-4 is the receptor of PPRV-infected host epithelial cells. In this study, Nectin-4 gene was synthesized to construct the recombinant plasmid pLOV-eGFP-Nectin-4. To obtain retrovirus-like particles,the recombinant plasmid was co-transfected into 293T cells with the packaging plasmid pSPAX2 and the outer membrane plasmid pMD2.G. To construct the MDBK (Madin-Darby bovine kidney) cell line stably expressing Nectin-4 receptor, the MDBK cells were firstly infected with the supernatant of cell culture which containing the retrovirus-like particles, and then screened and purified using puromycin. The MDBK cell line stably expressing Nectin-4 protein was obtained, which was named as MDBK-Nectin-4. The fluorescence of 293T cells co-transfected with pLOV-eGFP-Nectin-4, pSPAX2 and pMD2.G, respectively. Plasmids showed that the lentivirus was successfully packaged. The results of the minimal lethal concentrations of puromycin for MDBK cells showed that the best screening concentration of puromycin was 1 μg/mL. RT-PCR analysis showed that the Nectin-4 gene could be transcribed into mRNA in MDBK-Nectin-4 cells. The results of Western blot assay showed that Nectin-4 protein could be expressed stably in the MDBK-Nectin-4 cells. The results of indirect immunofluorescence assay (IFA) demonstrated that the expressed Nectin-4 protein was mainly distributed on the cell membrane. After continuous passaged to the 20th generation, the MDBK-Nectin-4 cell line still stably expressed the Nectin-4 protein, which indicated a good genetic stability of this cell line. MDBK-Nectin-4 cell line infected with PPRV was more rapidly cytopathic than the MDBK cells. The established cell line provides experimental materials for further investigation of the interaction mechanism between PPRV and receptor Nectin-4, as well as clinical rapid isolation of PPRV.
Key wordsPeste des petits ruminants virus    Receptor    Nectin-4    MDBK cell line    Stable expression
收稿日期: 2018-03-05      出版日期: 2018-09-26
基金资助:基于CRISPR/Cas9的羊口疮病毒dUTPase基因缺失株构建及其生物学特性
通讯作者: 王勇   
引用本文:   
杨侃侃 张成 王元红 殷冬冬 俞赵荣 刘光清 朱杰 李刚 赵长城 李永东 王勇. 稳定表达小反刍兽疫病毒受体Nectin-4的MDBK细胞系的建立[J]. , 2018, 26(10): 1814-1820.
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http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I10/1814
 
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