| 李玲玲,余敏君,邓湘赢,戴佩,朱翠明,罗丹,廖雅婷,曾焱华.利用膜蛋白酵母双杂交系统构建人尿道上皮细胞cDNA文库.[J].中南医学科学杂志.,2018,(5):478-481. |
| 利用膜蛋白酵母双杂交系统构建人尿道上皮细胞cDNA文库 |
| Construction and Identification of SV-HUC-1 cells cDNA Library Based on Split-ubiquitin Membrane Yeast Two-hybrid System |
| 投稿时间：2018-05-29 修订日期：2018-07-15 |
| DOI：10.15972/j.cnki.43-1509/r.2018.05.008 |
| 中文关键词: 膜蛋白酵母双杂交 人尿道上皮细胞 cDNA文库 |
| 英文关键词:membrane yeast two-hybrid SV-HUC-1 cells cDNA library |
| 基金项目: |
| 作者 | 单位 | | 李玲玲 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 余敏君 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 邓湘赢 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 戴佩 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 朱翠明 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 罗丹 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 廖雅婷 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 | | 曾焱华 | 南华大学病原生物学研究所,特殊病原体防控湖南省重点实验室,湖南省子靶标新药研究协同创新中心,湖南 衡阳 421001 |
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| 中文摘要: |
| 利用基于分离的泛素介导的膜蛋白酵母双杂交技术,构建人永生尿道上皮细胞(SV-HUC-1)cDNA文库。Trizol法提取人尿道上皮细胞总RNA,并分离纯化其mRNA；以mRNA为模板,合成cDNA第一链、第二链；通过T4 DNA聚合酶将DNA链两端加上5′接头；并将其大于1 kbp的片段与膜蛋白酵母双杂交载体pPR3-N连接,经电转化大肠杆菌感受态细胞,以构建基于分离的泛素介导的膜蛋白酵母双杂交cDNA文库,并检测文库的库容量和随机性。本研究成功构建了人尿道上皮细胞的基于分离的泛素介导的膜蛋白酵母双杂交cDNA文库,为进一步研究泌尿生殖道感染病原体与宿主尿道上皮细胞的相互作用奠定了实验基础。 |
| 英文摘要: |
| The split-ubiquitin membrane-based yeast two-hybrid system was used to construct the split-ubiquitin membrane yeast two-hybrid cDNA library of human urothelium cell (SV-HUC-1). The whole RNA of SV-HUC-1 cells was extracted by using Trizol and the mRNA was isolated and puried by Oligotex mRNA Kits. The first strand of cDNA was synthesized using?reverse transcriptase and the double-strand DNA was synthesized using DNA polymerase. After the 5′ adaptors were added, the cDNA products were electrophoresised. The DNA fragments that were longer than 1,0 bp were collected and then ligated into the pPR3-N vector. The recombinant vectors were electrotransformed into Escherichia Coli to construct the SV-HUC-1 cells cDNA Library based on split-ubiquitin membrane yeast two-hybrid system. The quality of library was then identified. The capacity of the library was approximately 1.2×107pfu/cm3 and the recombination rate was about 100%, and the average inserts were about 1,0bp. These results demonstrated that the split-ubiquitin membrane yeast two-hybrid cDNA library of SV-HUC-1 cells with high quality was successfully constructed, which lays an experimental basis for the further study on the interaction between urogenital pathogens and SV-HUC-1 cells. |
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