期刊论文

Construction and Identification of SV-HUC-1 cells cDNA LibraryBased on Split-ubiquitin Membrane Yeast Two-hybrid System

中文

中南医学科学杂志

2095-1116

2018

46

5

478-481

10.15972/j.cnki.43-1509/r.2018.05.008

NO31370207:国家自然科学基金 81871256:国家自然科学基金

本校为第一机构

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p PR3-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。

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 tha...