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 than 1,000 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 1.2×10 7pfu/cm 3 and the recombination rate was about 100%, and the average inserts were about 1,200bp. 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.
作者
李玲玲
余敏君
邓湘赢
戴佩
朱翠明
罗丹
廖雅婷
曾焱华
LI Lingling;YU Minjun;DENG Xiangying;DAI Pei;ZHU Cuiming;LUO Dan;LIAO Yating;ZENG Yanhua(Institute of Pathogenic Biology,University of South China,Hunan Provincial Key Laboratory forSpecial Pathogens Prevention and Control,Hunan Province Cooperative Innovation Center ofMolecular Target New Drug Study,Hengyang 421001,Hunan,China)
出处
《中南医学科学杂志》
CAS
2018年第5期478-481,共4页
Medical Science Journal of Central South China
基金
国家自然科学基金(NO:31370207
81871256)
