title: Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.
abstract: Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research.
authors.affiliation: Liaoning Medical University, Jinzhou 121200, Liaoning, China. Electronic address: zhaowei8072@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: 2nd Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Liaoning Medical University, Jinzhou 121200, Liaoning, China.
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Liu
authors.affiliation: Liaoning Medical University, Jinzhou 121200, Liaoning, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Liaoning Medical University, Jinzhou 121200, Liaoning, China.
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Jin
authors.affiliation: Liaoning Medical University, Jinzhou 121200, Liaoning, China.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Sui
mesh_terms: D018938:Caco-2 Cells; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D001257:Mamastrovirus / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
keywords: Caco-2; Human astrovirus; SMART technology; Yeast two-hybrid; cDNA library