Screening of proteins interacting with retinoic-acid-inducible gene-I by yeast two-hybrid technique

Objective:To screen proteins binding with the protein encoded by retinoic-acid-inducible geneⅠ(RIG-Ⅰ) from human splenic cDNA library by yeast two hybrid technique.Methods: The DNA sequence of 2CARD domain which was a part of RIG-Ⅰgene was amplified by polymerase chain reaction (PCR) using HEK 293T cell RNA as the template and linked to the pGBKT7 vector as a bait plasmid ,called pGBKT7-RIG-Ⅰ2CARD. The plasmid was transformed into yeast competent cell AH109. Positive clones were continued to be transformed with the human splenic cDNA library and plated on synthetic dropout nutrient medium containing X-α-gal for selection.The blue colonies were incubated and then the plasmids were extracted. The plasmids were transformed into E.coli DH5α for amplification. After the analysis of restriction endonuclease digestion analysis and sequence analysis, the sequences of the plasmids were analyzed by bioinformatics.Results:RIG-Ⅰ2CARD gene was successfully cloned and expressed in yeast cells.Fifty-nine positive colonies were obtained using yeast two-hybrid technique. After sequence analysis, twelve clones which might have interaction with RIG-Ⅰ2CARD protein were found.Conclusion:RIG-Ⅰ2CARD gene was successful cloned and twelve proteins that might interact with RIG-Ⅰ2CARD protein were also screened.