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Objective:To screen the proteins interacting with the Treg specification factor forkhead box protein P3 (FOXP3) by yeast two-hybrid system.Methods:Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells (PBMC) and inserted into plasmid pGBKT7 to construct the bait vector,then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed.Thereafter,a human liver cDNA library was screened by the bait vector.The positive clones were selected out by nutrient-deficient culture and back-hybridizing.The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods.Results:The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109.Three proteins which interacted with FOXP3,including tumor protein D52,splicing factor 3b subunit 1 and hypothetical protein,were identified.Conclusion:Three new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library,which may facilitate the further study of FOXP3 in Treg.
Objective: To screen the proteins interacting with the Treg specification factor forkhead box protein P3 (FOXP3) by yeast two-hybrid system. Methods: Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells (PBMC) pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed.Thereafter, a human liver cDNA library was screened by the bait vector.The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods. Results: The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109.Three proteins which interacted with FOXP3, including tumors protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified. Confluence: Three new candidate proteins interacting with FOXP3 are selected out by this yea st two-hybrid system and library, which may facilitate further study of FOXP3 in Treg.