Construction and identification of yeast two-hybrid expression vector containing human transforming growth factor-beta 1 and different fragments of transforming growth factor-beta receptor type Ⅱ

Objective To clone human transforming growth factor-beta 1 (TGF-β1) and 4 genome fragments of its receptor and to construct the yeast expression vector to provide technical support for using yeast two-hybrid in identification of interaction domain of TGF-β type Ⅱ receptor (TβRⅡ) with TGF-β1.Methods The gene TGF-β1 and gene fragments of TβRⅡ from the RNA of human peripheral blood were amplified with regular PCR using specific primers,and then cloned into yeast two-hybrid expression vector pGBKT7 and pGADT7.The recombinant plasmids pGBKT7-TGF-β1 and pGADT7-TβRⅡ-A,pGADT7-TβRⅡ-B,pGADT7-TβRⅡ-C,pGADT7-TβRⅡ-D sequences were confirmed with PCR,restriction endonucleases digestion,and automated DNA sequencing.Results The yeast two-hybrid expression vector pGBKT7-TGF-β1 (1 200 bp) and pGADT7-TβRⅡ-A (453 bp),pGADT7-TβRⅡ-B (366 bp),pGADT7-TβRⅡ-C (276 bp),pGADT7-TβRⅡ-D (165 bp) were successfully constructed and the sequences of the plasmids’ DNA were correct.Conclusion The yeast two-hybrid expression vectors pGBKTT-TGFβ1 and pGADT7-TβRⅡA,B,C,and D were successfully constructed.