Construction of recombinant plasmid pcDNA3.1myc-HisA-Smad2/3/4 and its protein expression

Objective To construct the expression plasmid of pcDNA3.1myc-HisA-Smad2/3/4 and identify its fusion protein expression.Methods pcDNA3.1-Smad2/3 and pGEX2T-Smad4 were used as templates,and the special primers were designed.The Smad2/3/4 coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into pcDNA3.1myc-HisA vector.After the target region was sequenced,the plasmid was transfected into HEK293 cell line.The expression of the recombinant plasmid in HEK293 cells was detected by Western blotting.Results Smad2/3/4 was constructed into expression vector pcDNA3.1myc-HisA successfully.The lengthes of the fragments were 1 401,1 275 and 1 656 bp,and they were identified by restriction enzymes digestion.The expression of pcDNA3.1myc-HisA-Smad2/3/4 fusion protein was proved by Western blotting.Conclusion The eukaryotic expression plasmid pcDNA3.1myc-HisA-Smad2/3/4 is successfully constructed,and the expression of pcDNA3.1myc-HisA-Smad2/3/4 fusion protein is identified.