The new method for transfection of macromolecules into cells using protein transductions domain / 白血病·淋巴瘤

ABSTRACT

Objective To construct a fusion protein to transfect some cell lines that were difficult to be transfected such as neoplastic cells, nerve cells, stem cells. Methods PCR was performed to amplified protein transductions domain(PTD),G4S and streptavidin (Strep).Enzymatic digestion and ligation were used to construct pAYZ-PTD-Strep plasmid. Fusion protein was induced to express by AP5 medium and was isolated by E-tag affinity chromatography. Fusion protein was identified by Western blot. eGFP was trasfected into U937 cells by pAYZ-PTD-Strep. FACS was performed to detect transfection percentage. Results Fusion protein PTD-G4S-Strep was expressed as soluble protein.The concentration of fusion protein was about 0.7 mg/L,and purity was over 90 ％. The protein could carry plasmid into a suspended cell line, U937 cells. The transfection-efficiency of protein was higher than monometer PTD.Conclusion The protein PAYZ-PTD-Strep could carry macromolecules into blood tumor cells,and its biological activity may be expected to develop into a highly efficient and reliable transfection method.