PCR-induced Modification of C Terminus of HPV-16 E7 and Expression of Mutational E7 in Eukaryotic Cells / 中华皮肤科杂志

ABSTRACT

Objective To induce the mutation of HPV-16 E7 in two zinc-binding motifs near the C terminus by polymerase chain reaction (PCR) and evaluate the effect of this mutation on the antigen-specific immunity of HPV-16 E7. Methods HPV-16 E7 fragment was amplified by PCR and cloned into pGEM-3zf vector. Two site mutations at 58 and 91 animo acid sites in the open reading frame of HPV-16 E7 were induced by PCR, and then the molecular clones of HPV-16 E7 wild type (pcDNA3.1/E7) and mutant (pcDNA3.1/ME7) were successfully reconstructed. Western blot and immunofluorescence were used to detect the expression of E7 protein. Results Intracellular fluorescence signals were observed in the cells transfected with pcDNA3.1/E7 and pcDNA3.1/ME7 24 hours after transfection, but the signals in the cells transfected with pcDNA3.1/ME7 disappeared 48 hours after tansfection. Twenty-four and 48 hours after transfection with pcDNA3.1/ME7, E7 protein was not detected by Western blot. Conclusions The stability of HPV-16 E7 protein is reduced by mutations (C58G, C91G) near two zinc-binding motifs. It is suggested that the zinc-binding motifs near the C terminus of HPV-16 E7 may be important for maintaining the stability of E7 protein.