Objective      To design and synthesize a new sequence of cell-penetrating peptide (CPP)

CGRLWMRWYSPWARRYGC

and to investigate its potentiality as gene vector

in vitro

.       Method      The new sequence of CPP was synthesized by the solid-phase synthetic method with florenl methyoxycarbonyl amino acids. N-terminal fluorescein isothiocyanate-labeled peptide was also synthesized. The purity and molecular weight of CPP were determined using HPLC and MALDI-TOF-MS. The cell-penetrating activity was examined with flow cytometry and confocal l

aser scanning microscopy. Transfection effect of CPPs-mediated enhanced green fluorescence protein plasmid (pEGFP) in HUVEC was observed using fluorescent microscopy and flow cytometry. The cytotoxicity of CPPs/plasmid DNA compound was evaluated by MTT assay. The affinity of plasmid DNA and CPPs and the protection of CPPs peptide to plasmid DNA were analyzed by agarose gel electrophoresis.       Results      The purity of synthesized CPPs was 96.13%

and molecular weight was 2 348.42. The results of flow cyto-metry showed that about 98.81% of HUVEC had been labeled

and confocal laser scanning microscopy showed that fluorescence-labeled CPPs transmembraned and localized in the cells. Transfection effect of CPPs-mediated enhanced green fluorescence protein plasmid (pEGFP) in HUVEC was 11.44%. MTT assay indicated that no significant difference of cell viability was found. The results of agarose gel electrophoresis showed that the plasmid DNA was unable to migrate when the charge ratio of CPPs to plasmid DNA was larger than 2∶1

and the complex could resist digestion of DNase Ⅰ when the charge ratio of complex was 4∶1

8∶1 and 16∶1

especially 8∶1.       Conclusion      Our new designed CPP

CGRLWMRWYSPWARRYGC

can be used as a potential gene vector.