Study Of Peptide Vector-mediated Dna Delivery

In order to enhance the efficiency of exogenous DNA delivering the nucleus, a polyfunctional peptide vector--K,:RNLS, KKKKKKKKKKK-KACDCRGDCFCGPKKKRKV, was designed and synthesized. This peptide comprises three fragments, an arginime-glycine-aspartic acid tripeptide motif to recognize the integrin receptor in the cell membrane, a nuclear localization signal sequence to recognize the nuclear pore complexes in nuclear envelope and a short stretch of 12 lysine residues to enhance the peptide binding to DNA. When the peptide was compounded with plasmid DNA in the solution, the complexes of peptide-DNA formed spontaneously because of the static electricity of role between the positive charge of Ki;RNLS and the negative charge of plasmid. 0.5|ag of plasmid was compounded with the serial dilutions of peptide in a volume of 20uL serum-free medium. The mixtures were vortexed and incubated at room temperature for 20 minutes before the complexes were analyzed on 1% agarose gels. It was found that DNA was fully retarded when Ijag of DNA was mixed with 2.2ug of peptide, which was defined as one unit of retardation. Under the transmission electron microscope it was observed that most of the complexes formed in 2 retardation units were about 20 to 50nm in diameter.The plasmid pIRGFP containing a reporter gene which expresses green fluorescent protein was compounded with the K1;RNLS in an appropriate ratio and incubated at room temperature for 20 minutes, then their complexes were transfected into the culture cells. After 48h posttransfection the cells expressing the green fluorecent protein could be observed under the fluorecent microscopy. An optimal transfection would be produced by the DNA-peptide complexes formed in 2 retardation units.In addition, the transfection in vivo also was. carried out, which the K12RNLS and plasmid pCDLacZ (encode p-lactase Z gene) were compounded and incubated at room temperature for 20 minutes, then the complexes were injected into the rectus femoris muscle of the mice. Four days later, the muscles were taken, fixed and dyed and the blue on the membrane of myofibros and myofiber was obviously observed compared with the control groups.It was initially proved in this study that the designed peptide could deliver the reporter gene into the cells in vitro and in vivo where the gene could be expressed.