Preparation And Transfection Performance Of LK6 Antibacterial Peptide And Modified Peptide Gene Vector

Gene therapy as a potential effective method to solve human genetic diseases have arouse great concern.Gene vectors are divided into viral vectors and non-viral vectors.Viral vectors have some disadvantages,such as high transfection efficiency but difficult preparation,small target gene capacity,poor targeting specificity,strong immunogenicity,easy to produce wild type virus and so on.Therefore,its clinical application is greatly limited.At present,the key to gene therapy is to develop a safe,effective and cheap non viral gene delivery system.In this paper,we selected the antibacterial peptide L-K6 as a template for modification.L-K6 is a modified peptide obtained from the natural antibacterial peptide Temporin-1 CEb which is isolated and purified from the skin secretions of Rana chensinensis.The antimicrobial peptide containing Trp has a good killing effect on cancer cell membranes,and can interact with intracellular substances through cell membranes.Based on the previous work,a series of new antibacterial peptides I1 W,L5W,and L12 W containing Trp were synthesized by replacing and designing single Trp residues at different positions.It was found that their cell affinity and hydrophobicity were increased,and it was speculated that they may have good transfection ability.So in this paper,we explored the ability of antibacterial peptide L-K6 and its analogs to bind to exogenous GFP-1 DNA,and its intracellular transfection performance.First,the secondary structure of the peptides L-K6,I1 W,L5W,and L12 W interacting with DNA is detected by circular dichroism(CD)experiments.The results show that compared with L-K6,the ?-helix content of I1 W,L5W,L12 W.The ?-helix contents of I1 W,L5W,L12 W and L-K6 are: 67.68%,44.14%,42.91% and 36.99% respectively;the results of dynamic light scattering detection of peptide molecular size and peptide Zeta potential show that the particle size of L-K6,L12 W,L5W,and I1 W become smaller one by one;the potential increased;and the inhibitory effect of four peptides on human cervical cancer cells and mouse renal epithelial cells was tested by CCK8 experiment.When it is lower than 25?M,it has little effect on the growth of human cervical cancer cells and mouse kidney epithelial cells;the cell viability is higher than 90%,so selecting peptides with a concentration of less than 25?M in the transfection experiment can exclude the effect of peptides on cell growth.In order to study the binding mode and ability of peptides and DNA,the results of agarose gel electrophoresis experiments show that L-K6,L12 W and ct DNA are completely bound when the mass ratio is 1: 1,while I1 W and L5 W were completely bound to ct DNAwhen the mass ratio is 0.5: 1,indicating that L-K6 and its analogs can bind to ct DNA.are126.4nm,118.7nm,112.9nm and 86.4nm all of which are below 200 nm,the substance is beneficial to the uptake of cells.The Zeta potential of L-K6 and its analogs with ct DNA complex can neutralize the negative charge on the surface of DNA when the mass ratio is 0.25:1,and its zeta potential becomes positive,and the zeta potential increases with the increase of the mass ratio of peptide to DNA.The experiments of UV spectrum and circular dichroism indicate that the combination of L-K6 series peptides and ct DNA is electrostatically combined,and the binding capacity is positively related to the mass ratio of peptide to DNA.We study the efficiency of transfection of GFP-1 plasmid with antibacterial peptide L-K6 and its analogs as non-viral gene vectors.First,we prove that L-K6 and its analogs have no toxic effect on cells through hemolysis experiments;The GFP-1 expressed in human cervical cancer cells and mouse renal epithelial cells is observed by laser scanning confocal microscopy;flow cytometry is used to detect the expression efficiency of GFP-1 expressed green fluorescent protein in human cervical cancer cells and mouse kidney epithelial cells;the level of plasmid gene GFP-1 expressed green fluorescent protein in the cells show the following trends: L-K6 <L12W <L5W <I1W.The fluorescence microplate reader further show that the higher the mass ratio of L-K6 series peptides to DNA,the more obvious transfection effect.Compared with the commercial Lipo2000,the fluorescence intensity of I1 W and L5 W transfection increas by about 5%,indicating that the efficiency of gene transfection can be improved by using antimicrobial peptide L-K6 as template and replacing single Trp residues at different locations.