Furthermore, the antibacterial ability against of BD2/3 in VRB2B3-PEG-O-CS-PEI group was significantly higher than those in the other groups. China). Construction of prokaryotic expression plasmid of fusion gene of BD2/3 To construct a fusion gene of BD2/3 (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”AF071216″,”term_id”:”3818536″,”term_text”:”AF071216″AF071216, “type”:”entrez-nucleotide”,”attrs”:”text”:”AF301470″,”term_id”:”10717135″,”term_text”:”AF301470″AF301470), six oligodeoxynucleotide fragments were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China (Table I). The fused gene of BD2/3 was prepared by overlap extension PCR (23). The stop codon of BD3 and the start codon of BD2 were deleted, and restriction sites (DH5) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl–D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains (ATCC25922, ATCC 26112, ATCC49619 and ATCC10211). Minimum inhibition concentration (MIC), minimal bactericide concentration (MBC) of fusion BD2/3 protein expressed by E. coli Broth dilution methods were carried out to determine the MIC of fusion BD2/3 protein against bacterial cultures of 5105 CFU/ml (24). MBCs were determined by transferring 100 l samples from clear wells onto agar plates without antibiotics. The MBC was the lowest concentration at which there was no visible microbial growth. Large-scale preparation of recombinant VRB2B3 A single colony of containing the recombinant VRB2B3 plasmid was inoculated in Luria Bertani (LB) broth with kanamycin (100 mg/ml), with shaking at 37C overnight. Plasmid DNA was extracted following large-scale alkaline lysis and precipitation by the spermine method (19), then suspended in sterile saline BAY-u 3405 water and stored at 20C until use. Preparation of LP Lecithin, cholesterol, octadecylamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and dimethyldistearylammonium bromide (DDAB) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). A mixture of 3 mg lecithin, 1.7 mg cholesterol and 0.5 mg octadecylamine (30:17:5 by weight) in 10 ml chloroform was added to a 250 ml round bottom flask and evaporated under vacuum in a rotary evaporator at 37C, forming a thin film on the inner surface. The 20 ml of ddH2O was added at 37C and the flask was shaken with intermittent sonication in a bath sonicator. Preparation of nanoparticles using LP modified CS Five different delivery systems (CS, PEG-O-CS-PEI, LP, PCL and PCL-protamine) with and without entrapped VRB2B3 (VRB2B3-CS, VRB2B3-PEG-O-CS-PEI, VRB2B3-LP, VRB2B3-PCL, VR B2B3-PCL-protamine) were prepared by the ionotropic gelation method (26). Briefly, biomaterials (CS, PEG-O-CS-PEI, LP, PCL and PCL-protamine) were diluted, respectively, by buffer CH3COOH/CH3COONa (pH 5.5) containing triphosphate and heated for 10 min at 65C with mild magnetic stirring. Then, the solution of plasmid was added slowly to the solution of biomaterial drop by drop, BAY-u 3405 and the mixed solution was remixed and left for 5 min. The average diameter and zeta potential of the polymeric micelles were detected by Zetasizer 3000 HS/IHPL (Malvern Instruments Ltd., Malvern, UK). Polyamine cationic liposomes (PCL) were prepared using 30 mg DOPE, 10 mg cholesterol and 10 mg DDAB per round bottom flask and were used to produce PCL as described above. PCL/protamine formulations were prepared by adding protamine to the PCL (Vprotamine:VPCL = 1.5:1). CS (95% deacylated, MW = 150 Rabbit polyclonal to RB1 kDa) was supplied by Chengdu Organic Chemistry Institute of China Academy of Science; polyethyleneglycol-O-chitosan-polyethyleneimine (PEG-O-CS-PEI) was provided by the College of Chemistry of Sichuan University (25). Agarose gel electrophoresis assay of nanoparticles The DNA binding ability of biomaterials (CS, PEG-O-CS-PEI, LP, PCL and PCL-protamine) were evaluated by agarose gel electrophoresis. The nanoparticle solutions of plasmid DNA with biomaterials (CS, PEG-O-CS-PEI, LP, PCL and PCL-protamine) copolymer were loaded into individual wells of 0.7% agarose gel, electrophoresed at 100 V for 45 min and stained with 0.01% gold-view. The plasmid migration pattern was revealed under UV irradiation. Transfection and efficiency analysis of fusion BD2/3 gene in eukaryotic cells in vitro 293 cells (human embryonic kidney cells; ATCC no. CRL-1573TM) were BAY-u 3405 purchased from the Chinese Academy of Science Cell bank (Shanghai, China). 293 cells were cultured in 6 well plates (1.5106 cells/well) for 24 h and grown in 2 ml Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 4.0 mM L-glutamine, 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin (Thermo Fisher Scientific, Inc.), and maintained at 37C in a 5% CO2 humidified incubator (Sanyo Electric Co., Ltd., Tokyo, Japan) until the cell confluency of 293 achieved 80%. The complexes of nanoparticles containing CS, PEG-O-CS-PEI, LP, PCL and PCL-protamine each containing 5 g VRB2B3 plasmid were added into each well to transfect.