ABSTRACT
In order to study the important factors involved in cationic liposome-mediated gene transfer, Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies. Different properties of the two reagents were analyzed and compared by DNA arrearage assay and MTT assay. Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell. However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell. On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition. Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.
Subject(s)
Humans , Cell Line, Tumor , Cell Survival , DNA , Genetics , Fatty Acids, Monounsaturated , Chemistry , Toxicity , Gene Transfer Techniques , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins , Metabolism , Lipids , Chemistry , Toxicity , Quaternary Ammonium Compounds , Chemistry , Toxicity , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the immune responses and protection from virus challenge, induced by the coinjection of IL-2cDNA with herpes simplex virus type 1 (HSV-1) glycoprotein-D (gD) DNA vaccine.</p><p><b>METHODS</b>Two DNA vaccines (pgD and pIL-2) were constructed by inserting the gD gene and IL-2 cDNA into the eukaryotic expression vector pcDNA3.1, respectively. The BALB/c mice were inoculated intramuscularly three times at 2-week intervals. Two weeks after the final immunization, mice were bled for antibody assay and spleen cells were separated for Th cell proliferation and cytokine assays. Delayed type hypersensitivity (DTH) response was detected by the pinna-swelling test. Corneal protection under HSV-1 virus challenge was continuously observed with slit-lamp microscope.</p><p><b>RESULTS</b>IL-2 cDNA coinjection remarkably enhanced the specific IgG2a level when compared with gD plasmid vaccination alone. Th cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by IL-2 cDNA coinjection. However, the production of IL-10 was inhibited. The DTH response was also enhanced by IL-2 coinjection. When the mice were challenged with HSV-1, the cornea epithelial lesions were significantly alleviated by IL-2 coinjection as compared with gD vaccination alone.</p><p><b>CONCLUSION</b>IL-2 cDNA can enhance both the humoral and cellular immune responses, and thus increase the vaccine potency.</p>
Subject(s)
Animals , Female , Mice , Antibodies, Viral , Blood , COS Cells , Cell Proliferation , Chlorocebus aethiops , DNA , Genetics , Herpesvirus 1, Human , Virulence , Hypersensitivity, Delayed , Allergy and Immunology , Immunization , Immunoglobulin G , Blood , Interferon-gamma , Blood , Interleukin-2 , Genetics , Mice, Inbred BALB C , Random Allocation , Th1 Cells , Cell Biology , Transfection , Vaccines, DNA , Allergy and Immunology , Viral Envelope Proteins , Genetics , Viral Vaccines , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>The goal of this study was to construct a eukaryotic expression plasmid containing the gene encoding herpes simplex virus type I glycoprotein D (HSV-1, gD) and evaluate its utility for DNA immunization in mice.</p><p><b>METHODS</b>The gD gene was amplified from viral DNA using PCR with EcoR I and BamH I restriction sites encoded on 5' and 3' ends, respectively. The PCR fragment was inserted into the transfer vector pGEM-T Easy. gD was then cut from this vector and inserted into the EcoR I and BamH I sites in the pcDNA3.1 at the multiple cloning sites (MCS). The recombinant plasmid, pcDNA3.1-gD1, was transfected into COS-7 cells using Lipofectamine according to the manufacture's instructions. The expression of the glycoprotein D was analyzed by immunoblotting of the cell lysates. 4-6 weeks old BALB/C mice were given two injections at tibia anterialis muscle, each containing 100 micrograms of plasmid DNA, on days 0 and 15. pcDNA3.1 was used as negative control. Blood samples were taken from all mice at weeks 0, 2, 4, and 6 after the first inoculation. Standard indirect ELISA was employed to evaluate the levels of specific total Ig in serum.</p><p><b>RESULTS</b>The recombinant plasmid was confirmed with restriction digestion and sequencing to contain target gene segment and expressed in COS-7 cells in vitro shown by Western blotting. The pcDNA3.1-gD1 immunized group induced specific antibody response as compared to the negative control, and the titer was about 1:2000.</p><p><b>CONCLUSIONS</b>The recombinant plasmid pcDNA3.1-gD1 is potential to be used as a candidate vaccine, for the treatment of HSV-1 infection.</p>
Subject(s)
Animals , Humans , Mice , COS Cells , Metabolism , Escherichia coli , Genetics , Genetic Vectors , Mice, Inbred BALB C , Vaccines, DNA , Genetics , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and ImmunologyABSTRACT
The 125 strains of the symbiotic and epiphyte microorganisms were isolated from marine organisms (Sea cucumber, Sea urchin, Sea anemone, Sea actinia, Ulra, Sargassum, Undaria). Among them,21 strains of bacteria,8 strains of actinomycetes and 2 strains of fungi have shown to have antagonistic activity on bacterial or fungal growth. In the 21 strains of bacteria, 7 strains belong to Bacillus sp., 11 strains belong to Vibro sp., and 3 strains belong to Pseudomonas sp.. In the 8 strains of actinomycetes, 5 strains belong to Streptomyces sp., 3 strains belong to Micromonospora sp.. 2 strains of fungi belong to Penicillum sp..