OBJECTIVETo investigate the expression and analysis of its activity of anti-bacterial peptide gloverin in COS-7 cells.

METHODSThe appearance frequency of all genetic codes in the cDNA sequence from the same species of protein Attacin A was analyzed, and its cDNA sequence was synthesized by PCR overlapping extension method in conjunction with the designation of the known protein sequence of gloverin. The genes were inserted into pCDSI, an eukaryotic vector, after being identified correctly. As a result, the vector pBZHG was constructed. Thereafter, the liposome FuGENE( trade mark ) 6 was employed as the vector, and the COS-7 cells were transfected with liposome pBZHG and blank vector pCDSI. The normal cells were taken as the control. The supernatant was collected for the detection of its bactericidal activity after 72 PBHs.

RESULTSThe gloverin cDNA sequence designed artificially was expressed in COS-7 cells. The supernatant of the cells transfected by pBZHG exhibited bactericidal activity to E. coli J5 when compared with that from normal cells and in cells transfected with blank vectors.

CONCLUSIONThe designed cDNA sequence of gloverin was proved to be genuine, and it provided the basis for future study of its antibiotic and anti-endotoxin activities.

Animals ; Anti-Bacterial Agents ; pharmacology ; COS Cells ; Cercopithecus aethiops ; Proteins ; genetics ; pharmacology ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection.

METHODSIron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet.

RESULTSForeign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold.

CONCLUSIONIONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Animals ; COS Cells ; Ferric Compounds ; administration & dosage ; Genetic Vectors ; Magnetics ; Polylysine ; administration & dosage ; Transfection ; methods

The purpose of this research was to construct a lentiviral vector containing pir-b gene, and to detect the expression of pir-b gene in 293T cells. The open reading frame (ORF) of pir-b gene from the mRNA of mouse was cloned, then inserted into a sequencing vector. The pir-b gene was subcloned into the transfer plasmid of the lentivirus system, which was transfected together with the packaging plasmids into 293T cells by Lipofectin 2000, thereafter, the supernatant was collected and concentrated to transfect 293T cells. Western blot was used to detect the expression of the exogenous PIR-B after 293T cells were infected by the virus, while the lentivirus harboring egfp gene was packaged as the control group. The result indicated that the ORF of the pir-b gene was successfully cloned, the sequence of which was consistent to that was expected and the PIR-B protein could be expressed in 293T cells normally. It is concluded that the lentiviral vector containing pir-b gene was constructed successfully, which would contribute to illustrating the important role of pir-b gene in the immunological regulation.
Animals ; COS Cells ; Cercopithecus aethiops ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; RNA, Messenger ; genetics ; Receptors, Immunologic ; genetics

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by coexpression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Suprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4- dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evlauate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.
Adsorption ; Animals ; Antibodies ; COS Cells ; DNA ; Helper Viruses ; HIV-1* ; Membranes ; Plasmids ; Product Packaging ; RNA ; Virion

TREK (TWIK-RElated K+ channels) and TRAAK (TWIK-Related Arachidonic acid Activated K+ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K+ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.
Animals ; Arachidonic Acid ; Chimera ; COS Cells ; Dithionitrobenzoic Acid ; Dithiothreitol ; Membranes ; Oxidants

This study inquired about the influences on the gene delivery efficiency of polyethylenimine. pSVbeta plasmids were transferred into COS-7 and NIH3T3 cells with polyethylenimine. Influences of plasmids factor, albumin, serum, cell density, operation methods and polyethylenimine/DNA preserve factors on transfection efficiency were investigated. Inhibitors of biological activities in plasmids could be removed by ultrafiltration with cutoff molecular weight of 3000 or 10000. Linear plasmids lowered transfection efficiency. Serum and albumin in the culture medium decreased the transfection efficiency of polyethylenimine/DNA. Cell density was associated with PEI/DNA transfection efficiency. Incubation of PEI/DNA complexes with the cells for 8 h and then aspiration for removal of the complexes could obtain an optimal transfection effect. Freezing of PEI/DNA complexes significantly decreased transfection efficiency. In conclusion, polyethylenimine could obtain optimal and reduplicate transfection results by controlling related factors.
3T3 Cells ; Animals ; COS Cells ; Cercopithecus aethiops ; DNA ; genetics ; Genetic Vectors ; genetics ; Mice ; Plasmids ; genetics ; Polyethyleneimine ; chemistry ; Transfection ; Ultrafiltration

OBJECTIVEThis study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.

METHODSEukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.

RESULTSIt proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.

CONCLUSIONThe eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

Animals ; Bacterial Proteins ; genetics ; COS Cells ; Cercopithecus aethiops ; Cysteine Endopeptidases ; genetics ; Genetic Vectors ; Plasmids ; Porphyromonas gingivalis ; genetics ; Transfection

In order to explore the effects of 35C > T and 682A > G mutations on the activity of alpha-(1,2) fucosyltransferase, the coding region of fut1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA. PCR product was ligated into expression vector using TOPO TA cloning kit to obtain the recombinant plasmids. The recombinant plasmids were transfected into COS-7 cells by liposome method. After screening by using G418, H antigen expression on the COS-7 was tested by flow cytometry and fut1 mRNA was detected by real-time PCR. The results indicated that three kinds of recombinant plasmids pcDNA3.1/V5-His-wild (35C + 682A), pcDNA3.1/V5-His-35T and pcDNA3.1/V5-His-35T-682G were successfully constructed. After transfection, the H antigen expressed on membrane of COS-7 cells at the second day, with the maximum level of expression at the fourth day. When compared with pcDNA3.1/V5-His-wild transfected cells, the H antigen expression level of the 35T and 682G + 35T recombinant plasmids in the transfected cells was 52.7% and 13.3% on the fourth day, respectively. Although the level of fut1 mRNA decreased with prolonging of time, the mRNA expressed on the pcDNA3.1/V5-His-35T-682G transfected cells reached to 14% of the wild plasmids on the first day. It is concluded that 682A > G mutation obviously reduces the activity of alpha-(1,2) fucosyltransferase, while 35C > T mutation leads to partial reduction of H antigen in vitro expression.
Animals ; Antigens, Bacterial ; genetics ; COS Cells ; Cercopithecus aethiops ; Fucosyltransferases ; genetics ; Genetic Vectors ; Mutation ; Plasmids ; RNA, Messenger ; genetics ; Transfection

In order to construct an eukaryotic expression vector for gene of multiple myeloma mucin1 (muc1-2vntr) gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine. muc1-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind III and XbaI restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector, and the resulted recombinant vector was transformed into E.coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3.1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using muc1 monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr/myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.
Animals ; Base Sequence ; COS Cells ; Cercopithecus aethiops ; Genetic Vectors ; Molecular Sequence Data ; Mucin-1 ; genetics ; Multiple Myeloma ; genetics ; Plasmids ; Transfection

BACKGROUND: In oxidative stress, reactive oxygen species (ROS) production contributes to cellular dysfunction and initiates the apoptotic cascade. Autophagy is considered the mechanism that decreases ROS concentration and oxidative damage. Propofol shows antioxidant properties, but the mechanisms underlying the effect of propofol preconditioning (PPC) on oxidative injury remain unclear. Therefore, we investigated whether PPC protects against cell damage from hydrogen peroxide (H₂O₂)-induced oxidative stress and influences cellular autophagy. METHOD: COS-7 cells were randomly divided into the following groups: control, cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂) for 24 h without propofol; H₂O₂, cells were exposed to H₂O₂ (400 µM) for 2 h; PPC + H₂O₂, cells pretreated with propofol were exposed to H₂O₂; and 3-methyladenine (3-MA) + PPC + H₂O₂, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to H₂O₂. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) reduction. Apoptosis was determined using Hoechst 33342 staining and fluorescence microscopy. The relationship between PPC and autophagy was detected using western blot analysis. RESULTS: Cell viability decreased more significantly in the H₂O₂ group than in the control group, but it was improved by PPC (100 µM). Pretreatment with propofol effectively decreased H₂O₂-induced COS-7 cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol during apoptosis. Western blot analysis showed that the level of autophagy-related proteins was higher in the PPC + H₂O₂ group than that in the H2O2 group. CONCLUSION: PPC has a protective effect on H₂O₂-induced COS-7 cell apoptosis, which is mediated by autophagy activation.
Animals ; Apoptosis* ; Autophagy* ; Blotting, Western ; Cell Survival ; COS Cells* ; Hydrogen Peroxide ; Methods ; Microscopy, Fluorescence ; Oxidative Stress ; Propofol* ; Reactive Oxygen Species