RESUMEN
A reliable, low-cost, and highly efficient nonviral gene delivery system using lower molecular weight polyethylenimine (LMW-PEI) is provided. LMW-PEI was linked to an expressing plasmid with green fluorescence protein gene (gfp), the transfection activity mediated by PEIs were examined in the CM7721 cell line and the skin tissue of mouse, respectively. The cytotoxicity of PEIs, the localization and continuance time of gfp expressed in the skin tissue of mouse were also studied. Results showed that the transfection rate of gfp mediated by LMW-PEI in the CM7721 cell line was about 55% . However, with the increasing PEI molecular weight, the cytotoxicity of PEI increased, but its transfection activity decreased. The tissue transfection results showed that LMW-PEI induced a significant expression of the gfp in the cells of hair vesicle and sweat gland of mouse skin tissues following transfection of 24 h, and the expression of gfp lasted 7 - 9 d. When the tissue of mouse was treated with retinoic acid and nitrogenous ketone, respectively, gfp was transferred to the granule layer of mouse skin tissue. The LMW-PEI described here is a new, highly efficient vector; it would be a useful nonviral vector for gene delivery technology.
Asunto(s)
Animales , Ratones , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Genética , Metabolismo , Inmunohistoquímica , Microscopía Fluorescente , Peso Molecular , Plásmidos , Química , Genética , Polietileneimina , Química , Toxicidad , Piel , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B-EGFP fusion protein and obtain a stable transfected HepG2 cell line.</p><p><b>METHODS</b>The coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho I/Kpn I. This fragment was inserted into pEGFPN3 with T4 ligase and transformed E. coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectin AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level NS5B-EGFP fusion protein was obtained.</p><p><b>RESULTS</b>The eukaryotic expression vector named pEGFPN3-ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B-EGFP fusion protein was obtained.</p><p><b>CONCLUSION</b>The stable transfected HepG2 cell line could express NS5B-EGFP fusion protein, could be used for anti-HCV infection with ns5b gene as the target.</p>
Asunto(s)
Humanos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Genética , Proteínas Fluorescentes Verdes , Genética , Metabolismo , Microscopía Fluorescente , Proteínas Recombinantes de Fusión , Genética , Metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas no Estructurales Virales , Genética , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To identify and characterize the epitope associated with the virus attachment protein (VAP) of hantaan virus.</p><p><b>METHODS</b>The monoclonal antibody 3G1 was used as the ligand to biospan from a phage-displayed 12-amino acid peptide library, then the positive phage clones were chosen and sequenced. The amino acid sequences of them were compared with that of hantaan virus G2 in homology. The characteristics of positive phage were studied by IFA and ELISA. A decapeptide combining to cell membrane was observed under laser scanning confocal microscope (LSCM).</p><p><b>RESULTS</b>The conservative motif PX(1-2) HX(0-2) H displaying on positive clones shared homologous amino acid sequence with G2 96YPWHTAKCHY105.</p><p><b>CONCLUSION</b>G2 96YPWHTAKCHY105 might play some roles in virus binding to host cell, and might be a possible key epitope of hantaan virus VAP.</p>
Asunto(s)
Animales , Cricetinae , Células CHO , Membrana Celular , Metabolismo , Chlorocebus aethiops , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Epítopos , Alergia e Inmunología , Metabolismo , Virus Hantaan , Alergia e Inmunología , Microscopía Confocal , Oligopéptidos , Alergia e Inmunología , Metabolismo , Biblioteca de Péptidos , Células Vero , Proteínas Virales , Alergia e Inmunología , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To detect succinate dehydrogenase (SDH) of sperm mitochondria by an improved method, and to evaluate the relationship between SDH and the motility and viability of sperm.</p><p><b>METHODS</b>In 46 fertile and infertile men (aged from 25 to 36), the motility, viability and mitochondrial SDH of sperm were detected by computer-assisted semen analysis system, dead/live sperm molecular fluorescent probe and an improved cytochemical method. Then, the correlation between the motility and viability of sperm and the positive percentage of sperm mitochondrial SDH were analyzed.</p><p><b>RESULTS</b>In the 46 fertile and infertile men, the motility and viability of sperm were (67.33 +/- 7.37)% and (79.78 +/- 7.65)%, and the positive percentage of sperm mitochondrial SDH was (74.74 +/- 8.29)%. The motility and viability of sperm and positive percentage of sperm mitochondrial SDH had significant correlation (r = 0.901, P < 0.01; r = 0.876, P < 0.01; r = 0.917, P < 0.01).</p><p><b>CONCLUSIONS</b>Detection of sperm mitochondrial SDH has significance in evaluation of sperm mitochondrial function and may serve as an assisted marker of sperm viability.</p>